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TP009 Tópicos Especiais em Alimentos e Nutrição

Food Safety

PROGRAMA FINAL As palestras da manhã serão sempre no ITAL. Local: Auditório Central do ITAL Endereço: Av. Brasil, 2880 - Jardim Chapadão, Campinas - SP, 13070-178, Campinas. Avisos importantes:

Os alunos poderão almoçar no ITAL, pelo preço de R$10,00. Confirmar interesse junto ao professor um dia antes. Avaliação da disciplina:

Presença mínima de 75% às aulas teóricas. Não será cobrada presença às visitas.

Apresentação de um seminário no dia 28/04, das 14 às 17h no Salão Nobre da FEA-UNICAMP. o No dia 24/04, os alunos da FEA deverão se organizar em 4 grupos de aproximadamente 6 alunos. o Cada grupo fará a apresentação de um dos artigos abaixo. o O critério de avaliação será: forma, conteúdo e tempo de apresentação.

Forma: qualidade do formato (Power Point) Conteúdo: qualidade da informação da palestra Tempo: 30 minutos +/- 3 minutos.

o A apresentação ocorrerá no dia 28/04, sexta-feira, a partir das 14h no Salão Nobre da FEA. o Os alunos terão a manhã do dia 28/04 livre para prepararem a apresentação. o Cada grupo terá 30 minutos para a apresentação + 5 minutos para perguntas. o As notas são individuais; portanto, todos os elementos do grupo devem falar na apresentação do seminário.

Calendário detalhado: Monday 24th: Day organized by ITAL (Institute of Food Technology). 8:30 – 12:00 (ITAL)

Protein-polysaccharide interactions: applications in food dispersions Dr Mitie Sonia Sadahira Use of residues and by-products from fruit and vegetable industry: potentials and challenges

Dr Sílvia P. M. Germer & Dr Cristhiane C. Ferrari

Sustainability & Ethics – key issues for packaging and food trends Dr Anna Lucia Mourad Food safety in low acid canned foods Dr Maria Isabel Berto

(ITAL) Afternoon: visits to CETEA, TECNOLAT e Cereal Chocotec Students from FEA-UNICAMP will organize themselves in 4 groups and

will

Tuesday 25th Day organized by UNICAMP (University of Campinas) 8:30 – 12:00 (ITAL)

Nutrition and bioactive substances Dr Mario Marostica –Dep. Food and Nutrition Processing food by high pressure technology Dr Marcelo Cristianini- Dep. Food Technology Microbial production of bioactice compounds Dr Juliano Bicas -Dep. Food Sciences Improving the yeast-based biorefinery Dr Andreas Gombert –Dep. Food Engineering

14:00 (FEA-UNICAMP)

Afternoon Visit to FEA Meeting point: central square of the School of Food Engineering (FEA)

Wednesday 26th: Day Organized by Institute of Biosciences, USP, Sao Paulo: Food quality in a world with Global Climate Change 8:30 – 12:00 (ITAL)

Climate Change and food production Dr Marcos Buckeridge Using a systems biology approach to assess plant responses to the environment

Laboratory of Plant Physiological Ecology - LAFIECO

Laboratory versus field experiments: Can food quality really change? Afternoon visit to LNBIO, Synchrotron (to be confirmed)

Thursday 27th Day organized by School of Pharmacy, Dep Nutrition and Food Sciences USP, Sao Paulo Microbial control through innovative approaches Dr. Uelinton Pinto Nutrition in early life and developmental programming of chronic disease:

focus on epigenetic mechanisms Dr. Thomas Prates Ong

Biological effects of fruit pectins: a new perspective Dr. João Paulo Fabi Afternoon : visit to Fazenda Santa Elisa, Agronomic (to be confirmed)

Friday 28th - School of Food Engineering – UNICAMP Morning

The students are free to prepare the Seminar for the afternoon.

14:00 – 17:00 FEA Salão Nobre UNICAMP

Seminars: Group1 - Improving conversion yield of fermentable sugars into fuel ethanol in 1st generation yeast-based production processes. Group 2 - Bioaromas e Perspectives for sustainable development. Group 3 - Fructooligosaccharide intake promotes epigenetic changes in the intestinal mucosa in growing and ageing rats. Group 4 - High pressure processing (HPP) of pea starch: Effect on the gelatinization properties.

MARIO ROBERTO MAROSTICA JUNIOR

Improving conversion yield of fermentable sugars intofuel ethanol in 1st generation yeast-based productionprocessesAndreas K Gombert1 and Antonius JA van Maris2

Available online at www.sciencedirect.com

ScienceDirect

Current fuel ethanol production using yeasts and starch or

sucrose-based feedstocks is referred to as 1st generation (1G)

ethanol production. These processes are characterized by the

high contribution of sugar prices to the final production costs,

by high production volumes, and by low profit margins. In this

context, small improvements in the ethanol yield on sugars

have a large impact on process economy. Three types of

strategies used to achieve this goal are discussed: engineering

free-energy conservation, engineering redox-metabolism, and

decreasing sugar losses in the process. Whereas the two

former strategies lead to decreased biomass and/or glycerol

formation, the latter requires increased process and/or yeast

robustness.

Addresses1 Faculty of Food Engineering and Bioenergy Laboratory, University of

Campinas, Rua Monteiro Lobato, 80, 13083-862 Campinas, SP, Brazil2 Department of Biotechnology, Delft University of Technology,

Julianalaan 67, 2628 BC, Delft, The Netherlands

Corresponding author: van Maris, Antonius JA

([email protected])

Current Opinion in Biotechnology 2015, 33:81–86

This review comes from a themed issue on Energy biotechnology

Edited by E Terry Papoutsakis and Jack T Pronk

http://dx.doi.org/10.1016/j.copbio.2014.12.012

0958-1669/# 2014 Elsevier Ltd. All rights reserved.

Introduction90 billion liters of fuel ethanol are currently produced

worldwide (Renewable Fuels Association; URL: www.

ethanolrfa.org) using almost exclusively starch or sucrose

containing feedstocks. The hexose sugars released from

for instance corn starch (by industrial hydrolytic enzymes)

or sugar-cane derived sucrose (by yeast invertase) can

directly be fermented to ethanol by yeast. These process-

es are referred to as 1st generation (1G) fuel ethanol

production. With a high contribution (up to 70%) of

the feedstock to the final production cost [1], high pro-

duction volumes and small profit margins, the overall

conversion yield of the raw material into ethanol is crucial

for the process economy. This review focuses specifically

www.sciencedirect.com

on recent scientific advances with the potential to

improve the ethanol yield on sugar. Strategies that

aim at increasing ethanol titers, such as very high

gravity fermentation, resulting in decreased distillation

costs, decreased contamination risks, and decreased

vinasse production [2], are beyond the scope of this

review.

Conversion of 1 mol of hexose sugar into 2 mol ethanol

and 2 mol CO2 is a redox-neutral conversion (Figure 1).

This makes the maximum theoretical yield 0.51 g ethanol

per g hexose sugar. Industrial ethanol production operates

at >90% of this theoretical yield [3]. Yeast biomass and

glycerol are the two main by-products of ethanol produc-

tion, besides the unavoidable production of CO2. Under

anaerobic conditions alcoholic fermentation of sugars is

the sole pathway in yeast that provides energy in the form

of ATP for cellular maintenance and, if sufficient ATP is

available, for growth. When ATP is used for growth, yeast

biomass and accompanying glycerol (see below) are

formed at the expense of feedstock that is not converted

to ethanol (Figure 1). Any reduction in yeast biomass

production and glycerol formation will result in increased

ethanol yields.

Industrial ethanol processes are often carried out without

complete asepsis. Growth of contaminating microorgan-

isms can divert sugar away from ethanol formation or even

result in incomplete fermentations due to accumulation

of toxic compounds. Use of robust yeast strains that can

operate at high ethanol concentrations and at decreased

pH creates a selective advantage over potential contami-

nants and decreases these losses.

Engineering free energy conservation toincrease ethanol yieldGrowth of yeast and the accompanying glycerol formation

diverts carbon away from ethanol production. The extent

of this growth is dependent on the availability of energy in

the form of ATP (Figure 1). If the ATP yield on sugar is

decreased, this increases the ethanol yield on sugar in two

ways [4]. Firstly, more sugar has to be converted solely to

ethanol and CO2 to provide the same amount of ATP for

cellular maintenance. Secondly, in a strain with a de-

creased ATP yield on sugar, but with identical biomass

yield on ATP, an increased fraction of the sugar is con-

verted to ethanol, simultaneously decreasing the biomass

and glycerol yields.


http://crossmark.crossref.org/dialog/?doi=10.1016/j.copbio.2014.12.012&domain=pdf

mailto:[email protected]

http://dx.doi.org/10.1016/j.copbio.2014.12.012

http://www.ethanolrfa.org/

http://www.ethanolrfa.org/

http://www.sciencedirect.com/science/journal/09581669

82 Energy biotechnology

Figure 1

sugar

ethanol + CO2glycerol

yeast biomass

ATP growth

mai

nten

ance

NADH

Robustness & diversity

NADH

Current Opinion in Biotechnology

Schematic representation of the distribution of sugar for ethanol

production, formation of yeast biomass, and formation of glycerol as a

by-product. To achieve a high ethanol yield on sugar, the robustness

of the process and yeast strains are essential.

Replacing the Embden–Meyerhof glycolysis, which

yields 2 ATP per hexose, by a heterologous Entner–Doudoroff pathway that yields 1 ATP per hexose would

decrease the ATP yield on sugar. To investigate this

possibility, Benisch and Boles [5�] constructed a yeast

strain containing 6-phosphogluconate dehydratase and 2-

keto-3-deoxygluconate-6-phosphate (KDPG) aldolase

from Escherichia coli. High activities were shown for

KDPG-aldolase. However, activities of the heterologous

6-phosphogluconate dehydratase were insufficient for

functional replacement of the Embden–Meyerhof glycol-

ysis by the Entner–Doudoroff route, which was attributed

to poor assembly of the [4Fe–4S] iron–sulfur cluster of the

6-phosphogluconate dehydratase in yeast. These findings

illustrate that functional expression of bacterial proteins

containing iron–sulfur clusters remains a challenge in

yeast metabolic engineering [5�,6].

Engineering the stoichiometry of sugar transport provides

another opportunity to decrease the ATP-yield on sugar.

Wild type Saccharomyces cerevisiae strains hydrolyze su-

crose extracellularly and use facilitated diffusion to take

up glucose and fructose. When this mechanism is

replaced by sucrose uptake via proton symport and intra-

cellular hydrolysis, the ATP requirement for subsequent

proton extrusion decreases the anaerobic ATP yield on

Table 1

Selected strategies to increase ethanol yield on sugar in first generat

Breeding/biodiversity

Desired traits of different strains into one strain [25�,53]

Decrease glycerol [14�,15]

Decrease free-energy conservation

Increase stress tolerance in yeast

Decrease contamination


sucrose from 4 to 3. Requiring a combination of metabolic

and evolutionary engineering, this strategy resulted in an

11% increase of the ethanol yield on sucrose [7�](Table 1). This same strategy can in theory be applied

to replace the facilitated diffusion of the hexose sugars

with transport via proton-symport, resulting in a 50%

decrease in the ATP yield from 2 to 1 mol per mol of

hexose. That this strategy not necessarily requires het-

erologous transporters, was shown by the characteriza-

tion of the fructose/H+ symporter Fsy1 from a wine strain

of S. cerevisiae [8,9].

Whereas the abovementioned strategies all rely on chang-

ing the stoichiometry of ATP formation in sugar metabo-

lism, other strategies apply non-stoichiometric ATP

drains by intervening in ATP or H+ homeostasis. A classic

example of this strategy is introduction of ATP-hydrolyz-

ing futile cycles in yeast through the deregulation of some

gluconeogenic enzymes [10]. A recent attempt to increase

ATP hydrolysis, thereby potentially decreasing growth

and increasing alcoholic fermentation, encompassed the

overexpression of ATPase [11]. Further studies are re-

quired to quantify the impact on the ethanol yield under

industrially relevant conditions. In another study, the

authors claim that overexpression of alkaline phospha-

tase Pho8 increased the ethanol yield on sugar by up to

13%, despite a small impact on intracellular concentra-

tions of ATP [12]. However, the challenge with the

introduction of such non-stoichiometric ATP drains,

especially for industrial implementation, is in the fine

tuning between the positive impact and decreased cel-

lular robustness.

Decreasing formation of glycerol as a by-product to increase the ethanol yieldGlycerol is the 3rd major by-product of alcoholic fermen-

tation after co-production of CO2 and yeast biomass. It is

estimated that in industrial fermentations approximately

4% of the sugar feedstock ends up as glycerol [13]. In

anaerobic yeast fermentations, formation of glycerol is

essential to re-oxidize surplus NADH resulting from

growth on sugars. Additionally, glycerol is the main com-

patible solute in yeast, produced in response to the high

osmotic pressure that can occur in some process config-

urations. A first approach to minimize the formation of

ion fuel ethanol production

Genetic strategies Process

strategies

Recombinant DNA Evolution Shuffling

[17,20,21�,24�]

[5�,7�,11,12] [7�]

[35,46,34] [36] [32,33]

[27,30,31�]


Improving ethanol yield in 1G yeast-based processes Gombert and van Maris 83

‘excess’ glycerol that is not strictly required for anaerobic

growth investigated the natural biodiversity amongst

yeasts and resulted in identification of S. cerevisiae strains

with lower than average glycerol production [14�]. With

rapid advances in whole genome sequencing, screening of

this natural biodiversity resulted in identification of mul-

tiple alleles of regulatory or structural genes conferring a

decreased-glycerol-formation-phenotype and concomi-

tant increase in ethanol yield [14�,15]. Another approach

used genetic modifications to directly intervene in glyc-

erol formation. Glycerol-3-phosphate dehydrogenase is a

key enzyme for glycerol production and yeast contains

two isoenzymes encoded by GPD1 and GPD2. Deletion of

both genes eliminates glycerol formation, but also results

in a yeast strain unable to grow under anaerobic condi-

tions [16]. One approach shown to successfully decrease

the formation of glycerol by up to 61%, is the fine-tuning

of the promoter strengths of GPD1 and/or GPD2 [17],

albeit at the expense of decreased anaerobic growth and

fitness [18].

The remainder of glycerol formation is required to re-

oxidize the excess biosynthetic NADH [19]. A first

category of approaches to decrease glycerol formation

provides alternative redox sinks through metabolic engi-

neering. Jain et al. [20] introduced alternative oxidore-

ductases resulting in sorbitol or 1,2-propanediol

formation. Of these options, formation of 1,2-propane-

diol (via NADH dependent methylglyoxal reduction)

has stoichiometric potential to improve the ethanol

yield, by requiring only 0.25 mol glucose and 0.5 mol

ATP per mol NADH re-oxidized. In comparison, reoxi-

dation of NADH through glycerol formation requires

0.5 mol glucose and 1 mol ATP. Another illustration of

this concept is formation of formic acid as a redox sink,

through the introduction of pyruvate formate lyase and

acetylating acetaldehyde dehydrogenase [21�]. Since

the resulting acetaldehyde can be further converted to

ethanol, excess NADH can be re-oxidized without re-

quiring extra sugar. A potential drawback of this ap-

proach is the role of formic acid as an inhibitor of yeast

fermentations [22,23], especially at low pH values pre-

ferred in industrial fermentations.

Conversion of 2 mol of CO2 and 6 mol NADH to 1 mol

ethanol and 3 mol of water is stoichiometrically the most

efficient way to re-oxidize excess NADH on glucose as

the sole carbon source. To use CO2 as electron acceptor,

Guadalupe et al. [24�] functionally expressed phosphor-

ibulokinase and Rubisco, the two key enzymes of the

Calvin cycle, in S. cerevisiae. This resulted in 90%

decrease of glycerol production and an accompanying

10% increase in the ethanol yield on glucose in anaero-

bic hexose-limited chemostat cultures. Although suc-

cessful as proof-of-principle, improvements in growth

rate and genetic constructs are required for industrial

implementation.


Improving process and yeast robustness toincrease the ethanol yieldThe low-cost requirement precludes full asepsis in in-

dustrial ethanol production. Contaminating microorgan-

isms can decrease the ethanol yield directly by consuming

sugars or because they produce metabolites that inhibit

yeast performance. In addition to process hygiene mea-

sures, such as centrifugation and sulfuric acid washing

during cell recycling in Brazilian ethanol processes, dif-

ferential tolerance of yeast and of contaminating micro-

organisms towards process conditions can be explored to

minimize the impact of contamination on fermentations.

High ethanol titers give a selective advantage to yeast

over contaminants. Pooled-segregant whole-genome se-

quence analysis was used to investigate ethanol tolerance

[25�] and can potentially improve ethanol tolerance of

industrial S. cerevisiae strains through reverse engineering.

Mapping QTL’s related to stress factors, as performed by

Greetham et al. [26], might also lead to superior pheno-

types. The use of bacteriocins such as nisin represents an

alternative approach to control levels of gram-positive

contaminants, such as Lactobacilli [27], thereby avoiding

the use of antibiotics and the undesirable emergence of

bacterial resistance. Investigating the physiology of com-

mon contaminant yeasts, such as Dekkera bruxellensis [28–30] or of contaminant bacteria, such as Lactobacilli [31�],might lead to new strategies to control contamination

levels. Other examples to increase stress tolerance of

yeast and thereby create a selective advantage over con-

taminants include the use of whole genome shuffling

[32,33], enhancement of acetic acid tolerance via over-

expression of HAA1 [34], thermotolerance enhancement

via overexpression of a RSP5-BY allele in a htg6 strain

background [35] or via laboratory evolution [36,37�].

Another consequence of the ‘open’ nature of industrial

ethanol fermentations, especially in the Brazilian setting,

is that no matter which yeast strain is inoculated, it might

be substituted by a different wild yeast strain during the

production season. In fact, the strain displaying the highest

specific growth rate under the particular process conditions

will always outcompete other strains, which complicates

the introduction of engineered strains in these processes.

Thus, understanding why and how some yeast strains are

naturally capable of persisting and eventually dominating

in the industrial environment is essential. In the first place,

there is a need to identify such strains and one of the

seminal works with this purpose was performed by Basso

et al. [38]. More recently, some DNA fingerprinting meth-

ods that can uniquely identify S. cerevisiae strains were

reported [39,40]. Whole-genome sequences are now avail-

able for three such wild isolates: the CAT-1 strain [41], a

haploid spore of the PE-2 strain [42] and a haploid deriva-

tive of the YJS329 strain [43].

Once identified, physiological analysis of these strains is

important. In one example, Ramos et al. [44] isolated



several S. cerevisiae strains from distilleries and evaluated

stress tolerance, flocculation, glycerol and ethanol yields

on substrate and biomass formation. Although mainly

concerned with wine strains, Henderson et al. [45]

reported interesting results correlating ethanol tolerance

to lipid composition. Zheng et al. [46] used comparative

functional genomics to understand differences in the

physiology of the industrial fuel ethanol strains YJS329

and ZK2. Recent studies using pure culture and labora-

tory media show that, quite surprisingly, fuel ethanol and

laboratory strains only behave differently under very

specific conditions, for example at very low pH in com-

plex media [47,48�]. Brown et al. [49] investigated the

transcriptome of fuel ethanol strains under industrial-like

conditions. None of the above mentioned reports have

yet provided a definitive answer on why and how some

yeast strains persist and/or dominate in this industrial

process.

ConclusionsAlthough the first 2G fuel ethanol factories have started,

1G ethanol production will probably remain either as a

standalone technology or as part of biorefineries. Howev-

er, most of the abovementioned improvements in the

ethanol yield, have hitherto only been demonstrated at

laboratory scale and/or in laboratory media. Competition

of genetically engineered yeast strains with wild yeast

strains in non-aseptic processes provides an important

challenge. Engineering such strategies in those yeast

strains that already persist in the industrial process might

(partially) prevent this. Therefore, the development of

recombinant DNA technology protocols suitable for in-

dustrial yeast strains, which are not necessarily as amena-

ble to genetic modifications as laboratory strains [50,51] is

of high importance. Interestingly, recent developments,

such as the CRISPR/Cas9 system and simultaneous inte-

gration of multiple genes [52,53], might result in in-

creased accessibility of industrial yeasts for metabolic

engineering.

AcknowledgementWe thank Maarten D. Verhoeven for his assistance with the lay out of themanuscript.

References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:

� of special interest�� of outstanding interest

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5.�

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7.�

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21.�

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31.�

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lable at ScienceDirect

Trends in Food Science & Technology 62 (2017) 141e153

Contents lists avai

Trends in Food Science & Technologyjournal homepage: http: / /www.journals .e lsevier .com/trends- in- food-science-

and-technology

Review

Bioaromas e Perspectives for sustainable development

Lorena de Oliveira Felipe a, Ana Maria de Oliveira a, Juliano Lemos Bicas a, b, *

a Department of Chemistry, Biotechnology and Bioprocess Engineering, Alto Paraopeba Campus, Federal University of S~ao Jo~ao Del-Rei, Rod. MG443, Km 7,Fazenda do Cadete, 36420-000, Ouro Branco, MG, Brazilb Department of Food Science, School of Food Engineering, University of Campinas, Rua Monteiro Lobato, 80, Cidade Universit�aria, 13083-862, Campinas, SP,Brazil

a r t i c l e i n f o

Article history:Received 24 May 2016Received in revised form16 January 2017Accepted 13 February 2017Available online 28 February 2017

Keywords:AromaBiotransformationFlavorSustainabilityTerpenes

* Corresponding author. Department of Food ScienceUniversity of Campinas, Rua Monteiro Lobato, 80, CidCampinas, SP, Brazil.

E-mail address: [email protected] (J.L. Bicas).

http://dx.doi.org/10.1016/j.tifs.2017.02.0050924-2244/© 2017 Elsevier Ltd. All rights reserved.

a b s t r a c t

Background: Aroma compounds can be produced using three main methods: chemical synthesis,extraction from nature, and biotechnological process (bioaromas). In the latter method, when comparedwith chemical synthesis and direct extraction from nature, the (bio)aroma compounds obtained presentnumerous advantages, in such a way that this approach meets two important demands of modern so-ciety: the first one refers to products obtained by biotechnological processes, which can be considered asnatural, and the second one is related to the concept of sustainable development, since such productionprocesses are aligned with the best practices in environmental preservation.Scope and approach: In this review we demonstrate that the technological development of the pro-duction of aroma compounds using microorganisms is effectively promising as a process that allows theinextricably approach of the three pillars of sustainability: environment, economics, and social aspects.Key findings and conclusion: This review shows that bioaroma production consists of renewable pro-cesses that employ mild conditions of operation, do not generate toxic waste, uses biodiversity rationally,and may also avail agro-industrial residues or by-products in a special way. Moreover, biological (e.g.,antioxidant, anticancer, anti-inflammatory) activities attributed to some terpene biotransformationproducts are increasingly being reported, indicating that their applications may transcend food industry.

© 2017 Elsevier Ltd. All rights reserved.

1. Introduction

Aroma compounds can be produced using three methods: (i)chemical synthesis, (ii) extraction from nature, and (iii) biotech-nology. In the case of chemical synthesis, this method is marked byhigh yields and low cost. However, it generates low quality prod-ucts. That is because, considering its low regio- and enantiose-lectivity, a mixture of products is obtained at the end of the process.In addition, aromas obtained using this method cannot be labeledas natural. Another questionable point about this method refers tothe process parameters, which generally require a high energy cost(high pressures and temperatures), in addition to generatingenvironmental liabilities (use of large volumes of organic solvents)(Akacha & Gargouri, 2014).

On the other hand, aroma compounds obtained by the method

, School of Food Engineering,ade Universit�aria, 13083-862,

of direct extraction from nature or by biotechnology can be labeledas “natural”. Thus, products obtained using such processes have anundisputed marketing appeal. However, the method of directextraction from nature is full of challenges, among which we canhighlight: (i) seasonality (the availability of a product is related tocertain periods of the year), (ii) ecological, social and political is-sues, and (iii) low yield, which results in a high price for theproduct. In the case of the last challenge, the vanilla essence pro-duced from the orchid Vanilla planifolia illustrates this scenario.According to Gallage and Møller (2015), it is estimated thatapproximately 500 kg of pods of the aforementioned orchid arerequired for 1 kg of essence, in a process that takes more thantwelve months.

Thus, the biotechnological production of aroma compoundsoutstands as a very promising option to overcome problems asso-ciated with these other methods of production. Among the mainadvantages of this method, we can highlight: (i) high enantiose-lectivity, which allows obtaining aromas of high optical purity,beneficially impacting sensory characteristics of the product; (ii)continuous production throughout the year and without seasonal


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L.O. Felipe et al. / Trends in Food Science & Technology 62 (2017) 141e153142

interference; (iii) adoption of parameters of processes that are lessstringent (thus reducing energy costs and the use of reagentsharmful to the environment) (Berger, 2015); and, (iv) controllableand optimizable process conditions.

Therefore, the production of aroma compounds by biotech-nology meets two major demands of modern society. The first onerefers to the supply of natural products, meeting the expectationsof consumers and contributing to a higher quality of the finalproduct. The second one is related to the concept of sustainabledevelopment, since such production processes align the companywith the best practices in environmental preservation, in additionto increasing the credibility of the consumer regarding such com-pany (Manget, Roche, & Münnich, 2009). This last aspect, inparticular, deserves great prominence for being one of the mostimportant topics in the modern world.

1.1. Sustainable development

The sustainable development concept was created from theneed to combine industrial activities with the environment in aharmonious way. Therefore, it is a relatively new term that stillfinds barriers to its comprehensive understanding. That is because,often, sustainability is directly associated with the fulfillment oflaws allowing only environmental protection (Cristina & Diana,2014).

However, the creation of technologies that seek sustainabledevelopment must inextricably address three aspects that effec-tively provide the sustainability of this new process, namely:environmental, economic, and social aspects. In fact, the concept ofsustainable development would be subjected to a considerablelimitation if only the environmental aspect was necessary to thedetriment of economic and social development (Ciegis,Ramanauskiene, & Startiene, 2009).

Because of this, the triple bottom line was created (Fig. 1), basedon the inseparability of the aspects previously mentioned. Thus, atechnology is only recognized as sustainable if it broadly andsimultaneously meets the three pillars of that tripod (Gimenez,Sierra, & Rodon, 2012). Several indexes have been proposed tomeasure sustainability. However, as it is a complex quantificationdue to the different aspects involved and the related contexts, eachindex has positive and negative points, being better shaped

Fig. 1. Articulation of the triple bottom line that must be fully covered by a technology tha

depending on the situation involved (Ciegis et al., 2009).Currently, the trend of industrial processes e mainly chemical

ones e is focused on the replacement of all or part of the procedurefor new technologies that use microorganisms (bioprocess). Thisfact is widely justified based on the best interests of industries inoffering products that are environmentally friendly, socially equi-table, involving high added value and thus meeting marketingtrends (Toldr�a, 2015).

Therefore, the objective of this review is to demonstrate thattechnological development for the production of aromas usingmicroorganisms is a promising idea from the point of view of thesustainable development, since it allows the inextricably approachof the three pillars of sustainability, each being detailed in the nextsections.

2. Environmental aspect

2.1. Operations conditions

Bioprocesses are used bymankind since ancient times, mainly infood and beverage production (Kwon, Nyakudya, & Jeong, 2014).However, over the years, the use of microorganisms as biologicalfactories for the production of various products went beyond thefood sector and, currently, this tool is used in distinct areas such asenvironmental remediation, pharmaceutical industry, and others(Heux, Meynial-Salles, O'Donohue, & Dumon, 2015). This factclearly demonstrates the versatility, adaptability, and potential ofusing microorganisms in providing products with commercial in-terest. Moreover, industrial biotechnology might be advantageousin environmental terms, since they occur under mild conditions(process close to room temperature and atmospheric pressure) andthere is a possible reduction in the volume of environmental lia-bilities (Boukroufa, Boutekedjiret, Petigny, Rakotomanomana, &Chemat, 2014).

In the case of the aroma industry, the production process ofvanillin is a good example to understand the reasons for the ten-dency to replace classical processes with biotechnological pro-cesses (Gallage & Møller, 2015).

Vanillin is one of the main aroma compounds in commercialterms, with annual demand of approximately 2.0� 104 tons (Fache,Boutevin, & Caillol, 2015). In the second half of the 19th century

t seeks sustainable development. Adapted from: Sustainability e What do we mean?.

L.O. Felipe et al. / Trends in Food Science & Technology 62 (2017) 141e153 143

(1874e1875), the vanillin chemical synthesis was proposed fromeugenol (clove essential oil) as a starting substrate. Subsequently,the chemical synthesis of that compound was made from the ligninpresent in black liquor, a residue of the pulp and paper industry.However, the production based on eugenol and black liquor assubstrates for conversion were abandoned considering the volumeof effluents with high polluting potential generated during theprocess. For example, to produce 1 kg of vanilla aroma, havinglignin as a conversion substrate, the demand of the process is160 kg of sodium hydroxide. Additionally, 150 kg of environmentalliabilities are generated (Lampman et al., 1976). Hence, consideringthe significant demand for caustic soda and the consequent gen-eration of liquid effluent, previously mentioned production routeshave fallen almost into disuse. Thus, currently, the synthetic routepreferably adopted for vanillin is made from the conversion of twosubstrates: guaiacol and p-cresol, both precursors derived from thepetrochemical industry (Fache et al., 2015). The preference for thisproduction method is due to the higher yield provided by the re-actions, generating the least amount of effluents. Furthermore,when compared with the production method from lignin, it haslower economic cost (Hocking, 1997). These and other productionprocesses for chemical synthesis are described in Table 1, in whichwe also present the operation parameters adopted in the chemicalsynthesis of two other commercially important aroma compounds:g-decalactone and 2-phenylethanol. Comparatively, the productionof these compounds may occur under milder conditions whenusing bioprocesses, such as the following examples:

i. Streptomyces sp. V-1, when cultivated at 30�C/120 rpm in aculture medium (aqueous broth) supplemented with 45 g/Lferulic acid and 8% macroporous adsorbent resin DM11 andpH 7.2, was able to produce 19.2 g/L vanillin after 55 h (Xuet al., 2009);

ii. Candida sorbophila, when cultivated in a bioreactor con-taining 2 L of culture medium (aqueous broth) supplementedwith 400 g ricinoleic acid and operated at 27 �C, pH 6.0,aeration of 1 L/min and 600 rpm, produced almost 50 g/L R-g-decalactone (ee � 99%) after 10 days of fermentation(Mitsuhashi & Iimori, 2006);

iii. Kluveromyces marxianus CBS 600, when cultivated in abioreactor containing a culture medium (aqueous broth)supplemented with 50 g/L L-phenilalanine and poly-propylene glycol 1200 and operated at 30 �C, pH 5.0, aerationof 1-1.5vvm and 1000 rpm, could produce 26.5 g/L 2-phenylethanol after 30 h (Etschmann & Schrader, 2006).

Therefore, the replacement of classical aroma production pro-cesses using biotechnology may be seen as a more environmentallyfriendly approach, since it minimizes the use of eventually toxicreagents and solvents, in addition to often employing weakerreactional conditions (temperature and pressure). Therefore, not

Table 1Parameters of processes used by the chemical industry for the production of synthetic v

Product Conversion substrate Re

Vanillin Lignin Sop-cresol PoGuaiacol Fo

g-decalactone Gamma-bromocapric acid/9-decen-1-oic acid So2-phenylethanol Friedel e Crafts Reaction of Benzene and Ethylene Oxide A

Hydrogenation of Styrene Oxide RaN

*References: 1. Surburg and Panten (2006) and Harold & Tomlinson Jr (1937); 2. Surburg(2006) and Kamlet (1953); 4. Burdock, 2010; 5. Surburg and Panten (2006), Thomas, Nic

only vanillin, but several other aroma compounds have had theirproduction methods progressively replaced bymicrobial processes,and 22 biotechnology companies around the world already sellaromas of biotechnological origin (UBIC Consulting, 2014), such asthe examples shown in Table 2. To illustrate this scenario, Fig. 2shows the increase in patent records, indicating a growing trendin the adoption of microbial processes for the aroma compoundsproduction.

In fact, many biotech companies are investing in syntheticbiology to produce aroma compounds. Evolva, for instance, iscurrently producing biotech vanillin de novo from glucose using amodified yeast (Schizosaccharomyces pombe) (Hansen et al., 2009).Similarly, Amyris is using bioprocesses for the de novo synthesis ofsesquiterpenes (Clearwood™ e patchouli e and Biofene™ e far-nesene) with genetically modified Saccharomyces cerevisiae in largescale using sugarcane as substrate (Renninger, Newman, Reiling,Regentin & Paddon, 2010; Schalk & Deguerry, 2015). In the begin-ning of 2016, Ambrox® (ambroxide), produced by Firmenich using asimilar approach (Schalk et al., 2012), was also announced to be inthe market in the following months (Firmenich, 2016). These andother examples (Table 2) demonstrate that the production of flavorcompounds using genetically modified organisms is already acommercial reality, and such strategy will be increasingly used infuture.

However, despite the advantages inherent to the previouslymentioned bioprocesses, it is necessary to consider factors thatdecrease their commercial competitiveness, including: (i) the en-ergy cost spent in aerobic processes, (ii) the purification steps forthe target product, (iii) the demand or the characteristics of thereagents used in the downstream step, and (iv) the yield of productthat enables its economic viability (Najafpour, 2015). Moreover, theincreasing use of genetically modified microorganisms for theproduction of “natural” bioflavors might have commercializationproblems, as a reflex of legal and social constrains (i.e., consumerrejection), even though the regulations of use and labeling ofgenetically modified organisms are not applied to fermented in-gredients, since the microorganism itself is not present in the finalproduct (Hayden, 2014). As mentioned by Berger (2015), althoughthe legal status of “natural” flavors derived from recombinant hostsis not clear, the flavor industry is being pressured by the depletionof petrochemicals.

Thus, considering the growing trend of replacement of classicalprocesses for processes that uses biological catalysts, it is necessaryto recognize the need for greater investment in the research andtraining of human resources (Woodley, Breuer, & Mink, 2013).Specifically, higher yields and lower process costs can be achievedfrom the implementation of experimental planning tools to deter-mine the optimum production conditions, with the use of strainsresistant to process conditions, and appropriate systems to over-come problems related to toxicity and volatility of the substrate(Molina, Bicas, Moraes, Mar�ostica, & Pastore, 2013). Additionally, a

anillin, g-decalactone, and 2-phenylethanol.

agents Temperature (�C) Ref*

dium hydroxide/calcium hydroxide. 125e160 1tassium hydroxide/sodium hydroxide/methanol 60 2rmaldehyde 120e125 3dium carbonate/H2SO4 80% 90 �C 4luminum chloride 10e15 5neyickel, sodium hydroxide

10e110 6

and Panten (2006) and Nishizawa, Hamada & Aratani (1984); 3. Surburg and Pantenholl & Bitler (1949); 6. Surburg and Panten (2006) and Wood (1971).

Table 2Examples of commercially relevant processes for the biotechnological production of biotech aroma compounds (Gallage & Møller, 2015; Leffingwell & Leffingwell, 2015).

Product Company Microorganism* Substrate Ref**

Vanillin Evolva - IFF GM Schizosaccharomyces pombe Glucose 1Mane GM Streptomyces strain Eugenol 2Shanghai Apple Streptomyces sp. V-1 Ferulic acid 3Solvay Streptomyces setonii Ferulic acid 4BASF GM Pseudomonas strains Ferulic acid 5

Patchouli Amyris - Firmenich GM Saccharomyces cerevisiae Sugarcane 6Farnesene Amyris GM S. cerevisiae Sugarcane 7Ambroxide Firmenich GM Escherichia coli Sugarcane 8Nootkatone Allylix (Evolva) GM yeasts Valencene 9

*GM: Genetically Modified. **References: 1. Hansen et al. (2009, 2014); 2. Lambert, Zucca&Mane (2013); 3. Xu et al. (2009); 4. Muheim, Müller, Münch&Wetl (2001); 5. Graf& Altenbuchner (2016); 6. Schalk & Deguerry (2015); 7. Renninger, Newman, Reiling, Regentin & Paddon (2010) and Meadows et al. (2016); 8. Schalk et al. (2012); 9. Saran &Park (2016).

Fig. 2. e Patent search on the platform Google Patents for different periods and keywords: “vanillin and microbial process” (black), “2-phenylethanol and microbial process” (gray),“gamma-decalactone and microbial process” (white).


detailed study on the parameters of scale-up and downstreamsteps of the target product is also necessary (Najafpour, 2015). Inthis context, multidisciplinarity is another point of great impor-tance, since the understanding of satisfactory conditions to culti-vate microorganisms requires knowledge on different areas such asengineering, microbiology, and biochemistry (Velayudhan, 2014).

2.2. Use of agro-industrial waste

Another significant point of interest for the use of microorgan-isms in the production of inputs for the industry is the growingtrend to use alternative media for cultivation in bioprocesses (Wen,Liao, Liu, & Chen, 2007). These alternative cultivation media arerepresented by by-products of low commercial value or wasteoriginated from different industrial activities, mainly in the agri-cultural sector, which is characterized by the great generation ofsuchwaste (Table 3) (Madeira, Nakajima, Macedo,&Macedo, 2014).This strategy has some advantages from the point of view of wastemanagement, such as: (i) reduced financial expenditure for thesuitability of environmental liabilities to current standards, and (ii)added value to what was considered as unusable (Mirabella,Castellani, & Sala, 2014). In addition, the use of agro-industrialwastes and by-products as the culture medium of microorgan-isms represents a potential significant reduction in the costs ofbioprocesses, given that the formulation of such medium can affectfrom 38 to 73% the total product cost (Stanburry, Whitaker, & Hall,

1995).In this sense, there are several reports in the literature on the

production of bioaromas using different agro-industrial wastes andby-products. Such studies have been especially focused on solidstate fermentation (SSF), as in the following examples.

Christen, Meza, and Revah (1997), using the fungus Ceratocystisfimbriata, have studied three different substrates: wheat bran,cassava bagasse, and sugarcane bagasse. By using such wastes,those authors have shown that the profile of volatile compoundsobtained is closely linked to the type of substrate used and thesupplementation available. Thus, the enrichment of sugarcanebagasse with 200 g L�1 of glucose culminated in the production of afruity aroma, while the supplementation of this same substratewith leucine or valine produced volatile compounds with a sharpbanana aroma. Differences in the volatile profile according to thesupplementation of substrates were also observed in the study ofSoares, Christen, Pandey, and Soccol (2000), who have used coffeehusks as solid support for fermentation by C. fimbriata. Supple-mentationwith 20 and 35% of glucose resulted in a sharp pineapplearoma. On the other hand, the supplementationwith 46% of glucoseand 10 mmol of leucine resulted in a strong banana aroma.

Years later, in studies with the same species (C. fimbriata), Rossiet al. (2009) investigated the influence of the supplementation ofcarbon sources (cane molasses and soybean), as well as the sup-plementation of nitrogen (urea or soybeanmeal), in the productionyield of fruity aroma using citrus pulp as fermentation solid

Table 3Annual generation of agro-industrial waste or by-products with potential application as nontraditional cultivation medium for biotechnological production of variousbioaromas.

Culture Volume of global annual generation (tonne)a Type of agro-industrial waste generated Estimated volume of waste (tonne) Note

Cassava 2.77 � 108 Manipueira(cassava wastewater)

2.7 � 109 m3 b

Cassava peel 1.48 � 106 b

Sugarcane 1.91 � 109 Sugarcane bagasse and trash 2.8 � 108 c

Apple 8.08 � 107 Apple pomace 2.0 � 107 d

Rice 7.41 � 108 Rice husk 1.5 � 108 e

Coffee 8.92 � 106 Coffee husk and pulp 3.6 � 106 f

Orange 7.14 � 107 Orange bagasse 2.9 � 107 g

Milk (cow) 6.36 � 108 Whey 4.07 � 107 h

a Data obtained from FAOSTAT (2016), for the global production of each of the products presented.b Processing of 250e300 tonnes of cassava results inz2655m3 (with 1% solids) of liquid effluent or manipueira. The processing of 250e300 tonnes of cassava tubers results

in approximately 1.6 tonnes of solid peels and approximately 280 tonnes of bagassewith highmoisture content (85%). Value calculated considering that all cassava produced isprocessed (Pandey et al., 2000).

c Source: del Río et al. (2015).d The volume of apple pomace corresponds to 20e30% of the initial weight of the apple (Dhillon, Kaur,& Brar, 2013). Value calculated considering that all apple produced is

processed.e The amount of rice husk corresponds to 20% of the total produced (Gul, Yousuf, Singh, Singh, & Wani, 2015). Value calculated considering that all rice produced is

processed.f The amount of coffee husk and pulp corresponds to 30e50% of the weight of the total production (Oliveira & Franca, 2015). Value calculated considering that all coffee

produced is processed.g Peel, pulp, and seeds are z50% of the fruit (Macagnan et al., 2015). It was considered that ~ 80% of the fruits produced are processed (Companhia Nacional de

Abastecimento (CONAB), 2013).h Source: Prazeres, Carvalho, and Rivas (2012).


support. The best yield of volatile compounds e 99.60 mmol L�1 g�1

(120 h) e was achieved when the citrus pulp was supplementedwith 25% of cane molasses, 50% of soybean meal, and mineral saltsolution.

Larroche, Besson, and Gros (1999) have also noted the impor-tance of enriching the medium to increase the production of aromacompounds in solid state fermentation. Using crushed soybeansenriched with L-threonine and acetoin, they have achieved a yieldof 2 g L�1 in the production of pyrazines (2,5-dimethylpyrazine andtetramethylpyrazine) by the bacterium Bacillus subtillis IFO 3013.

A similar conclusion was obtained by Fadel, Mahmoud, Asker,and Lotfy (2015). These same authors used sugarcane bagasse asa carbon source for the fermentation with Trichoderma virideEMCC-107. The production of lactones was evidenced, in particularfor 6-pentyl-a-pyrone (character impact compound of coconut),and their production reached 3.62 mg after five days of fermenta-tion. Furthermore, with an established confidence level of 95%, itwas possible to conclude that the yield in production and the in-crease in microbial biomass were directly related to the supple-mentation of sugarcane bagasse to the culture medium.

Aggelopoulos et al. (2014); Mantzouridou, Paraskevopoulou,and Lalou (2015); Medeiros, Pandey, Freitas, Christen, and Soccol(2000) and Rodríguez Madrera, Pando Bedri~nana & Su�arez Valles(2015) have investigated the use of yeasts in the production ofvolatiles from different agro-industrial wastes. In the case ofAggelopoulos et al. (2014), mixed agribusiness effluents (cheesewhey, cane molasses, malt rootlets) were used as substrate ofconversion for Saccharomyces cerevisiae, Kluyveromyces marxianus,and kefir. The best microbial fermentation yield was achieved usingkefir, having been calculated a production of 4 kg of ε-pinene foreach tonne of waste used.

Mantzouridou et al. (2015) have assessed the volatile productionby de novo synthesis catalyzed by Saccharomyces cerevisiae havingcitrus pulp as solid support. Among the volatile compounds iden-tified as fermentation products after 72 h, we can highlight: isoamylacetate (48.7 mg kg�1), ethyl dodecanoate (25.2 mg kg�1), ethyldecanoate (9.3 mg kg�1), ethyl octanoate (6.3 mg kg�1), and phenylethyl acetate (4.5 mg kg�1). The authors also have observed anintense production of ethyl hexanoate, whose maximum

concentrationwas 154.2 mg kg�1 after 48 h. The sum of the volatileesters production (z250 mg kg�1) showed that this productionprocess is a viable way to add value to an agribusiness waste ofCitrus.

On the other hand, Medeiros et al. (2000) have studied the in-fluence of five agro-industrial wastes in the production of volatilecompounds by Kluyveromycesmarxianus using the response surfacemethodology. Cactus meal and cassava bagasse, both supple-mented with 10% of glucose, were assessed as the most suitablesubstrates. Ethanol (418 mmol L�1) and ethyl acetate(1395 mmol L�1) were the major components obtained from cactusmeal and cassava bagasse, respectively.

Finally, Rodríguez Madrera et al. (2015) have tested the rela-tionship between the production of volatile compounds and themicroorganism used. Apple peel was used as solid support and fouryeasts were used: Saccharomyces cerevisiae (obtained commer-cially) and three others isolated from cider (S. cerevisiae, Hanse-niaspora valbyensis, and H. uvarum). Altogether, from thefermentation of the four strains, 132 volatiles were identified fromdifferent families, having been possible to conclude that theamount of volatiles produced is strain-dependent.

However, despite the clear advantages demonstrated by the useof agro-industrial wastes as a means of nontraditional cultivationfor the production of bioaromas or for any other bioprocess, thereare some challenges that permeate such application. Among them,we highlight the heterogeneity of such substrates and the possibleneed for pretreatments or supplementations. Additionally, there isa great challenge to be overcome in terms of downstream pro-cessing, which may entail additional costs, because the purificationof products in these cases can be much more complex. For thisreason, the value added in the final chain of product must justifyadjustments in the use of this strategy.

Other issues that also deserve to have their economic viabilityassessed in the final cost of the by-product are: financial demandswith the transport of such waste to the place where they will bereused, and the storage costs of these alternative cultivationmeans.Furthermore, we must think that the wide availability of a partic-ular industrial waste is segregated to certain areas. In Brazil, forexample, the availability of sugarcane bagasse is particularly


abundant in the state of S~ao Paulo; whey is mainly found in thestate of Minas Gerais. Therefore, these logistical issues must beconsidered when studies on the added value of agro-industrialwaste and by-products are proposed.

3. Economical aspect

According to Leffingwell & Associates, the global sale of aromasand fragrances in 2014 was approximately US$ 24.9 billion, withexpected growth rate from 5 to 6% per year. Regarding marketsegmentation, the American and European continents are respon-sible for more than half the consumption of the aromas and fra-grances sold worldwide (UBIC Consulting, 2014). Despite this, Asiahas emerged as a growing market. An example that illustrates thisscenario is the increased market pressure for menthol (characterimpact compound of mint) in Asian countries, whose demand hasincreased at a rate of approximately two digits for years. This factcan be explained from the awareness of the benefits of the routineuse of dentifrices by Chinese and Indians (McCoy, 2010).

Despite the clear economic potential of the aroma market,another interesting point about this sector is the ability to add valueto commodities. Brazil, for example, has an important participationin the world market for the export of primary products. Amongthese products, we can highlight the significant production of or-anges in Brazil (approximately 30% of the world production)(FAOSTAT, 2016), whose oil (annual production of 30,000 tonnes(Schwab, Fuchs, & Huang, 2013)) contains >90% of the mono-terpene R-(þ)-limonene. This compound is commonly used in thesynthesis of resins, adhesives, paints, solvents, cleaning productsetc.

Similarly, pulp and paper industry generates 330,000 tonnes ofthe by-product turpentine (Schwab et al., 2013), whose composi-tion presents large amounts of a-pinene, a bicyclic monoterpenethat is very important as starting substrate in industrial syntheses(Surburg & Panten, 2006). However, such compounds arecommonly underutilized in the formulation of low value-addedproducts such as cleaning products in general, personal hygieneproducts, and solvents (Surburg & Panten, 2006).

Therefore, from an economic point of view, by-products of thosecommodities represent a promising alternative to stimulate theindustry of aroma production. This is because the microbialbiotransformation of R-(þ)-limonene and a-pinene (Berger, 2015)is able to produce products with high added value, with a marketvalue from 10 to 30 times higher than the starting substrate. Usingthe Molbase database (www.molbase.com) as reference, forexample, it is possible to illustrate this scenario: while R-(þ)-limonene has a reference price of US$34/L, its oxygenatedcounterparts, perillyl alcohol, carveol, and carvone present refer-ence prices of US$405, US$529, and US$350, respectively. Similarly,the possible derivatives of a-pinene (reference price of US$64/L),i.e., myrtenal, myrtenol, verbenol, and verbenone, have referenceprices of 913, 1939, 1926, and 906, respectively. Therefore, there isan important economic opportunity to be explored in the additionof value to commodities.

In recent years, a similar scenario has emerged with anothercommodity produced in Brazil. Currently, the company Amyrismanufactures and markets trans-b-farnesene (Biofene®) to be usedin the production of the so-called “sugarcane diesel”. Althoughthere are no reports of the biotransformation of trans-b-farnesenein its pure form, a few reports describe fungi (Aspergillus niger andPenicillium solitum) as being capable of performing oxy-functionalization of other farnesene isomers, obtaining aromacompounds with very peculiar sensory profile (Krings et al., 2006).Thus, trans-b-farnesene can be regarded as a great starting sub-strate for biotransformation studies, since, in addition to the

possibility of producing new compounds with great interest to thearoma industry, there is still ample space for scientific and tech-nological innovation, with the dissemination of novel researchstudies.

Therefore, it is important to emphasize that the economic aspectof any technology is closely tied to profit generation, market de-mand, and turnover of products. If this scenario is not widelycovered, the new technology will, possibly, not be perpetuated.Thus, the creation of ideas (translated into patents, for example)does not necessarily mean that it will be immediately adopted bythe business sector. Therefore, innovative processes should befirmly focused on the viability of an industrial scale from the benchscale (Shimasaki, 2014). That is why, when it comes to the pro-duction of bioaromas, it is essential that the economic part isprofitably met, favoring the commercial feasibility and demand forthese same products (Otte & Hauer, 2015).

The available data on the commercial values of aromas ofbiotechnological origin, compared with the natural and syntheticones, indicate that this demand can be achieved. To illustrate thisscenario, the prices of three different aroma compounds obtainedby different methods are shown:

- Vanillin: synthetic ¼ US$ 15; natural ¼ US$ 1200e4000;“biotech” ¼ US$ 1000 (Gallage & Møller, 2015);

- g-Decalactone: synthetic ¼ US$ 150; natural ¼ US$ 6000;“biotech” ¼ US$ 300 (Dubal, Tilkari, Momin, & Borkar, 2008);

- Ethyl butyrate: synthetic ¼ US$ 4; natural ¼ US$ 5000;“biotech” ¼ US$ 180 (Dubal et al., 2008);

In this way, it is possible to see clearly the production potentialof the so-called “biotechnological” aromas. This is because aromasobtained by this method have a clear cost-benefit ratio. Accordingto Janssens, De Pooter, Schamp, and Vandamme (1992), aromasobtained from biotechnology have commercial competitivenesseven when their market prices are 10e100 times higher than theirsynthetic analogues. In fact, flavor compounds biotechnologicallyproduced (i.e., fermentations) might be labeled as “natural”, such asin EU and US legislations (Regulation (EC) no. 1334/2008; US Codeof Federal Regulations, Title 21 Section 101.22). This is commerciallyinteresting, since consumers tend to prefer foods formulated with“natural” components (Carocho, Morales, & Ferreira, 2015). On theother hand, technology-based innovations, such as the use of irra-diation and genetically modified organisms, might be rejected byconsumers (Ronteltap, van Trijp, Renes, & Frewer, 2007). In Brazil,for instance, a survey showed that one third of those interviewedconsidered that the consumption of transgenic food could beharmful (Folha de S~ao Paulo, 2016). Therefore, from an economicpoint of view, the production of bioaromas is very promising inrelation to the market acceptance of such products when weconsider the commercial value and the benefit associated withtheir use in different sectors. However, as alreadymentioned in thistext, it is important to recall the possible social issues and regula-tory constrains that might come from the increased use of geneti-cally modified organisms (Hayden, 2014).

4. Social aspect

4.1. BIOPROSPECTING

4.1.1. Rational exploration of the environmentBioprospecting is an activity that consists in the investigation of

the environment aiming at finding biological agents that could beused in the production of goods of commercial value (Artuso, 2002).These biological agents can be quite varied as well as the applica-bility found for each of them. Among the main ones, common

http://www.molbase.com


agents explored by humanity since antiquity, we can mention plantextracts and biomolecules isolated from certain animals or micro-organisms such as fungi and bacteria (Verpoorte, 2015).

According to Morales (2010), Brazil has approximately 70% ofthe world's species, integrating the group of seventeen countriesconsidered as megadiverse (de Lima, Fortes-Dias, Carlini, &Guimar~aes, 2010). Despite the Amazon presenting much of thisheritage, biomes, such as the Brazilian Cerrado and Atlantic rain-forest, have also been commonly pointed out as hotspots of sig-nificant value for the search for new genetic potentials that can beused in an industrial scale (Marchese, 2014). Therefore, consideringthis context, Brazil presents a huge window of possibilities for thebioprospecting of different biological resources (Adenle, Stevens, &Bridgewater, 2015).

Hence, in general, bioprospecting activities are quite attractiveaccording to the three main aspects that go beyond the contribu-tion to innovation, namely: (i) appreciation of natural resources, (ii)access to associated traditional knowledge, and (iii) potential forcontribution to the financial sustainability of certain localities.Thus, bioprospecting assumes an important value for theimprovement of social conditions of a population, which presentsnatural resources as environmental heritage (Lewandowski, 2014).

Regarding social improvement, the appreciation of natural re-sources of a given region can clearly contribute to the increase inthe quality of life of individuals who inhabit it (Sandifer, Sutton-Grier, & Ward, 2015). This fact can be easily justified by: (i) pres-ervation of green areas contributing to air quality, (ii) maintenanceof water sources (lately considered as one of themost critical pointsof the indiscriminate environmental exploration), and, finally, (iii)preservation of endemic species (with concomitant reduction inthe rate of extinction of species) that represent a wealth ofexpressive value because of the low probability of being found inother locations. Therefore, in this case, we can explore the envi-ronment, without, however, degrading it (Barrett & Lybbert, 2000).

Another point of great importance is the fact that bio-prospecting is seen as an activity that values the traditionalknowledge associated with the knowledge of the local population(Toledo, 2013). Therefore, it gives relevant value to learning linkedto the experience of individuals who already enjoy the use of acertain biological potential for more noble uses such as the use oftypical herbs for the production of infusions with medicinal prop-erties, the manufacture of natural dye from the bark of trees, or theextraction of aromatic substances from the native flora (Cox& King,2013).

In addition, access to the associated traditional knowledge oftenrepresents a clear positive impact on the financial sustainability ofcertain populations. This is enabled by sharing this knowledge fortechnological innovation and development of new products forcommercial purposes. From this, the increase of the per capita in-come of low-income communities is possible with the creation ofcooperatives and/or associations, which promotes the allocation ofbenefits from the exploration of natural resources of the placewhere this population lives (Weiss & Eisner, 1998).

Therefore, bioprospecting, despite the challenges permeatingthis activity, shows that the exploration of the biodiversity togetherwith social development is possible, valuing popular knowledge, inaddition to increasing the quality of life and the economic quality ofthese populations (Artuso, 2002).

4.1.2. Selection of strainsAmong strategies, bioprospecting is used by some authors in an

attempt to identify new strains of microorganisms for the pro-duction of bioaromas (van der Werf & de Bont, 1998). Particularly,soil samples have been frequently used for the prospection of mi-croorganisms that are potential bio-transformers of terpene

compounds. To illustrate this approach, some examples of micro-organisms isolated from soil may be cited: Bacillus fusiformis, whichwas able to convert isoeugenol to vanillin (production of 8.10 g L�1

after 72 h) (Zhao, Sun, Zheng, & He, 2006); B. pumilus, which wasalso able to convert isoeugenol to vanillin (production of 3.75 g L�1

after 150 h) (Hua et al., 2007); Pseudomonas putida, which was ableto convert isoeugenol to vanillic acid (98% of molar conversion after40 min) (Furukawa, Morita, Yoshida, & Nagasawa, 2003); Bacillussubtillis, which was able to convert isoeugenol to vanillin (pro-duction of 0.9 g L�1 after 48 h) (Shimoni, Ravid, & Shoham, 2000);and Chrysosporium pannorum, which was able to convert a-pineneto verbenone and verbenol (Trytek, Jedrzejewski, & Fiedurek,2015).

Other isolation and selection strategies, as further exemplified,can also be adopted, thus affirming the importance of prospectionefforts in the biotechnological production of aromas.

Ferraz et al. (2015) have isolated Penicillium crustosum from acheese sample. Lipase produced by said microorganism wasimmobilized in the fermentation system, being responsible forcatalyzing the conversion of geraniol/propionic acid in geranylpropionate. The optimization of fermentation parameters showedthat lipase produced by Penicillium crustosum has the potential tobe widely applied in the production of geranyl propionate.

Pastore, Park, and Min (1994), from samples of beiju (a typicalfood of the North/Northeast region of Brazil produced from cassavastarch), have isolated eight different strains of Neurospora sp.identified as capable of producing pleasant fruity aromas.

Dai, Cheng, He, and Xiu (2015) isolated a strain of Bacillus subtilisDL01 from marine sediment in China. The prospection of thatbacteria was encouraging because of the tolerance shown on sys-tems with low aeration rate (0.4 vvm) and high concentration ofsugar (210 g L�1 of glucose) to produce acetoin (76 g L�1/1.0 g L�1

h�1/60.9 g L�1 d-acetoin).van der Werf, Keijzer, and van der Schaft (2000), from sediment

of the Rhine river, insulated Xanthobacter sp. C20 using cyclohexaneas the sole source of carbon and energy. Such strain has proved tobe able to quantitatively biotransform both enantiomers of limo-nene into limonene-8,9-epoxide (0.8 g L�1). This study proved to bea novel one for describing a new metabolic pathway for limonene,which had not been described before.

Krings et al. (2006) have isolated a strain of Aspergillus nigerfrom mango. This microorganism was able to biotransform a-far-nesene into two main products: p-menth-1-en-3-[2-methyl-1,3-butadienil]-8-ol (aroma that refers to apricot) and 2,6,10-trimethyldodeca-2,7,9,11-tetraen-6-ol (citrus scent).

Rottava et al., 2010 and Bicas and Pastore (2007) have isolatedstrains from effluents of citrus industry as well as other samples(soil from the plantation of citrus, citrus fruits, and citrus leaves). Ofthe 405 strains isolated by Rottava et al. (2010), eight were able tobioconvert R-(þ)-limonene and fifteen converted (�)-b-pinene,generating a-terpineol as a product in both cases. Bicas and Pastore(2007) have obtained 248 microorganisms, of which seventy weredeveloped in medium containing limonene as the sole source ofcarbon.

However, as reported by Molina et al. (2013), there are severalchallenges to be overcome to enable the production of aromas bybiotransformation, among them the high toxicity of both the sub-strate and the product and the low yields obtained. According tothese authors, such difficulties can be overcome with a good workin the isolation and selection of strains as the studies mentionedabove. This is justified by the constant difficulty of identifying mi-croorganisms that are actually able to convert terpene substrates ateconomically viable concentrations. Moreover, bioprospectingshows itself as a key factor for detecting strains that convert sub-strates not yet explored to biotransformation processes, among


them, the previously mentioned b-farnesene.Finally, the search for new microbial “factories” is essential to

reaffirm the enormous potential of the environment. This isbecause access to this biological richness has the clear ability ofoffering solutions to numerous demands in different bioprocesses.

4.2. Biological potential of bioaromas

In this section, we will discuss potential applications of aromacompounds that can transcend food industry, reaching the phar-maceutical industry. For now, there is no reason to believe thatthese compounds will replace the traditional pharmaceuticalscurrently in use. However, we anticipate that this seething area ofresearch might evolve in such a way that these compounds, in thefuture, might found use in medical applications rather than food,supposing that they might eventually have higher specificity andlower toxicity than conventional chemicals.

Used by traditional medicine, essential oils (rich in terpenecompounds) are recognized for assisting in the treatment ofdifferent health problems. Among the applications widelydescribed in the literature for them, we can mention anti-inflammatory, antispasmodic, anticancer, antimutagenic, antibac-terial, antifungal, antiviral, and vermicide activities (Raut &Karuppayil, 2014; de Sousa, Hocayen, Andrade,& Andreatini, 2015).

Among the most promising terpenes for these applications,which are also important aroma compounds, we can mentionlimonene and its derivatives (Kaur & Kaur, 2015). Between thederivatives, we highlight: perillyl alcohol (Imamura et al., 2014),carvone (Carvalho & Fonseca, 2006), and a-terpineol (Pinto et al.,2014).

Regarding the anticancer activity associated with terpenecompounds in particular, according to Crowell (1999), it can beexplained by the following factors: (i) blocking effects (initiationphase), characterized by enzyme induction of phase I and phase IIof the metabolism of xenobiotics, which is associated with thedetoxification of the carcinogenic agent; and, (ii) suppressing ef-fects (promotion phase), characterized by (ii.a) the inhibition of cellproliferation, induction of apoptosis or differentiation or by (ii.b)the inhibition of post-translational isoprenylation of cell growthregulatory proteins.

Additionally, considering the overall magnitude of oncogenicdiseases, there is an increasing demand for new drugs that can helpin the treatments of these disorders (Khazir, Mir, Pilcher, & Riley,2014). Moreover, such a scenario is reinforced by data released onthe World Cancer Report (2014) by the World Health Organization.According to this document, 14 million individuals were diagnosedwith this disease in 2012. Of this amount, 8.2 million have died.Furthermore, estimates suggest that the number of cancer di-agnoses may suffer an increase of 70% over the next two decades(L�opez-G�omez, Malmierca, de G�orgolas, & Casado, 2013). Thus,considering the demands of attention of oncogenic diseases,different studies are described in the literature focused on thechemopreventive action of different terpene compounds (Bicas,Neri-Numa, Ruiz, de Carvalho, & Pastore, 2011). Thus, next weconsider some important studies explored in this and other areas.

4.2.1. Perillyl alcoholIn relation to in vitro studies, Sundin, Peffley, Gauthier, and

Hentosh (2012) have shown that perillyl alcohol, as well as rapa-mycin, presented chemopreventive activity for prostate cancer.Thus, after an incubation period between 1 and 16 h, perillyl alcoholwas able to reduce the rate of activity of telomerase from 65 to 95%.The importance of this study lies in the fact that telomerase is oneof the enzymes responsible for acting in the process of cellimmortalization. In another study, Afshordel et al. (2015) have

investigated the action of perillyl alcohol and lovastatin. Theelucidation of the mechanism of action of both drugs demonstratedthat they are able to affect the post-translational modification ofisoprenoids (which are responsible for enabling the invasiveness,migration, and proliferation of brain gliomas). While lovastatin wasable to suppress the substrates of the pathway, perillyl alcoholinhibited enzymes that catalyze the reactions of that pathway. In aresearch developed by Lebedeva et al. (2008), the synergy of perillylalcohol was investigated with gene therapy as a tool for the che-moprevention of pancreatic cancer. On the other hand, Wagner,Huff, Rust, Kingsley, and Plopper (2002) have found that theadministration of perillyl alcohol presents significant potential asprophylactic therapy in the treatment of breast cancer. Its effectshave been attributed to the intervention in the migration ability oftumor cells.

However, it is in in vivo studies that we can see that the bio-logical potential of perillyl alcohol is really outstanding. Moreover,according to Chen, Da Fonseca, and Sch€onthal (2015), the pre-liminary results obtained from clinical studies are significantlypromising. In this context, a group led by researchers at the Flu-minense Federal University and Federal University of Rio de Janeirohave conducted clinical assays on terminally ill patients withdifferent malignant gliomas. The therapy adopted was based on thedirect inhalation of 0.3% perillyl alcohol, four times a day. Resultsshowed that such therapy was well tolerated and that some pa-tients showed regression of the tumor (da Fonseca et al., 2006a,2006b, 2008). Another pilot study, also conducted by the sameteam previously mentioned, has investigated the administration ofperillyl alcohol in eight patients with pancreatic cancer (one of themost deadly forms of the disease). It was observed a reduction inthe size of the tumor cell from the mechanism of cell apoptosis.Although a statistical significance was not demonstrated, patientshad a higher survival rate of 84 days when compared with thecontrol group (Matos et al., 2008).

In another study, Cho et al. (2014) have tested a new drugtherapy for the treatment of brain gliomas resistant to temozolo-mide. This drug is a tumor agent widely used as reference therapyin the treatment of this type of neoplasia. Thus, the so-called NEO212 (a conjugated drug of perillyl alcohol and temozolomide) wasadministered to mice and results were analyzed with 95% confi-dence (p < 0.05). Data analysis and tomography exams of the ani-mals showed that NEO212 was effective in the regression ofresistant tumors. Furthermore, this drug showed synergistic effectwhen compared with the separated administration of perillylalcohol and temozolomide. Therefore, research studies highlightedhere demonstrate the use of perillyl alcohol as a promising alter-native in cases of failure of oncogenic treatments using conven-tional methods.

Additionally, administration of perillyl alcohol in other areas hasalso been extrapolated, as shown in the study of Tabassum et al.(2015). In this latter case, they have investigated the effect oftherapy with perillyl alcohol on ischemia-reperfusion injuries(medical terminology applied to describe the changes that occurafter a period of ischemia). Results showed that perillyl alcohol wascritical to the mitigation of the oxidative stress and inflammatoryprocesses. Mitigation of these processes gave a neuroprotectiveactivity to that terpene.

4.2.2. CarvoneCarvone has demonstrated several applications from its bio-

logical potential (Carvalho & Fonseca, 2006). Thus, according toPatel and Thakkar (2014), in assays conducted in vitro, carvone wasable to control the proliferation of breast cancer tumor cells byapoptosis. Results also showed that apoptosis was accompanied bythe increase in the level of excretion of glutathione and reactive


oxygen species (ROS). In an in vivo study, Vinothkumar et al. (2013)have shown that the administration of carvone proved to beeffective in the chemoprevention of chemically induced colorectalcancer. The carvone dose of 10 mg kg�1 of body weight was satis-factory for reducing the rate of formation of polyps. The latter isgenerally deemed as responsible for the progression of intestinalneoplasms. According to studies developed by Zheng, Kenney, andLam (1992), the anticarcinogenic effect of carvone is mainly asso-ciated with its ability in stimulating detoxifying enzymes, amongwhich we can mention glutathione S-transferase.

Carvone has also been explored in other fields of study of sci-ence. Among these research studies, we can highlight the studydeveloped by Karanisa, Akoumianakis, Alexopoulos, and Karapanos(2015). These authors have verified the inhibition of post-harvestpotato sprouting from the application of carvone in these vegeta-bles instead of synthetic suppressors. Results showed that carvonewas effective in promoting bud dormancy when the potatoes werestored at 10 �C, even if for a long period. In another study, Holbanet al. (2014) have investigated the potential of carvone as antimi-crobial agent. This study has tested a bioactive system of conju-gated nanoparticles of magnetite and carvone (Fe3O4@CAR).Results showed that the nanosystem was able to inhibit coloniza-tion and biofilm formation of S. aureus ATCC 25923 and E. coli ATCC25922. In addition, in vitro tests showed that the nanostructuredbioactive showed no cytotoxic effects on eukaryotic cells.

On the other hand, Peixoto et al. (2015) have studied the po-tential of carvone as a natural insecticide. This pilot study hasaimed to study the toxicity of carvone against two species of in-sects: Sitophilus zeamais and Tribolium castaneum. These species areresponsible for heavy losses in grain storage silos. Results werepromising for the production of a natural insecticide from carvoneas repellent. In the study case carried out by Souza, da Rocha, deSouza, and Marçal (2013), it has been demonstrated that carvonehas a powerful antispasmodic action, acting preferentially blockingcalcium channels.

Finally, de Sousa, de Farias N�obrega & de Almeida (2007) havestudied the effect of two carvone enantiomers on the central ner-vous system. The in vivo study was conducted with mice. The LD50(median lethal dose) was 484.2 mg kg�1 for (S)-(þ)-carvone and426.6 mg kg�1 for (R)-(�)-carvone. Both enantiomeric formsshowed decreased brain activity. These effects were deducted bythe reduced touch sensitivity and increased level of sedation andantinociception (described as the decrease in the capacity of painperception). However, (S)-(þ)-carvone applied at 200 mg kg�1

significantly increased the latency to seizure, while (R)-(�)-carvonedid not.

4.2.3. LimoneneAmong most recent studies with this monoterpene, we can

highlight Zhang, Wang, Liu, Tang, and Zhang (2014). Using cellstrains of human gastric carcinoma, those authors have shown that,in vitro, the administration of limonene with barberine showedsynergistic effect when compared with other drugs used alone. Thescope of these results was assigned to the induction of apoptosis,increase in the production of reactive oxygen species, and cell cycleinhibition. In another study, Vandresen et al. (2014) have proposedthe syntheses of a new drug and its respective derivatives con-taining limonene in their formulation. The formulations weretested against several types of tumor strains (gliomas, melanomas,leukemia etc.). Among the 22 derivatives prepared, 4-fluorobenzaldehyde proved to be especially selective for prostatetumor strains. On the other hand, 2-hydroxybenzaldehyde provedto be the most active compound, with potent antitumor activityagainst all cell strains tested.

In the case of Miller et al. (2013), an in vivo pilot study has been

developed to investigate the bioactivity of limonene. The chemo-therapeutic action of this terpene has been studied in a group offorty-threewomen diagnosed with breast cancer in the early stagesof the disease. Despite early diagnosis, these women had surgicalindication for organ extraction. Hence, two to six weeks beforesurgery, each of the patients ingested two grams of limonene perday. Histological analysis of breast tissue showed that limonenewas able to homogeneously distribute itself in that organ. Althougha control group was not adopted and the population sampleadopted was reduced, it was still possible for the authors toconclude that cell proliferation was inhibited because of the sub-expression of cyclin D1 (a cell cycle regulatory protein).

Later, a new study was developed by Miller et al. (2015). How-ever, in this latter case, the sample population of women diagnosedwith breast cancer and with surgical indication was equal to 39. Toinvestigate the antitumor activity of limonene, the mapping of themetabolomic profile of the patients was used. The key changesidentified were related, again, to the subexpression of cyclin D1.Other scientific studies have also contributed to elucidate the otherapplications of limonene such as illustrated in the sequence.

Bacanlı, Basaran, and Basaran (2015), in experiments conductedin vitro, highlight the antioxidant action of limonene and narigin.Such potential is attributed to the protective effect against peroxideradicals. In addition, especially because of this antioxidant activitydemonstrated by limonene, Bai, Zheng, Wang, and Liu (2016) havecarried out in vitro assays. Of such assays, limonene proved to bepromising in the search for more effective therapies for the treat-ment of ocular degeneration associated with age such as cataracts.Antimicrobial activity of limonene has been investigated by Zahi,Liang, and Yuan (2015). In this case, limonene was encapsulatedin a nanoemulsion and tested against different microorganismsthat cause foodborne illness. Damage to the integrity of the cells ofsuch microorganisms was the main target of action of limonene.

Rossi et al. (2015) have conducted an in vivo pilot study on ninepatients with psoriasis. Oral and/or topical administration oflimonene was performed for 45 days. The good results obtainedfrom both patient satisfaction and the reduction in the extent of thedisease point out the need for extrapolation of this study to doubleblind assays. Tan, Chua, Ravishankar Ram, and Kuppusamy (2015)have also signaled to limonene the potential to be used in thecontrol of obesity and type 2 diabetes mellitus. This is because itstimulated the absorption of glycolysis and lipolysis. On the otherhand, Hajagos-T�oth, H�odi, Seres, and G�asp�ar (2015) have warnedabout the awareness of the use of medicinal herbs during preg-nancy. That is because, often, this type of therapy deemed as nat-ural seems to have a safe use. However, in vitro studies on the twoenantiomers of limonene, demonstrated that it raises the rate ofuterine contractility. Thus, these authors draw attention to the riskof the use of medicinal therapies, which can affect pregnant womenwith premature births.

4.2.4. a-TerpineolIn relation to the antitumor activity of a-terpineol, Hassan, Gali-

Muhtasib, G€oransson, and Larsson (2010) have assessed the cyto-toxicity of this terpene against cancer cells, in vitro. Results of theassays have shown that a-terpineol was able to reduce theexpression of the nuclear transcription factor NF-kB. Thus, ac-cording to this interference, a-terpineol inhibited the proliferationof different cancer cell strains. However, carcinoma cells of thesmall cell lung cancer were those that showed greater sensitivity tothe treatment. Bicas et al. (2011) have also investigated the anti-oxidant and antiproliferative action of some bioaromas, in vitro.Results showed that, among the tested terpenes (limonene, perillylalcohol, carvone, and a-terpineol), a-terpineol presented the bestperformance. In terms of antioxidant activity, a-terpineol


presented results similar to BHA (butylated hydroxyanisole, asynthetic antioxidant). Additionally, such a compound presented amechanism of inhibition of cell proliferation against six strains ofdifferent tumor cells. The most significant results were achievedwith breast carcinoma and chronic myelogenous leukemia, withcytostatic effect (Total Growth Inhibition) for the concentrations of181 mM and 249 mM, respectively. Because of this, in vivo studieshave been encouraged to further this promising potential. Wu et al.(2014) have also developed an in vitro experiment with a liver tu-mor strain. The suppression of cell proliferation was mainlyattributed to the triggering of mechanisms of apoptosis. However,g-terpineol was used in this latter study.

Other research studies have pointed out other applications of a-terpineol. Prakash, Singh, Goni, Raina, and Dubey (2015) haveassessed the antimicrobial activity from the combination of An-gelica essential oil, 2-phenyl ethanol, and a-terpineol in the pro-portion of 1:1:1. This compound has been tested against eightdifferent moldy fungi responsible for the production of mycotoxins(responsible for the oxidative deterioration of nuts). Resultsshowed that the inhibition of the synthesis of ergosterol in thefungal wall was the main target of the action of the proposednatural insecticide.

Held, Schieberle, and Somoza (2007) have conducted in vitrostudies with epithelial cells of the mouth. Results showed that a-terpineol suppressed the formation of IL-6, thus presenting anti-inflammatory activity. In another similar study, Nogueira, Aquino,Rossa Junior, and Spolidorio (2014) have achieved a similarconclusion about the anti-inflammatory activity of a-terpineol.However, in this latter case, a reduction in the production of IL-10and IL-1b, in addition to IL-6, was observed.

Mukherji and Prabhune (2015) have proposed the application ofa-terpineol as the precursor for the synthesis of a glicomono-terpenol. This compound was obtained by the biotransformationpromoted by Candida bombicola ATCC 22214 from a culture me-dium supplemented with linalool and a-terpineol. That glicomo-noterpenol proved to be very efficient as antagonist of the quorum-sensing mechanism. The importance of this study focuses on thefact that, with the development of bacterial multidrug resistance toseveral drugs, the intervention in the quorum-sensing mechanismcan be an alternative to solve such a problem.

In tests conducted in vivo by de Sousa, Quintans-Jr. & Almeida(2008), it has been shown that a-terpineol presented good resultsas an anticonvulsant agent. Activity in the central nervous systemhas also been demonstrated by Quintans-Júnior et al. (2011). In thiscase, the administration of a-terpineol (25, 50, and 100 mg kg�1) inmice presented analgesic activity without apparent interference inthe motor ability of those animals. On the other hand, Choi, Sim,Choi, Lee, and Lee (2013) have conducted a study with mice, inwhich a-terpineol was orally administered for twoweeks. After thisperiod, the assessment of hepatocytes showed a steatosis result (fataccumulation in the liver). However, in this latter case, specifically,no studies have been developed on humans. On the other hand, aspointed out by Bhatia, McGinty, Foxenberg, Letizia, and Api (2008),studies related to the toxicity of a-terpineol show that it presentsoral toxicity of 4.3 g kg�1 in rodents and dermal toxicity above3.0 g kg�1 in rabbits. In addition, there have been no reports in theliterature about the carcinogenic potential presented by thisterpene.

Despite the aforementioned research studies and the biologicalpotential presented by the terpenes and their derivatives, reportson the activity of other derivatives of biotransformation of thesecompounds are still scarce in the literature, among which we canmention limonene-1,2-diol. Thus, we consider of utmost impor-tance the exploration of these promising biological activitiesdemonstrated by different terpene compounds.

5. Final remarks

The concept of sustainable development is a subject of extremerelevance in modern society. However, despite the trendiness ofthis subject, it is important to understand its systemic approach,the actual applicability of this concept, and the benefits obtainedfrom such a practice. Especially when it comes to creating newtechnologies, clear perspectives must be well delineated in order toadapt innovative processes to new demands vividly discussedabout sustainability. In this sense, the growing trend of replace-ment of classical processes for bioprocesses emerges as a promisingopportunity to contemplate, at the same time, the three aspects ofsustainability, i.e., social, economic, and environmental aspects. Inour study, we suggest that this scenario is also applicable for theflavor industry, a point of view grounded in numbers.

Currently, the global market for fermentation-derived productsworth $24.3 billion and it is expected to grow at a compoundannual growth rate (CAGR) of 7.7% in the following years to reach avalue of $35.1 billion by 2020 (PRNewswire, 2015). If we include thedata for bioethanol, this market exceeds US$ 120 billion (Deloitte,2014). Thus, considering that the greatest impact of whitebiotechnology may be on the fine chemicals segment (Europabio,2003), the production of bioflavor not only has considerablyincreased in the last decades, but it is projected to have anincreasing role in the food industry to supply the consumers' de-mand for more natural flavors, whose market (excluding season-ings and flavoring materials) is expected to grow with a CAGR of9.1% (UBIC Consulting, 2014).

Acknowledgements

The authors would like to thank Minas Gerais Research Foun-dation (FAPEMIG) for financing the masters scholarship of L. O.Felipe (identification no. 11761, 11762), National Council for Scien-tific and Technological Development (CNPq) for the financial sup-port to the project related to this subject (473981/2012-2), andEspaço da Escrita (Unicamp) for the English edition of the text.

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lable at ScienceDirect

LWT - Food Science and Technology 76 (2017) 361e369

Contents lists avai

LWT - Food Science and Technology

journal homepage: www.elsevier .com/locate/ lwt

High pressure processing (HPP) of pea starch: Effect on thegelatinization properties

Thiago S. Leite a, *, Ana Laura T. de Jesus a, Marcio Schmiele a, Alline A.L. Tribst b,Marcelo Cristianini a

a Department of Food Technology (DTA), School of Food Engineering (FEA), University of Campinas (UNICAMP), Brazilb Center of Studies and Researches in Food (NEPA), University of Campinas (UNICAMP), Brazil

a r t i c l e i n f o

Article history:Received 1 February 2016Received in revised form11 July 2016Accepted 14 July 2016Available online 16 July 2016

Keywords:High pressure processingParticle Size DistributionDifferential Scanning CalorimetryPasting propertiesPea starch

* Corresponding author. DTA/FEA/UNICAMP, R. MUniversit�aria, CEP: 13083-862, Campinas, SP, Brazil.

E-mail address: [email protected] (T.S. Leite

http://dx.doi.org/10.1016/j.lwt.2016.07.0360023-6438/© 2016 Elsevier Ltd. All rights reserved.

a b s t r a c t

High pressure processing (HPP), an emerging technology, can be used to promote gelatinization of starchgranules. This phenomenon is highly dependent on the source of starch, pressure level, time and tem-perature applied as well as the dispersion medium. This work evaluated the effect of HPP (up to 600MPa/15 min/25 �C) on particle size distribution, optical microscopy, differential scanning calorimetry andpasting properties of pea starch. Results showed no difference between control samples and processedones up to 400 MPa (water dispersion) or all samples dispersed in ethanol, except for the thermalproperties at 400 MPa that showed 31% of gelatinization in water dispersion. Samples processed atpressures higher than 500 MPa showed changes on particle size and distribution (increase at 500 MPaand a slight reduction at 600 MPa), and no detected gelatinization enthalpy at DSC. The optical micro-scopy observation indicated that HPP (>400 MPa) caused the loss of birefringence. Regarding the pastingproperties, the initial viscosity increased from 8 cP at 0 MPa to 34 cP at 600 MPa. All results indicated thatHPP can be used to promote “cold gelatinization” on pea starch water dispersion, achieving a specifictechnological profile and possibly leading to new ingredients.

© 2016 Elsevier Ltd. All rights reserved.

1. Introduction

Starch is the main carbohydrate reserve in higher plants and themost important source of energy for humans. Industrially, it hasbeen widely used for numerous applications in various industries,including food and non-food, due to its functional properties, suchas dispersion of ingredients, texturizing agent, fat replacer, andmouth feel enhancer (Adebowale, Afolabi, & Olu-Owolabi, 2005;Perez-Pacheco et al., 2014; Wang & Copeland, 2013).

Starch structure and properties of phase transitions are impor-tant once they influence viscosity, appearance, texture, water-holding capacity and enzyme digestibility of processed starchbased food products (Wang & Copeland, 2012). The physicochem-ical properties of the starch and thus its applications depend onfactors such as the amylose/amylopectin ratio, granule size andshape, degree of polymerisation, diffraction pattern and the dif-ferences in crystalline/amorphous regions of granules, as well as its

onteiro Lobato, 80, Cidade

).

botanical origin and source (Błaszczak et al., 2003; Sankhon et al.,2014).

Pea starch is mainly available as a by-product from pea proteinextraction, being a relative cheaper source of starch compared towheat and potato. Pea starch is characterized by its high amylosecontent (35e65%), fast retrogradation, resistance to shear thinningand high resistant starch content (Wang & Copeland, 2015). Due toits high amylose starch percentage, pea starch is mainly industriallyused to obtain flexible films with good mechanical properties andgas barrier (Ratnayake, Hoover, & Warkentin, 2002).

Chemical and physical modifications of starch are commonlyemployed to obtain starches with special functional properties,increasing its range of use. Although chemically modified starchesare available for industrial uses, most industries (especially foodand pharmaceutical industries) prefer starches that have beenphysically altered (heat, moisture, shear, radiation, high pressureprocessing) due to their relative safety (Adebowale et al., 2005).

High pressure processing (HPP) is a non-thermal emerging tech-nology that subjects aproduct tohighpressures (upto1000MPa) foracontrolled time and temperature. HPP affects only non-covalentbonds and can cause serious structural damage to biopolymers,


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T.S. Leite et al. / LWT - Food Science and Technology 76 (2017) 361e369362

including protein denaturation and starch gelatinization (Balny,Masson, & Heremans, 2002; Hu et al., 2011). In this context, HPP hasbeen employed to gelatinize or physically modify different types ofstarch dispersions (Li et al., 2011, 2012; Yang, Gu, & Hemar, 2013).Starch granules could be gelatinized completely at room temperaturebyHPPand the impact of the process in starch gelatinization dependsmarkedly on starch type, treatment pressure, temperature, time andwater content (Bauer & Knorr, 2005; Li et al., 2011, 2012). All datarelative to HPP on starch were obtained in aqueous media; being notpreviously established if the HPP can induce changes on starchdispersed in other dispersion media, or if water (hydration) has acentral role in the modifications observed in starches after HPP.

Several studies have been carried out with starch processed byHPP (Li et al., 2011, 2012; Yang et al., 2013), however, nothing hasbeen done to verify its effects on Particle Size Distribution (PSD),optical microscopy, Differential Scanning Calorimetry (DSC) andpasting properties on the same study, which disrupts the wellunderstanding of the phenomenon on the starch. Additionally,there are studies on the structure and functionality of starches frompeas (Bogracheva, Morris, Ring, & Hedley, 1998; Chung & Liu, 2012;Ratnayake, Hoover, Shahidi, Perera, & Jane, 2001; Wang, Sharp, &Copeland, 2011), but little information is available for starchesfrom peas processed by HPP. In respect to that, Le Bail et al. (2013)observed that HPP (500 MPa; 20 min; 20 or 40 �C) induced gela-tinization of several starches including pea starch, lacking a deepevaluation about the process impact on the pea starch molecularstructure. Considering the lack of information about HPP effects onpea starch and on starches dispersed in non-aqueous media, thiswork aimed to evaluate the impact of HPP on pea starch dispersedin water and ethanol by using structural and functional evaluationof the processed sample.

2. Materials and methods

2.1. Pea starch and dispersions preparation

Pea starch (33% of amylose) was obtained from Emsland-St€arkeGroup - Food Division, Germany. Starch samples were preparedusing 4% (w/w) of starch dispersed in distilled water or in ethanol(99.9%), stirred at 25 �C (using magnetic mixer) within 24 h prior toprocessing.

2.2. High pressure processing (HPP)

A high pressure equipment (QFP 2L - 700 Avure Technologies,OH, USA) with a chamber of 2 L volume, maximum pressure workof 690 MPa and temperature controlled from 10 to 90 �C was usedin the assays. Samples were packaged in sealed LDPE-Nylon-LDPEbags (16 mm thickness - TecMaq, Brazil). Processes were per-formed at 300, 400, 500 and 600 MPa for 15 min at 25 �C. Thepressurization rate was about 6 ± 0.5 MPa/s; the depressurizationtime was almost instantaneous. After process, all samples weredistributed in glass petri dishes, cooled down quickly, lyophilized(water dispersion) or air dried (ethanol dispersion) and storedbefore analysis. All processes were carried out in triplicate.

2.3. Visual observation and optical microscopy observation

Visual observation was conducted in graduated cylinders of25 mL, all samples were shaken for 1 min, and placed on a table torest during 1 min, and a photo was taken showing samples side byside, for comparison purpose.

Optical microstructure was observed using an optical micro-scope (Carl Zeiss Jenaval, Carl Zeiss Microimaging GmbH, Germany)with an 20x, 40x or 100x objective and 10x optovar, coupled to a

digital camera and software (EDN2 Microscopy Image ProcessingSystem). Before the observation, a small amount of sample wascarefully placed on a glass slide, and placed a droplet of distilledwater and covered with a cover glass.

2.4. Particle Size Distribution (PSD) analysis

PSD was evaluated by light scattering (Malvern Mastersizer2000 with Hydro 2000s, Malvern instruments Ltd, UK). A littleamount of dry sample was slowly added into a sample compart-ment, previously filled with ethanol (99.9%; room temperature),until obscurity reaches values around 10. The mean diameter wasevaluated based for both particle volume (D[4,3]; Equation (1)) andthe particle surface area (D[3,2]; Equation (2)). This is useful sincethe particles are not ideal spheres, and D[3,2] is more influenced bysmaller particles, whilst D[4,3] is more influenced by larger ones(Bengtsson & Tornberg, 2011; Lopez-Sanchez et al., 2011). Theseanalyses were carried out in triplicate.

D½4;3� ¼

Pinid4iP

inid3i

(1)

D½3;2� ¼

Pinid3iP

inid2i

(2)

2.5. Differential Scanning Calorimetry (DSC)

Thermal transition of starch samples was evaluated using a DSC(TA Instruments, New Castle, DE, USA). Samples (control and pro-cessed) were weighed (3 mg) in aluminium pans and water (7 mL)was added. Then, the pans were sealed, rested for 30 min at roomtemperature and heated at temperatures from 30 �C to 95 �C at therate of 10 �C min�1. An empty pan was used as reference, and theDSC equipment was calibrated with indium for temperature andheat capacity. From the DSC data it was obtained the thermaltransitions of starch dispersions - according to the parameters To(onset), Tp (peak gelatinization) and Tc (conclusion) - DH value(referred to enthalpy of gelatinization), temperature range (DT ¼ Tc- To) and degree of gelatinization (%G), that was calculated using thefollowing Equation (3) (Blaszczak et al., 2007):

%G ¼�DHcs � DHps

DHcs

�,100% (3)

Where DHcs and DHps were the gelatinization enthalpies of controlstarch and pressurized starches, respectively.

2.6. Pasting properties

The pasting properties of pea starch samples were evaluatedaccording to the method 162 of ICC (1996) using a Rapid ViscoAnalyser (RVA) (model RVA 4500, Peter Instruments, Warriewood,Australia) and the curves were analysed by the softwareTCW3.15.1.255 through the profile ‘Extrusion-1’ and 3.0 g (db) ofstarch sample. The parameters analysed were the viscosity (cP) andthe pasting temperature (�C). The ‘Extrusion-1’ profile was chosenby analysing the cold viscosity.

2.7. Statistical evaluation

When relevant, the effect of pressurewas evaluated by using the

T.S. Leite et al. / LWT - Food Science and Technology 76 (2017) 361e369 363

analysis of variance (ANOVA) and the Tukey test at 95% confidencelevel using Statistica 7.0 (StatSoft, Inc., USA) software.

3. Results and discussion

3.1. Visual observation and optical microscopy

Fig. 1 shows a picture of pea starch processed HPP in waterdispersion taken immediately after the process. Fig. 1 clearly il-lustrates that the high pressure process can affect the pea starchdispersion inwater. It was observed that process up to 400 MPa didnot change the appearance of the dispersion. However, after pres-surization at 500 MPa, the sediment phase presented a highervolume, and, after process at 600 MPa, it was clearly observed a gelphase. This indicates that the HPP can lead to formation of peastarch gel at room temperature, and this process can be called ‘cold-gelatinization’ as did not requires a conventional cooking step.

According to Santos et al. (2014), high pressure can increase thewater hold capacity (WHC) of starch. The authors found that maizestarch at 500 MPa for 15 min showed a 35-fold higher WHC, thanthe non-pressured sample. On the contrary, at 300 MPa sample didnot show difference with the non-processed, being the resultscompatible with the obtained in the present research.

The results of starch pressurized dispersed in ethanol (data not-showed) highlighted the importance of the water to promotegelatinization process. Fig. 2 shows the optical microscopy usingregular and polarized light of pea starch dispersed in water. It canbe noticed that no visual changes were observed for the ethanoldispersion pressurized up to 600 MPa.

Fig. 3 shows the optical microscopy only for the control and600 MPa processed samples on ethanol dispersion, since the otherdid not show any difference.

As can be seen in Fig. 2, as pressures increases more effects canbe noticed in the starch granules. Control sample showed thegranules nearly rounded shaped on regular light, while on polar-ized light was observed a “Maltese-cross”, due to the starch thatwas not gelatinized. For samples processed up to 400 MPa, nodifferences between the shape of the granules, size and also thepresence of “Maltese-cross” where observed in comparison withcontrol sample. At 500 MPa, granules showed an increase in size,and became a swollen granule, at polarized light almost none re-gion of the granule showed a difference in birefringence. Finally, at600 MPa, granules were reduced and converted from a roundedshape to an irregular shape, and at polarized light, the starchshowed no birefringence pattern. These results are in accordance

Fig. 1. Effect of HPP on pea starch in water dispersion.

with Fig. 1, corroborating the hypothesis that the HPP promotesslight and intense ‘cold-gelatinization’ at 500 and 600 MPa,respectively.

For sample dispersed in ethanol (Fig. 3) starch structure waspreserved even after process at 600 MPa, with granule roundedshaped and similar size of the non-processed samples. The differ-ences between the results obtained for starch dispersed in waterand ethanol clearly indicates that the ‘cold-gelatinization’ causedby HPP occurred by forcing water molecules into the granule,promoting a hydration process of starch without heating, which at600 MPa leads to complete gelatinization.

Several authors evaluated microscopically the impact of HPP onother sources of starch (Bauer, Hartmann, Sommer, & Knorr, 2004;Błaszczak, Fornal, Valverde, & Garrido, 2005; Li et al., 2015; Oh,Pinder, Hemar, & Anema, 2008; Pei-Ling, Qing, Qun, Xiao-Song, &Ji-Hong, 2012; Santos, Saraiva, & Gomes, 2015; Vallons & Arendt,2009). Santos et al. (2015) did not verify major change in maizestarch processed at 400 MPa at 27 �C for 5 min. Generally, gelati-nization occurs between 450 and 600 MPa for different types ofstarch (red adzuki bean e Li et al., 2015, sorghum e Vallons &Arendt, 2009, tapioca e Bauer et al., 2004; Oh et al., 2008; Le Bailet al. 2013, waxy corn starch e Pei-Ling et al. 2012; Oh et al.,2008, rice, waxy rice and corn e Oh et al., 2008, pea starch e LeBail et al. 2013, and broad beane Le Bail et al. 2013). However,starch from potato did not change its birefringence after 500 MPa(Le Bail et al. 2013) or even after 600 MPa (Oh et al., 2008).

The differences of starch resistance to gelatinization at highpressure can be attributed to the content of amylose in the granule.Comparing the results of gelatinization of rice,waxy rice, corn,waxycorn, tapioca and potato, Oh et al. (2008) observed that gelatiniza-tion was higher for samples with low amylose content (waxy rice,waxy corn and tapioca). Similarly, Błaszczak, Fornal, et al., (2005)verified that a sample with 68% of amylose did not show changeson birefringence and on the shape or size of granules after pro-cessing at 650 MPa for 9 min, but observed complete loss of bire-fringence afterprocess at 650MPa for3min for samplewith traces ofamylose. Therefore, as for thermal gelatinization, starch with lowamylase content requires low energy input (pressure) to gelatinize.

The pea starch studied has intermediate amylose content (33%),therefore it has some level of ‘cold-gelatinization resistance’. Thisexplains the small regions of birefringence observed for sampleprocessed at 500 MPa. Le Bail et al. (2013) studied HPP gelatiniza-tion induced of several starches, included pea starch, and observeda similar result as in this work, with extend, but not total, level ofbirefringence. In the referenced studied, the observed effect wasnot dependent on the process temperature (20 or 40 �C).

The results observed for samples processed at 500 and 600 MPaare in accordance with the two-step mechanism proposed byRubens, Snauwaert, Heremans, and Stute (1999), which describedthat the high pressure firstly hydrate the amorphous region of thegranules, and this induces a swelling of granules, that promotes adistortion and further destruction of the crystalline region of thegranule. On the second step, the crystalline region is easily accessedbywater, andmorewater penetrate into the granule, increasing theswelling and hydrogen bond formation between water and starch,finalizing the gelatinization process.

3.2. Particle Size Distribution (PSD)

The effect of HPP on mean particle diameter of pea starch onwater and ethanol dispersion are presented on Fig. 4. It can benoticed that, in general, the particles in water dispersion presenteda higher mean diameter than particles in ethanol dispersion,corroborating the requirement of water dispersion to promotestarch gelatinization under pressure.

Fig. 2. Optical Microscopy of Pea starch processed by HPP inwater dispersione comparison between pressures at 0 MPa e a, 300 MPa e b, 400 MPa e c, 500 MPa e d and 600 MPa e e.


Fig. 3. Optical Microscopy of Pea starch processed by HPP in ethanol dispersion e comparison between pressures at 0 MPa e A and 600 MPa e B.

Fig. 4. Effect of HPP on the mean particle diameter of pea starch processed in water and ethanol dispersion (vertical lines are the standard deviation, same lowercase letters on thesame parameter means no difference at 5% of significance).


Regarding the starch in water dispersion, the mean particle sizeshowed no difference up to 400 MPa, an increasing at 500 MPa andthen a slightly reduction at 600MPa, but remaining higher than thesize after processes up to 400 MPa. Regarding starch in ethanoldispersion, as stated above, no effect was observed in mean particlediameter with the increase of pressure level (P < 0.05).

The PSD of pea starch in both water and ethanol dispersion, afterseveral high pressure conditions (conditions that particle size issignificantly different from each other) is presented in Fig. 5. Again,is possible to notice that samples in ethanol dispersion showed noimpact due to pressure and that only processes carried out inwaterdispersion at pressures above 500 MPa showed differences withthe control sample. Starch size in ethanol dispersion showed amonomodal distribution, which confirms the fact the both D[4,3]and D[3,2] were equivalent. The distribution of particles in waterdispersion showed a monomodal distribution with an agglomera-tion of larger particles, especially at 500 and 600MPa. This explainsD[4,3] > D[3,2] for all samples in water dispersion, since largerparticles are associated with higher values of D[4,3]. For non-processed samples and processed samples up to 400 MPa, theselarger particles can be attributed to starch particles hydration thatwas induced in the 24 h of stirring preparation, since at roomtemperature the starch can increase its weight by approximately30% of water (Biliaderis, Page, Maurice, & Juliano, 1986). For sam-ples processed at 500 and 600 MPa, the increase in particle size canbe attributed to the effect of pressure making more water to enterthe granule, leading to gelatinization.

Pei-Ling et al. (2012), verified that high pressure processing didnot affect significantly the mean particle size of tapioca and waxy

corn starches, however, the distribution became broader. UsingSEM (Scanning Electron Microscopy) Li et al. (2015) estimate thesize of red adzuki bean starch granules processed at high pressureup to 450 MPa and observed that higher pressure samples showedpartially disintegrated clusters and bigger size, which is similarly topea starch results.

As observed, the presenceofwater clearly has a fundamental roleon this phenomenon. This statement is reinforced by the alreadydescribed two steps mechanism proposed by Rubens et al. (1999)and by the results of the study performed by Kawai, Fukami, andYamamoto (2012), who verified the impact of several ratios (%) ofstarch/water on the gelatinization induced by HPP. At concentra-tions up to 30%, the authors observed that the gelatinizationenthalpy decreased with the pressure and temperature increase,which are similar to diluted starch systems used in this work. Atratios of 40% and 50%, the temperature did not show any impact,however, the pressure still induced a reduction on gelatinizationenthalpy. At ratio higher than 60% no gelatinization and, conse-quently, no impact on gelatinization enthalpy was observed even at70 �C and 1000MPa. Therewas no sufficientwater on those samplesto hydrated the amylopectin, thus interfering in themelting processand consequently, unable the gelatinization. Pea starch at ethanoldispersion faced the same effect, withoutwater the amylopectin didnot hydrolyzed and would not gelatinize in this dispersion.

3.3. Pasting properties

The pasting properties of pea starch in water dispersion pro-cessed by HPP are presented in Fig. 6. The samples at ethanol

Fig. 5. Effect of HPP on the particle size distribution of pea starch processed in waterand ethanol dispersion.


dispersion were not presented since no effect was observed afterHPP processing. Table 1 shows several parameters obtained by RVAequipment. It is notable the effect of high pressure in pea starch inwater dispersion, in the same pattern: almost no effect up to400 MPa; different behaviours after process at 500 MPa and600 MPa.

The ‘Cold peak’, that represents the suspension viscosityobserved by the equipment before heating, is approximately 65%higher for sample processed at 500 MPa comparing to the coldviscosity of the samples processed up to 400 MPa. This was ex-pected since the PSD and mean diameter are higher for sampleprocessed at 500 MPa, leading to an increase in the friction be-tween particles with consequent increase in the viscosity. Also the

Fig. 6. Pasting profile of Pea Starch HP

sample processed at 600 MPa showed gelatinization prior RVAanalysis, which explains the consistency 4 times higher for peastarch processed at 600 MPa when compared to the controlsample.

Oh et al. (2008), observed a similar trend for starch of rice, corn,waxy corn and tapioca, with no changes on the initial viscosity forsamples processed at 400 MPa and increase of viscosity after pro-cess at 600 MPa. Using sorghum starch dispersion, Vallons andArendt (2009) also found that the initial complex viscosity washigher for samples processed at pressures higher than 500 MPa.

The ‘Raw peak’ also known as ‘peak viscosity’, is the maximumviscosity of the solution that occurs when the starch granules arecompletely hydrated and reach their maximum size, immediatelybefore the granules start to break in minor particles (Kaur, Fazilah,& Karim, 2011). This is considered a good parameter to indicate thethickening properties of the starch. The samples up to 400 MPashowed a lower viscosity at the maximum point than samplesprocessed at 500 and 600 MPa (which did not show statically dif-ference among them; p < 0.05).

The ‘Final viscosity’ is the consistency obtainedwhen the systemis cooled after the end of the gelatinization process. Again, thesample processed at 600 MPa showed the highest value (~20%higher) and all other samples showed similar lower values(Table 1). Therefore, it can be concluded the HPP, especially at600MPa, changes the pasting profile of the pea starch improving itscharacteristics and possibly opening alternatives for industrialapplication of this starch.

Other authors who evaluate the pasting profile of high pressureprocessed starch observed that pressurization reduces the viscosity(peak and/or final viscosity) of red adzuki bean starch (Li et al.,2015), waxy corn and tapioca starch (Pei-Ling et al., 2012). Thesource of the starch, the amylose content and the drying method of

P processed in water dispersion.

Table 1Values of pasting properties by RVA analysis of pea starch processed by HPP in water dispersion (same lowercase letters on the same parameter means no difference at 5% ofsignificance).

Pressure Cold peak Raw peak Hold Breakdown Final viscosity Setback Peak time

Control 8.00±<0.01a 302.67 ± 3.40ab 93.67 ± 2.62c 209.00 ± 5.72a 284.33 ± 3.09b 190.67 ± 0.94b 6.16 ± 0.08a300 MPa 8.33 ± 0.47a 285.67 ± 5.44a 89.00 ± 2.16bc 196.67 ± 7.41a 267.00 ± 2.16a 178.00±<0.01a 6.09 ± 0.06a400 MPa 9.00 ± 0.82a 315.33 ± 2.87b 94.67 ± 0.47c 220.67 ± 3.30a 288.00 ± 0.82b 193.33 ± 1.25b 6.18 ± 0.03a500 MPa 14.00 ± 0.82b 471.00 ± 15.94c 80.33 ± 1.89a 390.67 ± 17.56b 272.67 ± 3.86ab 192.33 ± 5.31b 6.13 ± 0.05a600 MPa 34.00±<0.01c 455.33 ± 2.05c 82.33 ± 3.30ab 373.00 ± 5.35b 333.00 ± 6.16c 250.67 ± 2.87c 6.22 ± 0.03a

Fig. 7. Differential Scanning Calorimetry of Pea Starch Processed by HPP in waterdispersion.


each study can explains the difference between these studies andthe results obtained for the pea starch.

3.4. Differential Scanning Calorimetry (DSC)

Fig. 7 presents the DSC curves of pea Starch processed by HPP inwater dispersion. Table 2 indicates some parameters obtained bythe analysis of the curves of Fig. 7.

The curves obtained for samples processed up to 400 showed apeak of DH, but samples processed at 500MPa and 600MPa did notshowany peak, indicating that higher pressures (�500MPa) inducetotal gelatinization of the starch. However, no difference wasobserved between the control and the processed sample at300 MPa, indicating that process at 300 MPa is unable to promoteany degree of gelatinization. Same trend was observed on sorghumstarch processed at 300 MPa for 10 min (Vallons & Arendt, 2009).The sample processed at 400 MPa showed a slightly reduction ofthe enthalpy, indicating 31% of gelatinization. For this sample HPPdid not change the pea starch gelatinization temperature, since thetemperatures were similar for non-processed and processed sam-ple at 400 MPa.

Incomplete gelatinization was observed on waxy maize (81%) at

Table 2Values of thermal properties by DSC analysis of pea starch processed by HPP in water dsignificance).

Sample Tonset (�C) Tpeak (�C) Tend (

Control 53.61 ± 0.44a 58.79 ± 0.73a 62.78300 MPa 53.35 ± 0.06a 58.33 ± 0.28a 62.22400 MPa 53.73 ± 0.14a 58.34 ± 0.45a 62.08500 MPa Not detected600 MPa Not detected

400 MPa for 30 min (Ahmed, Singh, Ramaswamy, Pandey, &Raghavan, 2014), potato starch (65% and 73%) processed at600 MPa for 2 and 3 min, respectively (Błaszczack, Valverde &Fornal, 2005). Sorghum starch showed 34% and 83% at 400 MPaand 500 MPa, respectively, for 10 min (Vallons & Arendt, 2009),waxy corn starch dispersion (30% and 50%), processed at 450 MPafor 30 min (Pei-Ling et al., 2012) showed respectively 84.5% and95.5% of gelatinization, and red adzuki bean (6%, 9% and 56%)processed at 150 MPa, 300 MPa and 450 MPa (Li et al., 2015). Forthese same studies, but using higher pressures and holding times, itwas reached total starch gelatinization in the different sources ofstarch: tapioca starch and waxy maize at pressure conditionshigher than 400 MPa and 600 MPa for 30 min, respectively (Ahmedet al., 2014), red adzuki bean at 600 MPa (Li et al., 2015), Tapioca at600 MPa for 30 min (Pei-Ling et al., 2012) and sorghum starch at600 MPa for 10 min (Vallons & Arendt, 2009). The different starchsources, starch types, amylose and amylopectin content and theprocess condition (time, temperature and pressure) explain thesedifferences observed in the degree gelatinization induced by HPP.

Additionally to changes on the degree of gelatinization, somestudies described that HPP was able to modify the temperature ofgelatinization, such as on red adzuki bean starch (Li et al., 2015),potato starch (Blaszczack, Valverde et al., 2005& Le Bail et al. 2013),modified tapioca and modified waxy maize starch (Ahmed et al.2014), tapioca (Pei-Ling et al., 2012 & Le Bail et al. 2013), waxycorn (Pei-Ling et al. 2012), broad bean (Le Bail et al. 2013) andmaize(Santos et al. 2015). This change can be associated with the type ofstarch and was not observed in this study. However, Le Bail et al.(2013) observed this phenomenon for pea starch, with increase of5 and 8 �C on the gelatinization temperature after processing sam-ples at 500 MPa for 20 and 500 MPa however at 40 �C, respectively.The observed differences among these results and the obtained inthe present research can be explained once in our study the samplewere freeze dried before DSC analyses, and on the referenced studythe dispersion was analysed after the pressurization.

High pressure can destroy, or at least damage, the orderedstructure of the starch granule, as can be noticed by the change inthe enthalpy, which is related to phase transition (Liu et al., 2009).Although the complete vitreous transition induced by pressure wasobserved for samples pressurized at 500 MPa and 600 MPa(DH300 ¼ 0), the results of the other assays (pasting profile, mi-croscopy and PSD) for these samples were different. This can beexplained by the two step mechanism proposed by Rubens et al.(1999), which described that the water needs to enter in the

ispersion (same small letters on the same parameter means no difference at 5% of

�C) DH (J/g) DT (�C) %G

± 0.50a 3.75 ± 0.06a 9.17 ± 0.17a 0± 0.34a 3.79 ± 0.05a 8.88 ± 0.40a 0± 0.27a 2.57 ± 0.05b 8.36 ± 0.36a 31

100100


granule, creating more hydrogen bonds, increasing the swellingand effectively inducing gelatinization in the second step. There-fore, the difference between samples processed at 500 MPa and600 MPa is that, although 500 MPa is enough to change the orga-nization of the pea starch and, consequently, its enthalpy, it is not apressure high enough to make the complex “starch þ water” tointeract in order to achieve the complete gelatinization, with themaximum volume reduction foreseen by the Le Chatelier's princi-ple (Douzals, Marechal, Coquille, & Gervais, 1996). This lower vol-ume probably was just reached after process at 600 MPa for 15 minfor pea starch, characterized by the results obtained for the opticalmicroscopy and pasting profile.

The results of all analysis performed indicated that all meth-odology employed are adequate to evaluate any direct or indirecteffect on the pea starch caused by HPP. Even the more simplemethodology (visualization in graduated cylinders) gives a goodidea about the impact of the process on the starch. The microscopyand PSD allows a structural evaluation of the granule, improvingthe discussion of the changes that occurs at each pressure level. TheDSC is the most sensitive method to evaluate the degree of gelati-nization, quantifying effects even for the samples processed at400 MPa (for which no other methodology showed alteration). Onthe other hand, this methodology cannot be used isolated to eval-uate the impact of HPP in starches once the results obtained by DSCdid not show difference between 500 and 600 MPa processedsamples while in the other methodologies such as pasting profile,these two process conditions resulted in modified starch withdifferent properties.

4. Conclusions

The high pressure processing caused the loss of the birefrin-gence of the pea starch granules in water dispersion, changed thestarch granule shape, size and particle size distribution, and thepasting properties. The relevant changes occurred with samplespressurized at 500 MPa/15 min, and the more significant changesoccurred after process at 600 MPa/15 min. All changes observedreflect the starch gelatinization induced by the high pressure pro-cessing. By these results, it was concluded that HPP can be used as aphysical methodology to modify pea starch (improving its perfor-mance and industrial application) and that the resistance of thiskind of starch to HPP is similar to the starch from rice, corn, waxycorn and tapioca.

Additionally, these results are useful to understand the phe-nomenon of gelatinization induced by HPP, which forces theentrance of water inside the starch granule, promoting the “coldgelatinization” just for starch dispersed in water. No physicalchange on the granule occurs in absence of water, even at 600 MPa,making the process completely ineffective at this condition.

Acknowledgments

The authors thank the S~ao Paulo Research Foundation forfunding project no.2012/13509-6 and A.L.T.J. scholarship (2015/01570-0); and National Counsel of Technological and ScientificDevelopment for T.S.L. scholarship.

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Vol.:(0123456789)1 3

Eur J Nutr DOI 10.1007/s00394-017-1435-x

ORIGINAL CONTRIBUTION

Fructooligosaccharide intake promotes epigenetic changes in the intestinal mucosa in growing and ageing rats

Glaucia Carielo Lima1 · Vivian Cristine Correa Vieira1 · Cinthia Baú Betim Cazarin1 · Rafaela da Rosa Ribeiro2 · Stanislau Bogusz Junior3 · Cibele Lima de Albuquerque2 · Ramon Oliveira Vidal4 · Claudia Cardoso Netto5 · Áureo Tatsumi Yamada2 · Fabio Augusto6 · Mário Roberto Maróstica Junior1,7

Received: 22 September 2016 / Accepted: 6 March 2017 © Springer-Verlag Berlin Heidelberg 2017

detectable changes in expression in the growing rats, while there were only 19 gene expression changes in ageing rats fed with FOS.Conclusion These results suggest that dietary FOS intake may be beneficial for some parameters of intestinal health in growing rats.

Keywords Prebiotic · Intestinal health · Youth · Ageing · Gene expression

Introduction

The physiology of the bowel and its microbiota composi-tion are targets of interest for many researchers in nutri-tional sciences [1]. Dysbiosis, characterized by an imbal-ance in the microbial community, has been associated with a large number of chronic diseases such as obesity, type 2 diabetes, inflammatory bowel disease and others [2]. The best known strategy to modulate the composition of the intestinal microbiota is the dietary use of prebiotics, pro-biotics and symbiotics. Prebiotic compounds primarily tar-get the bifidobacteria population of the colon microbiota to which various health benefits have been ascribed. Cur-rently, a high colonic bifidobacteria level has been consid-ered favourable at all ages [3]. In addition to modulating the microbiota, prebiotic intake has been associated with mucosal intestinal trophic effects through changes in their structure [4]. This effect could be attributed to the pro-duction of short-chain fatty acids (SCFAs) produced after prebiotic fermentation [4]. The production of SCFAs is one of the most important physiological processes mediated by colonic microorganisms in the large intestine [1].

Among the major colonic SCFAs, butyric acid appears to regulate immune functions, contributing to the maintenance

Abstract Purpose The aim of this study was to investigate the rela-tionship between fructooligosaccharide (FOS) intake at dif-ferent life stages of Wistar rats and its stimulatory effects on intestinal parameters.Methods Recently weaned and ageing female rats were divided into growing and ageing treatments, which were fed diets that partially replaced sucrose with FOS for 12 weeks.Results Dietary FOS intake induced a significant increase in the numbers of Bifidobacterium and Lactobacillus in growing rats. FOS intake was associated with increased butyric acid levels and a reduced pH of the caecal con-tents at both ages. Differential gene expression patterns were observed by microarray analysis of growing and age-ing animals fed the FOS diet. A total of 133 genes showed

* Mário Roberto Maróstica Junior [email protected]

1 School of Food Engineering, University of Campinas, Campinas, São Paulo, Brazil

2 Institute of Biology, University of Campinas, Campinas, São Paulo, Brazil

3 Institute of Chemistry, University of São Paulo, São Carlos, São Paulo, Brazil

4 Sainte-Justine University Hospital Center, Université de Montreal, Montreal, Canada

5 Department of Biochemistry, Biological Sciences and Health Center, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

6 Institute of Chemistry, University of Campinas, Campinas, São Paulo, Brazil

7 Laboratório de Nutrição e Metabolismo–Departamento de Alimentos e Nutrição, Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas, R. Monteiro Lobato 80, Campinas, SP 13083-862, Brazil

http://crossmark.crossref.org/dialog/?doi=10.1007/s00394-017-1435-x&domain=pdf

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of homeostasis in the large bowel mucosa. Gene expression analyses in cell lines have shown the effects of butyric acid on the downregulation of pro-inflammatory genes, such as IL-6 and COX-2 [5], increasing vascular endothelial cell adhesion [6]. Butyrate also modulates MUC gene expres-sion involved in cell proliferation and apoptosis [7] in intes-tinal epithelial goblet cells, which is dependent on glucose bioavailability [8]. Colonic administration of butyric acid in the same concentrations that can be achieved by the con-sumption of a high-fibre diet enhanced the maintenance of colonic homeostasis of healthy subjects by regulating fatty acid metabolism, electron transport and oxidative stress [9]. There is evidence that SCFAs may affect epigenetic gene regulation in the intestinal mucosa by a change in histone acetylation promoted by butyric acid [10].

There is evidence that non-digestible carbohydrates are substrates of the microbiota, leading to the production of short-chain fatty acids (especially butyric acid), which are associated with the regulation of gene expression [11]. However, there are many factors that contribute to the phys-iological benefits of these non-digestible carbohydrates, such as the ageing process. Morphological, enzymatic and metabolic conditions are important in obtaining good health. Given that the quality of dietary intake can modify transcription [12] and that ageing promotes physiological changes that alter gene expression, we investigated the rela-tionship between animal age and the stimulatory effect of fructooligosaccharides (FOS) on intestinal parameters and their effects on the modulation of the gene expression pro-file of growing and ageing female Wistar rats.

Materials and methods

Animals and diets

Experiment 1

Sixteen weaning (3 weeks) female Wistar rats were assigned to two groups according to experimental diet: standard AIN-93G diet [13] (control group) and AIN-93G diet with 50% of FOS (Orafti P95, Beneo-Orafti, Tienen, Belgium) substituting for 50% of sucrose (Table 1).

Experiment 2

Sixteen adult (47 weeks) female Wistar rats were assigned to two groups according to experimental diet: standard AIN-93M diet [13] (control group) and AIN-93M diet with 50% of FOS (Orafti P95, Beneo-Orafti, Tienen, Belgium) substituting for 50% of sucrose (Table 1).

In both experiments, the rats were individually housed in cages in an environmentally controlled room at 23 °C and

a relative humidity of 60% with a 12 h daylight/darkness cycle. Food and water were offered ad libitum. Food intake and body weight were recorded 2–3 times a week through-out the experiments. Intervention was carried out for 12 weeks in both experiments. The study was approved by the Institutional Animal Care and Use Committee (Protocol 20177-1 and 2213-1). Animals were cared for in accord-ance with institutional ethical guidelines.

Caecum collection

At the end of the experiments, the rats were euthanized by decapitation. The caecum was immediately extracted and its contents removed and weighed. The caecal content was divided into three fractions: (1) 100 mg was collected into a sterile assay tube for bacterial counting; (2) 1 g was frozen at −80 °C for SCFA analysis; and (3) 0.5 g was used for pH analysis. The caecum was flushed with ice-cold PBS, soaked in Trizol reagent (Invitrogen, Carlsbad, USA) and stored at −80 °C for gene expression analysis.

Determination of caecal content pH

Caecal contents were diluted with deionized water and homogenized. The pH of the homogenate was measured with a semiconducting electrode (TEC 5MP, Tecnal, Pirac-icaba, SP, Brazil) and accepted as the pH of the caecal contents.

Microbiological assays

Caecal luminal content samples were homogenized in pep-tone physiological saline (100 mg caecal content mL−1). Sixfold serial dilutions were made in peptone solution, and

Table 1 Composition of experimental diets

Ingredient (g/kg of diet) Experiment 1 Experiment 2

Control FOS Control FOS

Cornstarch 429.0 429.0 465.6 465.6Casein 150.0 150.0 140.0 140.0Dextrinized cornstarch 142.5 142.5 155.0 155.0Sucrose 108.0 54.0 100.0 50.0FOS – 54.0 – 50.0Soybean oil 70.0 70.0 40.0 40.0Cellulose 50.0 50.0 50.0 50.0Mineral mix (AIN-93G-MX/

AIN-93M-MX)35.0 35.0 35.0 35.0

Vitamin mix (AIN-93-VX) 10.0 10.0 10.0 10.0l-Cystine 3.0 3.0 1.8 1.8Choline bitartrate 2.5 2.5 2.5 2.5Tert-butylhydroquinone 0.014 0.014 0.008 0.008

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aliquots of 0.1 mL of the appropriate dilution were spread onto the following agar media: MRS agar for Lactobacilli and MRS agar supplemented with 0.5 mg L−1 dicloxacil-lin, 1 g L−1 LiCl and 0.5 g L−1 l-cysteine hydrochloride for Bifidobacterium [14]. Culture plates were incubated in anaerobic conditions (AnaeroGen; Oxoid, Basingstoke, Hampshire, England) at 37 °C for 24–48 h. After the incu-bation, the specific colonies grown on the selective culture media were counted and the number of colony-forming units per gram of caecal content (CFU g−1) was calculated. The average and range were calculated from the log 10 val-ues of the CFU g−1 caecal content.

Histological analyses

Immediately after sacrifice, the small intestine was removed from the abdominal cavity and dissected free from fat and mesentery tissues. The ileum was chosen to meas-ure the height of the villi, and this portion was defined as the distal segment before the ileocaecal junction. The distal part was sampled at 200 mm in proximity to the caecal bor-der. The samples were washed in a 0.1 M phosphate buffer (pH 7.4) and were then fixed with 4% paraformaldehyde in a 0.1 M phosphate-saline buffer at pH 7.4 for 24 h. The fixed samples were then dehydrated in a graded ethanol series (from 70 to 100%) and embedded in paraffin. Intes-tinal cross sections of 5-mm thickness were obtained in a rotary microtome, rehydrated and stained with haematox-ylin–eosin. The analyses were performed with an Eclipse 800 (Nikon, Japan), and digital images were obtained using a Media Cybernetics CoolSNAP camera and software. Villus height measurements were made using the Image J image analysis software [15].

SCFA concentration

The analysis of SCFA (acetic, propionic and butyric acids) content in the stool was performed according to Zhao and co-workers [16] with a few modifications. Three-hundred milligrams of the sample was thawed and suspended in at least 5 mL of deionized water and homogenized in a vor-tex for approximately 3 min. The pH of the suspension was then adjusted to 2–3 by adding 3 M HCl, and then kept at room temperature for 10 min with occasional shaking. The suspension was centrifuged for 10 min at 5433×g to pro-duce a clear supernatant. 2-Ethylbutyric acid was added to the samples as an internal standard, and 1 µL of the super-natant was immediately injected into the gas chromatogra-phy system.

Chromatographic analysis was carried out using an Agilent 6890N GC system equipped with a flame ioniza-tion detector (FID) and N10149 automatic liquid sampler (Agilent, Wilmington, DE, USA). The temperature of the

detector and injector were 250 and 230 °C, respectively. The injector was operated in split mode (1:10), and the injected sample volume was 1 µL. A 30 m × 0.25 mm i.d. × 0.25 mm Nukol™ capillary column (Supelco, Belle-fonte, PA, USA) was used. Hydrogen was supplied as the carrier gas at a flow rate of 1 mL min−1. The initial oven temperature was 100 °C, maintained for 0.5 min, raised to 180 °C at 8 °C min−1 and held for 1.0 min, then increased to 200 °C at 20 °C min−1, and finally held at 200 °C for 5 min.

Total RNA isolation

Total RNA was extracted from each caecum epithelial sam-ple (whole caecum wall) using Trizol reagent (Invitrogen, Carlsbad, USA). Total RNA was reverse transcribed to syn-thesize first-strand cDNA, which was then converted into a double-stranded DNA template for transcription. In vitro transcription synthesized amino-allyl labelled aRNA and incorporated a biotin-conjugated nucleotide. The aRNA was then purified to remove unincorporated NTPs, salts, enzymes, and inorganic phosphate. Following the frag-mentation of the biotin-labelled aRNA, the samples were prepared for hybridization onto GeneChip 230 2.0 expres-sion arrays (Affymetrix, Santa Clara, California, USA). All procedures were performed using the GeneChip 3ʹ IVT Express (Affymetrix, Santa Clara, CA, USA). The concen-tration of the total RNA was determined using a NanoDrop ND-1000 UV/Vis spectrophotometer (NanoDrop Technol-ogies, Wilmington, DE, USA), and the quality of RNA was assessed by agarose-formaldehyde gel electrophoresis.

Microarray hybridization, image acquisition, and statistical analysis

Total caecum RNA (100 ng) samples were labelled and hybridized using Affymetrix Rat Gene 230 2.0 arrays (Affymetrix, Santa Clara, CA, USA) in the Microarray Laboratory at the Brazilian Biosciences National Labora-tory (LNBio, CNPEM, Campinas, Brazil). Differentially expressed genes, as well as the fold-change, were deter-mined using R Bioconductor [17] libraries affy and limma. In summary, a background adjustment was made; the RNA expression values were computed, followed by quantile nor-malization and finally a data summarization. Using a linear model fit function (lmFit, available in limma library), we compared the treatment with the control replicates. A cut-off of fold-change >2 (for upregulated genes) or <−2 (for downregulated genes) and P value <0.05 were employed for subsequent analyses. Each probe ID was matched with their respective gene ID using the Affymetrix Rat Genome 230 2.0 Array annotation file. Gene ontologies were deter-mined using the DAVID online platform. The number of

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ontologies in each category was essential to determine the different functions of genes in each group.

Statistics

For statistical analysis, GraphPad Prism 5.0 (GraphPad Software, Inc. La Jolla, CA, USA) was used. Results are expressed as mean ± standard deviation (SD). Differences of means of the control and FOS groups were tested for significance using an unpaired Student’s t test, two-tailed, recognizing P < 0.05 as significant [6]. Body weight and food intake data from the rats were analysed by two-way ANOVA and Bonferroni tests; (P < 0.05).

Results

Biological assay and caecal characteristics

Body weight and food intake throughout time did not differ between the control and FOS groups in animals of different ages. Therefore, the replacement of sucrose by prebiotics in the diet did not influence these parameters, as shown in Fig. 1.

FOS intake increased Lactobacilli spp. and Bifidobac-terium spp. micro-organism counts in the caecal contents

of growing animals. However, this effect was not observed in ageing animals (Table 2). FOS intake significantly low-ered (P < 0.05) the caecal pH in both experiments (growing and ageing rats). Mean pH values of caecal content are pre-sented in Table 2.

The major SCFAs (acetic, propionic and butyric acids) were identified in the caecal contents of the animals. The concentration of acetic and propionic acids were not altered by adding FOS to the diet of growing or ageing rats. In con-trast, the concentration of butyric acid was 2.4-fold higher (P < 0.05) in growing rats and 2.3-fold higher in ageing ani-mals fed the FOS diet than those fed the control diet with-out FOS. These data are presented in Table 2.

Changes in villus height in different experimental ani-mals are observed during the growing stage [18]; however, growing rats fed the FOS diet did not show changes in vil-lus height compared with the control group (P = 0.8602) (Fig. 2).

Gene expression profiles obtained by microarray analysis

Gene responses were relative to expression in growing or ageing rats fed the control diets. More than 133 genes had detectable changes in expression in growing rats fed diets containing FOS (Table 3). Gene expression patterns of

Fig. 1 Body weight (a) and food intake (b) of the growing rats; body weight (c) and food intake (d) of the ageing rats throughout 12 experimen-tal weeks. The data were analysed by two-way ANOVA and Bonferroni tests (P < 0.05). All data were expressed as the mean ± SD (n = 8)

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growing rats were clustered according to their biological similarity in the ontology analysis (Fig. 2). The FOS diet was associated with the alteration of 19 gene expression (Table 4) patterns in ageing female rats (P < 0.05), wherein five of these genes were clustered in their respective path-ways by gene ontology analysis (Fig. 3).

Discussion

Effects of the FOS diet on food intake and growth

Dietary supplementation with FOS using a 50% replace-ment of sucrose did not change body weight and food intake of growing or ageing rats when compared to the control diet (Table 1). However, it is important to highlight the decreased food intake between ageing rats compared to the growing rats, independent of the diet. This observation could be in accordance with the decrease in gene expres-sion associated with energy metabolism observed by Ames et al. [19] and Lee et al. [20] during ageing.

However, Gudiel-Urbano and Goñi [21] observed a decrease in food intake and in the body weight gain when cellulose from the diet was replaced by FOS. These results suggest that the balance between soluble and insoluble fibres is important for subsequent beneficial effects on the

microbiota, as well as to achieve changes in food intake and body weight gain.

Effects of FOS intake in the intestinal environment

The effects of non-digestible oligossacharides (NDOs) in gut microbiota, specifically FOS, have been demonstrated in different experimental models, including studies on humans [22–24]. Lactobacillus and Bifidobacterium are two species of probiotics that have been investigated and related to many health benefits. Decreases in the Bifidobac-terium count in the microbiota of elderly people is reported in the literature, as well as the use of prebiotics and probi-otics to improve bacterial counts [25]. In the present study, we observed increased Bifidobacterium and Lactobacillus counts in the caecal contents of growing female rats fed a FOS diet. However, there was no change in Bifidobacte-rium and Lactobacillus counts in the ageing group after FOS intake.

The groups of different ages fed diets containing FOS had a significant reduction in the pH of the caecal content in relation to the control groups (Table 2). Accordingly, some studies have found a significant reduction in the cae-cal content pH using FOS in the diet [26, 27]. The reduc-tion in intestinal pH has been attributed to the increase of SCFAs produced during the fermentation of NDOs [1], and

Table 2 Microorganisms (Bifidobacterium spp. and Lactobacilli spp.), pH and short-chain fatty acids in the cecum contents of female Wistar rats in different ages fed control and fructooligosaccharide (FOS) diet

CFU colony-forming units. Values are means ± SDa,b Within the same row and for each experiment (growing rats or aging rats), values with different letter dif-fer significantly (P < 0.05) according an unpaired student’s t test, two-tailed

Experiment 1, growing rats Experiment 2, aging rats

Control FOS P value Control FOS P value

Cecal microorganisms, log10 CFU g− 1 contentes (n = 7) Bifidobacterium spp. 7.9 ± 0.39b 9.0 ± 0.68a 0.0044 8.6 ± 0.66a 9.2 ± 0.60a 0.0995 Lactobacilli spp. 8.0 ± 0.15b 8.7 ± 0.57a 0.0151 9.2 ± 0.38a 9.1 ± 0.67a 0.8440 pH (n = 8) 8.0 ± 0.30a 7.2 ± 0.37b 0.0004 8.3 ± 0.19a 7.3 ± 0.25b <0.0001

Cecal content SFCA, µmol g−1 (n = 6) Acetic acid 49.4 ± 14.1a 49.9 ± 23.1a 0.9632 13.9 ± 3.1a 14.8 ± 4.3a 0.7141 Propionic acid 7.5 ± 3.4a 7.5 ± 2.6a 0.9897 2.4 ± 0.7a 1.9 ± 0.5a 0.2976 Butyric acid 2.8 ± 1.0b 6.7 ± 2.1a 0.0028 2.7 ± 0.6b 6.2 ± 1.6a 0.0022

Fig. 2 Intestinal morphology of growing rats fed dietary FOS

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this decrease in intestinal pH has been attributed to a reduc-tion or inhibition in the growth of potential pathogenic microorganisms, improvements in nutrient absorption and a reduction in the absorption of ammonia and other amines

due to their dissociation [28, 29]. Moreover, in conditions of low pH, the formation of secondary bile acids, which are cytotoxic, is inhibited and its solubility decreased [30]. These effects can account for the protective effect of

Table 3 Changes in gene expression associated with FOS-based diets for 12 weeks in growing female Wistar rats as measured by Rat genome 230 2.0 array

Gene Name Pathway Fold-change

FCER1A Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide External stimulusInflammatory responseImmune responseStress response

2.00

GPX2 Glutathione peroxidase 2 (gastrointestinal) External stimulusImflammatory responseImmune response

−2.24

FCGR2B Fc fragment of IgG, low affinity IIb, receptor (CD32) External stimulusInflammatory responseImmune response

2.52

CXCR4 Chemokine (C-X-C motif) receptor 4 External stimulusImmune response

−2.02

CFH Complement factor H External stimulusInflammatory responseImmune response

2.07

CALCRL Calcitonin receptor-like External stimulusInflammatory responseImmune response

2.13

CXCL12 Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) External stimulusImmune response

2.11

CTGF Connective tissue growth factor Extracellular matrixImmune response

2.95

TEK Tyrosine kinase, endothelial Extracellular matrixImmune response

2.15

ITGB3 Integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) Extracellular matrixImmune response

−3.13

MUC4 Mucin 4 External stimulusExtracellular stimulusImmune response

−2.15

ITGBL1 Integrin, beta-like 1 (with EGF-like repeat domains) Extracellular matrix 2.21PIK3R1 Phosphoinositide-3-kinase, regulatory subunit 1 (alpha) External stimulus

Immune response−2.26

RT1-DA Histocompatibility 2, class II antigen E alpha External stimulusImmune response

−2.20

ATRX Alpha thalassemia Stress response 2.30MYO6 Myosin VI Stress response −2.37APOA1 Apolipoprotein A-I Stress response

Immune response2.10

CRYAB Crystallin, alpha beta Stress response 2.21DUOX2 Dual oxidase 2 Stress response −2.37SMC1A Structural maintenance of chromosomes 1A Stress response −2.72CD96 phosphatidylinositol-specific phospholipase C, X domain containing 2;

CD96 moleculeExtracellular matrix −2.40

PCDH7 Protocadherin 7 Extracellular matrix 2.04ITGBL1 Integrin, beta-like 1 (with EGF-like repeat domains) Extracellular matrix 2.21LDLR Low density lipoprotein receptor Cholesterol metabolic process −3.90HMGCR 3-Hydroxy-3-methylglutaryl-Coenzima A reductase Cholesterol metabolic process −2.50HMGCS1 3-Hydroxy-3- methylglutaryl-coenzima A synthase 1 Cholesterol metabolic process −2.49

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prebiotics in hypercholesterolemia, increasing cholesterol excretion by bile acid excretion and decreased fatty acid digestion [31].

The SCFAs produced by microbial fermentation of prebiotics promotes important physiological effects on the host. Luminal SCFAs, in particular butyric acid, are the preferred energy source for colonocytes [28]. An adequate energy supply of colonocytes is fundamental to the growth and differentiation of normal cells of the colonic epithe-lium, mucus production and increased blood flow in the intestinal mucosa, reducing the risk of mucosal injury [28, 32]. Growing and ageing animals fed FOS diets showed a significant increase (P < 0.05) of butyric acid in their cae-cal contents compared with the controls (Table 2). The capability of FOS to stimulate butyric acid production has been consistently reported [33, 34]. A butyrogenic effect

was observed in rats fed with FOS replaced with 9% starch at three different times (2, 8 and 27 weeks) [35]. Some authors observed increases in the amount of SCFAs over time and, consequently, the luminal pH decreased in the FOS group compared to that of the control group [35]. Some diseases, such as inflammatory bowel disease, have been related to the level of SCFAs in the faeces, especially butyric acid. The possible pathway in which butyric acid contributes to reducing intestinal inflammation was inves-tigated using NCI-H716 cells treated with 100 mM butyric acid to show a fivefold upregulation in MFG-E8 (milk fat globule-epidermal growth factor 8) expression [36]. This factor is associated with immune system homeostasis and could be a way to regulate inflammation in the bowel. In this way, the in vivo effects were tested in a dextran sodium sulphate-induced colitis model using C57BL/6N and

Table 4 Changes in gene expression associated with FOS-based diets for 12 weeks in aging female Wistar rats as measured by rat genome 230 2.0 array

Gene symbol Name Fold-change

Daf Decay-accelarating factor −2.12GR-LACS Gonadotropin-regulated long chain acyl-CoA synthetase −2.35Atf3 Activating transcription factor 3 −2.38Retnla Resistin like alpha −3.21Kdap Kidney-derived aspartic protease-like protein 3.70RT1.E MHC class I RT1.E protein mRNA 2.65Plaur Urokinase receptor mRNA −2.22Best5 Best5 protein −3.49Slc10a2 Solute carrier family 10 3.86Dtr Diphtheria toxin receptor (heparin binding epidermal growth

factor-like growth factor)−2.16

Thox2 NADHNADPH thyroid oxidase −2.25Hbb Hemoglobin 2.40Rev-ErbA Rev-ErbA-alpha protein mRNA 2.40RT1.Bb MHC class II antigen RT1.B beta chain mRNA 7.47RT1-A3 1.68RT1-T24-4 2.65LOC688090 7.46Duox2 Dual oxidase 2 −2.25RSAD2 Radical S-adenosyl methionine domain containing 2 −3.48

Fig. 3 Differential gene expression pattern in growing and ageing rats fed with dietary FOS

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MFG-E8KO mice [36]. A downregulation in proinflamma-tory cytokines was observed in the C57BL/6N strain com-pared to that in the MFG-E8KO strain, suggesting that the anti-inflammatory effects of butyric acid could be related to MFG-E8 expression [36]. In addition, some studies have shown the apoptotic effect of butyric acid in human colon cancer cells [37, 38].

Mucin promotes the protection of the epithelium against mechanical, chemical and microbiological aggressors through the maintenance of the mucus layer, and their pro-duction (such as MUC-2) is also stimulated by SCFAs [39]. Another protective effect of SCFAs in the intestinal barrier is based on the increase of tight junction protein expres-sion. Butyric acid has been shown to improve the barrier function and protection against dysfunction caused by etha-nol in a Caco-2 cell model [40, 41].

Morphological changes in the intestinal mucosa can be considered another effect attributed to prebiotics and SCFAs. SCFAs have been associated with histological changes in intestinal morphology, especially butyric acid, which showed an ability to inhibit colon adenocarcinoma HT-29 cell proliferation and induce apoptosis [42]. In addition, the authors observed that FOS supplementation improved SCFA formation and ameliorated inflamma-tion in the mucosa of colitic rats [42]. The trophic effect of SCFAs was evaluated by its gastric and rectal instilla-tion in rats, which showed increases in crypt cell prolifera-tion [43]. Male piglets fed diets supplemented with FOS showed extensions in the crypt depth [44]. Birds exposed to heat stress showed reductions in villus height, while man-nanoligosaccharide (MOS) intake increased villus height and surface area in the ileum [45]. Broilers fed diets sup-plemented with organic acids increased villus heights in the small intestines; however, no significant differences were observed in the ileum [46].

Maintaining the villous area is directly related to the intestinal absorptive surface area and, in this way, the intes-tinal nutrient absorptive capacity is directly related to the tissue morphology, especially the villus height and crypt depth [47]. Alterations in these parameters appears to have impacts on nutrient absorption, especially with regard to villus height [48].

The risk of developing osteoporosis increases in post-menopausal women, and magnesium deficiency is one of the factors that contributes to this risk. The effects of FOS intake on magnesium absorption were tested on healthy postmenopausal women who showed increases in the absorption and retention of this mineral [49]. The increase in mineral absorption may be related to the trophic effects produced by SCFAs from the process of FOS fermentation. The ileum and colon are sites in the intestine that absorb more magnesium [50], and the prebiotics are primarily fermented in this intestinal portion. In the present study,

FOS intake was not associated with changes in ileum villus height compared to that of the control group. However, we could not conclude that FOS did not have a trophic effect because our evaluation was restricted to the ileum portion and based only on villus height.

Effects of FOS intake on intestinal gene expression

Epigenetics is related to changes in gene expression which do not result from alterations in DNA sequence. The asso-ciation between altered epigenetic patterns in ageing and oncogenesis is well documented [51]. Significant differ-ences were observed in intestinal gene expression in ani-mals fed the FOS diet at different life stages compared to the control groups (Tables 3, 4). The comparison between growing and elderly animals showed that differential gene expression occurs during the ageing process.

There are many food constituents (nutrients and non-nutrients) that can promote epigenetic changes. Butyric acid generated during non-digestible carbohydrate fer-mentation is responsible for some of these changes in gene expression [52]. Microarray analysis showed the differen-tial regulation of 133 genes in the caecal tissue of grow-ing rats that were FOS fed, compared with control groups. Of these 133 genes, 76 were upregulated and 57 were downregulated. These genes were clustered based on their function and/or metabolic pathway. Genes related to bar-rier function and cholesterol metabolism were selected for discussion here. Growing rats showed changes in the expression of 7 genes related to stress responses, 10 genes related to external stimuli, 8 genes related to extracellular matrices, 5 genes related to inflammatory responses, 14 related to immune responses and 6 related to cholesterol metabolic processes (Fig. 3). On the other hand, ageing rats showed changes in the expression of two genes related to viral responses, four related to immune responses and three related to antigen processing and presentation. These dif-ferences in gene expression observed during the growing and ageing stages are related to natural changes in the gas-trointestinal environment during the animal’s lifespan [53]. In fact, an upregulated immune/inflammatory response is expected during ageing, especially due to physiologi-cal changes and age-related diseases observed during this life stage [54]. However, Miller [55] observed a failure in immune system response associated with senescence.

The development of the immune system is linked to the microbiota, and it is required to recognize, respond and adapt to foreign substances. In this way, the micro-biota composition can exert anti and/or pro-inflammatory responses in the gut [56]. The genes Fcer1a and Fcgr3 are associated with positive regulation of immune effects or processes, and they are part of the cluster of differen-tiation cell surface molecules [57]. These positive effects

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on the immune system and in the inflammatory process have been described in animals fed FOS diets [26, 58, 59]. Dietary food intake quality modifies the microbiota com-position, and the nutrients/substances absorbed can interact and modify the immune system response [60]. Butyric acid produced by non-digestible carbohydrate fermentation is used as a fuel by the enteric cells and has been associated with changes in cytokine production and intestinal barrier integrity [60]. The increased butyric acid concentration in the caecal contents of growing animals may be associated with these observed responses, since several studies have demonstrated the role of this SFCA as a modulator of the response of inflammatory-related gene expression [61, 62]. In this way, the consumption of food with prebiotic poten-tial could modify the gastrointestinal environment, as well as the luminal content and improve the gut-associated lym-phoid tissue function, through regulating inflammatory responses observed in some gut diseases [53, 63].

The gene encoding integrin β 3 (ITGB3) was downregu-lated by the FOS diet in growing rats. The overexpression of this integrin has been associated with the formation of some tumours, promoting their invasion and angiogenesis [64, 65]. Prebiotics are associated with decreased colon cancer risk and this protection could be related to the regu-lation of gene expression. Hsu et al. [66] observed a reduc-tion in colonic preneoplastic lesions in rats treated with 1,2-dimethylhydrazine with the intake of prebiotics. The effects of dietary FOS in cellular differentiation and angio-genesis requires additional investigation to identify the var-ious factors that can contribute to cancer development.

Mucins are glycosylated proteins distributed in the gastrointestinal tract between two types of proteins including gel formers and transmembrane [67]. The aber-rant expression of the transmembrane MUC4 has been associated with a variety of carcinomas [68]. In addition, MUC-4 is involved in intestinal epithelial cell differentia-tion, renewal and lubrication [69]. The increased expres-sion of MUC-4 in patients with inflammatory bowel dis-ease can stimulate the activation of nuclear factor kappa ß, which could contribute to the high incidence of malig-nancy and prolong the duration of the disease’s activity [70]. An imbalance in mucosal barrier functions was observed among patients with Crohn’s disease follow-ing MUC-4 overexpression [70]. Dietary FOS intake was associated with reduced MUC-4 expression compared to the control group in growing rats. Some studies have demonstrated a change in mucin expression by butyric acid, which could be related to the effects observed in the present study [71, 72]. This result indicates that the consumption of FOS could be protective against colorec-tal cancer development, a major cause of death world-wide [73]. In fact, the expression of other mucins such as MUC-2 and MUC-3, as well as trefoil factor-3 (TFF3)

is necessary to understand how these protective systems are modulated by FOS intake. However, the data contrib-ute to the statement that the nutritional state can modu-late mucosal cell proliferation, as well as that ageing can contribute to histological and functional tissue alterations [74].

Prebiotic intake has been associated with hypocholes-terolemic effects primarily by lowering cholesterol levels [75]. Growing rats fed a FOS-supplemented diet showed a downregulation in three key genes responsible for choles-terol biosynthesis and uptake, including HMGCR, HMG-GCS1 and LDLR. These results suggest a possible mecha-nism whereby FOS reduces serum cholesterol levels. In accordance with Thumelin et al. [76], dietary composi-tion can modulate HMG-CoA synthase gene expression, a key enzyme in lipid metabolism. Evidence that dietary fibre acts on lipid metabolism is well documented in the literature. Smith-Barbaro et al. [77] showed decreased HMG-CoA reductase activity in both the colon and small intestine of rats fed with diets containing wheat bran.

Conclusion

Our findings indicate that dietary FOS intake increases butyric acid levels in the caecum and concomitantly reduces the local pH, promoting health benefits to the host. The consumption of this oligosaccharide seems to be more effective in modulating the microbiota in grow-ing rats than in ageing rats, especially by increasing the Lactobacillus and Bifidobacterium counts in the caecum. The results confirm that there is differential gene expres-sion during the rat lifespan. Growing rats appear to be more responsive to dietary FOS intake than ageing rats in relation to changes in the gene expression profile. The gene expression profile identified in this study is the first step to elucidate some mechanisms used by FOS to pro-mote health benefits for the intestine.

Future analyses of FOS should address the effects of dietary concentrations in a larger sample size to deter-mine the effects of FOS intake in a group of subjects. In addition, efforts should focus on the effects of FOS in specific pathways, such as the immune system or lipid metabolism, to determine how FOS intake modulates gene expression and metabolic pathways.

Acknowledgements We acknowledge the Microarray Laboratory at the Brazilian Biosciences National Laboratory (LNBio), CNPEM, Campinas, Brazil for their support in the use of equipment, Fluid-ics station and Scanner GeneChip. We are grateful to Clariant SA (São Paulo, Brazil) for providing FOS (Orafti® P95). Finally, special thanks are also due to Espaço da Escrita, UNICAMP, Campinas, Bra-zil who reviewed this manuscript.

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Compliance with ethical standards

Financial support This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (Grant 2010/05681-8 and 2010/16752-3), CAPES, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 300533/2013-6) also provided finan-cial support. Fundação de Amparo à Pesquisa do Estado de São Paulo had no role in the design, analysis or writing of this article.

Conflict of interest On behalf of all authors, the corresponding au-thor states that there is no conflict of interest.

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