Food industry assessment, trends and current issues

In the food industry, enzymes can be classified according to the substrate from the food product, for instance I carbohydrate-active enzymes such as amylolytic, pectinolytic, celluloliti

F OOD AND B EVERAGE C ONSUMPTION AND H EALTH

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C ONTENTS

Cristiano José de Andrade, Ana Elizabeth C Fai B de Gusmão, Ana Paula Resende Simiqueli, Evandro Antonio de Lima, Guilherme Keppe Zanini, Joelise de Alencar Figueira Angelotti and Fabiano Jares Contesini

Chapter 2 Hygiene of Conveyor Belts for Food Production:

Sebastjan Filip, Martina Oder, Eva Stražar and Rok Fink

Chapter 3 Best Practices in Refrigeration Applications to

Promote Energy Efficiency: The Portuguese Case

P D Silva, P D Gaspar, L P Andrade,

J Nunes and C Domingues

Anatoliy G Goncharuk, Dr Habil and Aleksandra Figurek

Contents

vi

Industry in the Crisis: The Case of the Region

Francisco Andrés Díez-Zamudio and Silverio Alarcón

P REFACE

This book provides an assessment of the food industry It discusses trends and current issues Chapter One gives an overview of the main microbial enzymes in the food industry Chapter Two focuses on trends and challenges for the hygiene of conveyor belts in food production Chapter Three identifies energy-saving opportunities (technological, organisational or behavioural) and describes tailored energy-saving measures in the food and drink industry Chapter Four evaluates and compares the efficiency of winemaking in two developing countries (Ukraine and Bosnia and Herzegovina) from the perspective of their development Chapter Five makes a comparison between Denominations of Origins (DO) in Castilla y León in a determined period of time of economical crisis of the wine industry

Chapter 1 – Enzymes are one of the most important tools in the food industry due to their applications for the obtainment of important compounds such as sweeteners, flavors and bioactive compounds, among others They can also be applied in food modification, including galactose hydrolysis of dairy products and juice clarification, as well as for recovery and recycling of industrial byproducts In the food industry, enzymes can be classified according to the substrate from the food product, for instance (I) carbohydrate-active enzymes such as amylolytic, pectinolytic, cellulolitic and hemicellulolitic enzymes, (II) enzymes applied to lipids and hydrophobic compounds including lipases and esterases, (III) enzymes applied to proteins present in food, such as several types of proteases For instance, starch is a polysaccharide that presents several applications in the food industry Starch can be degraded/modified by amylases for process improvement; α-amylases randomly act on α-1,4-glycosidic bonds producing malto-oligosaccharides, maltose or even glucose, while β-amylases act on non-reducing end of starch

Doris Cunningham viii

producing maltose Other amylases are also important for starch modification, including isoamylase and pulullanases Hemicellulases like xylanases can be used to obtain sweeteners, such as arabinose Regarding lipases and esterases, several industrially relevant compounds can be obtained including mono- and diacylglycerols, and polyunsaturated fatty acids (PUFAs) Fatty acids, in particular short-chain fatty acids, have significant impact in the flavor of food products Lipases can be applied to release short chain fatty acids from triglycerides in order to result in flavor enhancement or cheese ripening processes Proteases correspond to an important group of enzymes in the food industry, since they can be used for the production of important bioactive peptides that present biological activity, such as anti-oxidant and anti-hipertensive activities Although there are several sources of enzymes for the food industry use, microbial enzymes are highlighted since they can be obtained through fermentation processes In addition, several genetic engineering techniques, such as recombinant production strains and protein engineering have been studied in order to enhance enzymes performance as well as improve their production Therefore, the application of enzymes by food industry covers a very wide range of food processing techniques, which

is fundamental for technological advances, resulting in more convenient and healthier products, with extended shelf life

Chapter 2 – Concerns for proper hygiene are greatest in food industry, where poor sanitation practices can lead to food related illnesses This is especially important in technology where foodstuff is in contact to surfaces for long period Conveyor belts are the linking element in many segments of the food industry and they are an important part of the process Cleaning methods can affect the amount of soil that is removed from the surface Choosing the right belt is vital for the efficiency and hygiene condition of the production line This chapter represent principles of conveyor belt for food industry, HACCP and cleaning and disinfection of conveyor surface Moreover, chapter

includes also results of testing different methods removing B cereus from the

surface of polyurethane conveyor belts Results of the research show that hurdle technology of cleaning agent and ultrasound is the most efficient Therefore cleaning in place system can maximize the cleanability of the belts

by combining two or more methods for bacterial adhesion control

Chapter 3 – The food and drink industry is the largest and most dynamic manufacturing sector of the European Union With its 286,000 companies (mostly SMEs) and turnover share of 15%, it provides jobs for over 4 million people There is an acute need to replace energy-intensive processes in this sector by new efficient ones The major energy consumption originates from

Preface ix

heating, cooling and drying processes, refrigeration, electrical drive systems, among others Refrigeration of food products can account more than 50% of the energy consumption This chapter identifies energy-saving opportunities (technological, organisational or behavioural) and describes tailored energy-saving measures Best practices and measures for energy efficiency improvement are disclosure, which can be applied to infrastructures, cooling chambers, vapour compression refrigeration systems, compressed air systems, steam generator/hot water systems, among others Additionally, the use of renewable energies and procedures for analysis of the electricity consumption and power management are discussed These best practices and energy conservation measures may substantially improve the energy efficiency and competitiveness of agrifood companies and will ultimately benefit the consumers and society by reducing food price, food waste and carbon emissions

Chapter 4 – Purpose – The paper is devoted to evaluation and comparison

of the efficiency of winemaking in two developing countries (Ukraine and Bosnia and Herzegovina) from the perspective of their development

Design/methodology/approach – In the research, four models of Data

envelopment analysis (DEA), correlation and other tools of the data analysis are used to analyse the efficiency of wineries in two developing countries Returns to scale, scale efficiency, super-efficiency and some other indicators are examined The research is based on the sample including 33 wineries from

Ukraine and Bosnia and Herzegovina Findings – Having the same average

efficiency and number of leaders, medium and large wineries in Ukraine are developing more efficiently than small wineries, whereas in Bosnia and Herzegovina, contrary, a small wine business is more efficient The authors found the high potential growth of efficiency in Ukrainian (up to 28.9%) and Bosnian wineries (up to 28.3%) The ways for its realization were suggested The cross-country efficiency analysis enabled us to find inter-country leaders

in the wine industry They grouped inefficient wineries, calculated the potential to reduce inputs and found main directions to improve efficiency for

each group Research limitations/implications – The research is limited to a

single industry in only two developing countries Future research can be devoted to comparison of the efficiency of wineries in developed and developing countries The results can determine which countries can be

leaders in the global wine market in the future Practical implication – This

study has a practical implication Its results provide useful information for: researchers of wine market in developing countries to understand the current state, basic problems and efficiency levels of wineries in Ukraine and Bosnia

Doris Cunningham

x

and Herzegovina; domestic policy-makers to improve regulation of wine industry to make it more competitive and efficient; wine producers in these countries to find the benchmarks with the best practice to adapt them in own

business and increase its efficiency Originality/value – This study on the

example of Ukraine and Bosnia and Herzegovina has shown that each such country has its own conditions of doing the wine business This is the first paper that compares the efficiency of wine industry in Ukraine and Bosnia and Herzegovina

Chapter 5 – The wine sector in Castilla y León is composed of many Denominations of Origins (DO) and other new wineries without DO, many of these areas were created in the beginning of this century However, some appellations have a long tradition in the elaboration of wine, which are recognized in Spain and also internationally, thus the industry has a relevantly important role in the region The aim of the study is to make a comparison between DOs in Castilla y León in a determined period of time of economical crisis In order to analyse the different types of enterprises, it is utilized economical-financial information The data was collected from SABI database (Sistema de Análisis de Balances Ibéricos, in English Iberian Balance Sheet Analysis System) which gathers accounting information from Spanish firms which present their financial statements The methodology consists of an analysis of 12 economic-financial ratios to examine the diagnosis of the wine sector by years and the different DO selected from Castilla y León All the economical-financial information that is obtained showed how the wine sector was affected by the economical crisis, which is expressed by the different trends obtained The profitability in the period time 2008-2013 presents a clear decrease, which is corroborated by the literature in other sectors and wine DOs

in the same period In the case of the DOs, it is possible to see a clear difference between the areas with more tradition in wine production with a specialization in certain types of wine and the areas with more concentration

of cellars Additionally, the wineries without DO permit to see the development of the sector in a new area Finally, the regression analysis was done by three different models; it is examined from less exactitude to the most trustable In this way it was possible to determine which financial ratios influence more the profitability in this sector In parallel it was done the same regression only for Ribera del Duero, because it represents around the 45% of all the autonomous community

In: Food Industry ISBN: 978-1-63485-792-5 Editor: Doris Cunningham © 2016 Nova Science Publishers, Inc

Chapter 1

Cristiano José de Andrade1, Ana Elizabeth C Fai B de Gusmão2,

Ana Paula Resende Simiqueli3, Evandro Antonio de Lima4, Guilherme Keppe Zanini5,6,

Joelise de Alencar Figueira Angelotti7

and Fabiano Jares Contesini5,6*

1Polytechnic School of the University of São Paulo (USP),

São Paulo, Brazil

2Department of Basic and Experimental Nutrition, Institute of Nutrition, Rio de Janeiro State University (UERJ), Rio de Janeiro, Brazil

3National Agricultural Laboratory - Brazilian Ministry of Agriculture, Livestock and Food Supply (LANAGRO-SP/MAPA), Campinas, Brazil

4Brazilian Biosciences National Laboratory (LNBio), Campinas, Brazil

5Institute of Biology, University of Campinas – Unicamp, Brazil

6Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM),

Campinas, Brazil

7Faculty of Food Egineering – University of Campinas –

Unicamp, Campinas, Brazil

* Corresponding author: fabiano.contesini@gmail.com

Cristiano José de Andrade, Ana Elizabeth C Fai B de Gusmão et al

2

Enzymes are one of the most important tools in the food industry due

to their applications for the obtainment of important compounds such as sweeteners, flavors and bioactive compounds, among others They can also be applied in food modification, including galactose hydrolysis

of dairy products and juice clarification, as well as for recovery and recycling of industrial byproducts In the food industry, enzymes can be classified according to the substrate from the food product, for instance (I) carbohydrate-active enzymes such as amylolytic, pectinolytic, cellulolitic and hemicellulolitic enzymes, (II) enzymes applied to lipids and hydrophobic compounds including lipases and esterases, (III) enzymes applied to proteins present in food, such as several types of proteases For instance, starch is a polysaccharide that presents several applications in the food industry Starch can be degraded/modified by amylases for process improvement; α-amylases randomly act on α-1,4-glycosidic bonds producing malto-oligosaccharides, maltose or even glucose, while β-amylases act on non-reducing end of starch producing maltose Other amylases are also important for starch modification, including isoamylase and pulullanases Hemicellulases like xylanases can be used to obtain sweeteners, such as arabinose Regarding lipases and esterases, several industrially relevant compounds can be obtained including mono- and diacylglycerols, and polyunsaturated fatty acids (PUFAs) Fatty acids, in particular short-chain fatty acids, have significant impact in the flavor of food products Lipases can be applied

to release short chain fatty acids from triglycerides in order to result in flavor enhancement or cheese ripening processes Proteases correspond to

an important group of enzymes in the food industry, since they can be used for the production of important bioactive peptides that present biological activity, such as anti-oxidant and anti-hipertensive activities Although there are several sources of enzymes for the food industry use, microbial enzymes are highlighted since they can be obtained through fermentation processes In addition, several genetic engineering techniques, such as recombinant production strains and protein engineering have been studied in order to enhance enzymes performance

as well as improve their production Therefore, the application of enzymes by food industry covers a very wide range of food processing techniques, which is fundamental for technological advances, resulting in more convenient and healthier products, with extended shelf life

Keywords: food industry, microbial enzymes, amylases, lipases, proteases

An Overview on the Main Microbial Enzymes in the Food Industry 3

In the past years, the advances in protein engineering technology have led to significant increase of enzyme applications by industries In general, enzymes have high specificity, are more efficient and also show higher catalytic activity compared to chemical catalysts For commercial enzyme production, a very wide range of sources are used The production of microbial enzymes is easier to control compared to the production of animal and plant enzymes (Soares et al., 2012) In the food industry, an emblematic example of the transition from animal enzymes to microbial enzymes is the production of cheese involving curdling of milk, which traditionally extracts enzymes from stomachs of calves, however, currently, can be produced by yeast (Kumar et al., 2010) The production of microbial enzymes contains also fewer harmful compounds, including phenolic and endogenous enzyme inhibitors In this sense, the food industry represents one of the major consumers of enzymes, and focuses its use on debranching (higher solubility and clarification) However, there is a trend toward researching on the enhancement of enzyme activity and enzyme stability against heat and organic solvents (considered the most critical parameters) by empirical and advanced computational (Rosetta@home) approaches and also for new uses (Choi et al., 2015)

Currently, due to advances in biotechnology and biochemistry, enzymes can be obtained in high purity form and can be used in the food industry to a) reduce the amount of sulfur and increase flavors in wines; b) clarify juices; c) increase the softness and durability of breads; d) reduce the alcohol content and calorie in beers; e) stabilize beer; f) nutritional additives; g) embedded texturing foods such as sausages (Kuraishi et al., 2001; Duran et al., 2002; Sponher et al., 2015; Soares et al., 2012) Besides the ability to act in various processes, the use of enzymes may be considered an environmentally correct strategy, as it eliminates certain by-products that should be processed in the food industry generating energy expenditure (Fernandes, 2010) Moreover, these molecules are considered natural products and have been used as part of the human diet for a long time and are, therefore, non-toxic and food compounds and are preferred by consumers rather than the use of chemical compounds for processing foods (James and Simpson, 1996; Duran et al., 2002)

Enzymes are classified by the substrate that they act upon, (I) carbohydrate-active enzymes such as amylolytic, pectinolytic, cellulolytic and hemicellulolytic enzymes (II) enzymes applied to lipids and hydrophobic

Cristiano José de Andrade, Ana Elizabeth C Fai B de Gusmão et al

Lipases and esterases (II) are responsible for the enzymatic modification

of lipids and hydrophobic compounds Their use in food industries encompasses both the development of new products, such as low calorie food and functional fatty acids, and quality improvement of well-known ones, like bread and cheese, increasing palmitic acid at sn-2 (higher absorption capability) and cocoa-butter-like lipid (Houde et al., 2003; Hasan et al., 2006) One of the most important advantages of using lipases instead of traditional chemical processes is the versatility of this group of enzymes, since, besides their hydrolytic activity in mild conditions, they may also catalyze esterification, interesterification and transesterification in low water content or in nonaqueous media (Hasan et al., 2006; Kotogán et al., 2016), except phospholipases that, due to their lack of affinity with free fatty acids, does not catalyze synthesis (Gerits et al., 2014) Moreover, they usually exhibit good chemioselectivity, regioselectivity and enantioselectivity, as well

as substrate specificity (Joseph et al., 2008)

Last but not least, proteases (III) (also known as peptidases, proteolytic enzymes and peptide bond hydrolases) are hydrolytic enzymes that catalyze the total hydrolysis of proteins into amino acids and represent one of the

An Overview on the Main Microbial Enzymes in the Food Industry 5

largest groups of enzymes (Tavano et al., 2013) Proteases are applied by the food industry in the curdling of milk (cheese production), in which proteases

by Aspergillus niger var awamori is commercially available) Proteases are

also applied in formulation of low allergenic infant food; flavor improvement

in dairy products; tenderization of meat; prevention of chill haze formation in beer; production of fish meals, fish/meat/vegetable extracts, or hydrolysates, among others (Ackaah-Gyasi et al., 2015; Li et al., 2013) Proteases represent approximately 60% of the industrial enzymes market (Ryder et al., 2016; Mayerhofer et al., 2015; Moreno et al., 2013; Rai and Mukherjee, 2010) Out of the ≈4.000 enzymes currently widely known, 200 are from microbial origin Nevertheless, only 20 enzymes are produced at industrial scale (Li et al., 2012) In addition, the market of enzymes is an oligopoly composed by three companies (≈ 75%), Novozymes, DuPont and Roche In this sense, carbohydrases, proteases and lipases are the most used enzymes, in particular in hydrolysis activity (Li et al., 2012) Therefore, there is a need for researches on screening for novel microbial enzymes and their applications, enhancement of activity enzymes, etc

Enzymes play an important role in many industrial processes The benefits provided by their use, both economically and environmentally, lead to

a great increase in demand for this type of molecule Currently, the two main production processes used to obtain enzymes are the Solid-State Fermentation (SSF) and the Submerged Fermentation (SmF)

by the industry (Thomas et al., 2013) This technique is based in a simple principle: cultivation of an isolated microorganism in controlled conditions of certain parameters (i.e., temperature, relative humidity, oxygen concentration)

Cristiano José de Andrade, Ana Elizabeth C Fai B de Gusmão et al

6

on a solid substrate in absence of free-water or near to it Therefore, the substrate must contain all the water and nutrients necessary for the microorganism’s development or it must be an inert material that serves as a support for nutrients (Singhania et al., 2009) This method does not result in a sudden change in the microorganism’s habitat since it represents almost their native growth condition As a result, the production level of enzymes is higher compared to the liquid culture medium method, reaching the necessary level to meet the industrial demand Moreover, by using this production method, industries can mitigate problems inherent to their production processes, such

as waste destination In SSF, industrial waste can be used for the production of commercially important enzymes, adding value to these compounds that would generate costs for proper disposal in addition to environmental problems related to it This is one of many reasons why several studies have been conducted in order to improve and optimize SSF For instance, fungi and yeast are most suitable for use at the expense of bacteria due to the physicochemical conditions that are found in SSF fermentation, as moisture and water available for use However, with the advancement in the understanding of the nutritional needs of microorganisms, the composition of the substrates, how the interaction between them is and advances in bioreactors itself, it is possible to overcome certain barriers, as shown by Sharma and Satyanarayana, (2012) with the production of α-amylase from a strain of bacteria on a SSF system

Several examples arise in this promising scenario for cost reduction and utilization of industrial waste for production of enzymes Rosés and Guerra (2009) performed surface response methodologies and empirical modeling to reach at optimal conditions of temperature, pH, moisture and the physical form that the substrate should be (granulated with specific particle size) for the

cultivation of the filamentous fungus Aspergillus nidulans U0-01 in order to

produce α-amylase The substrate used was sugarcane bagasse, which is an important industrial waste found in countries that produce ethanol from sugarcane Currently, the bagasse does not have any industrial application apart from being burned to generate electricity Roses and Guerra, (2009), showed that the optimum conditions for α-amylase production in that case was: sugarcane bagasse particle size of approximately 7 mm, pH 6.0 and temperature around 30ºC In these conditions, they produced 457.82 EU/g of dry suport Another example was elucidated by Coradi et al., (2013) in which

the lipase production system for the filamentous fungus Trichoderma

harzianum in a SSF system was for the first time described Coradi et al.,

(2013) tested various industrial residues for lipase production in a SSF and

An Overview on the Main Microbial Enzymes in the Food Industry 7

reached the conclusion that a substrate consisting of a mixture of sugarcane bagasse and castor oil cake in a 1:2 ratio supplemented with some other nutritional sources generated the best rate of production of this enzyme by the

fungus T harzianum in comparison with other substrates tested At that

time, the vast majority of the studies focused on the production of this class

of enzymes by other microorganisms in other fermentation system to be discussed in the next section Another example also involving lipases

production was published by Salgado et al., (201) The Aspergillus niger strains MUM 03:58, Aspergillus ibericus MUM 03:49, and Aspergillus

uvarum MUM 08:01 were selected for lipase production by SSF with the main

residue found in the olive mill industry, called Two-phase olive mill waste (TPOMW) The authors applied a Plackett–Burman experimental design in order to evaluate the effect of substrate composition and time on lipase production It was observed that the highest amounts of lipase were produced

by A ibericus on a mixture of Two-phase olive mill waste (TPOMW), urea,

and exhausted grape mark The optimal condition for lipase production by this species was found using 0.073 g urea/g of substrate and 25% of exhausted grape mark resulting 18.67 U/g of lipolytic activity Finally, knowing that the use of industrial waste is both an economical and environmental solution, Muthulakshmi et al., (2011) tested the production of proteases by SSF using different industrial waste, such as cottonseed, rice straw and bran wheat They found that the bran wheat residue generated better results with respect to enzyme production Therefore, further studies were made in order to optimize fermentation par ameters The cultivation was done at pH 5.0 for 7 days at 30°C with a supplement of 3% KNO3 as the nitrogen source, inoculum size 3% and 3% of substrate concentration that yielded 170 U/mg of protein using the

fungus Aspergillus flavus

2.2 Submerged Fermentation

The SmF is performed in a liquid nutrient medium, where the microorganism is submerged As it is held in liquid medium and not solid, some microorganisms such as bacteria end up being favored because they require greater degree of moisture to survive that is not present in solid fermentation (Singhania et al., 2009; Thomas et al., 2013) Furthermore, the extraction of the final product, enzymes or secondary metabolites, is easier and less costly than the SSF method, in which it is necessary to extract the product from the cultured microorganisms therein However, the use of industrial

Cristiano José de Andrade, Ana Elizabeth C Fai B de Gusmão et al

8

waste for production of enzymes by SmF is not as easy as SSF.Several studies have been published comparing the production of certain enzymes by these two methods of fermentation, suggesting that in each case, a preliminary study

is required to determine the most suitable one A study published by Hashemi

et al., (2013) showed significant differences between the production of

α-amylases from Bacillus ssp by SSF and SmF It was observed that for

solid-state fermentation, the optimum pH for enzyme activity was 4 and the temperature was 70°C whereas for the enzyme produced by SmF the optimum

pH was around 3.0 at 60°C Furthermore, it was observed that for the enzyme produced by SSF, pH and temperatures led to lower enzyme activity in 45.8 ºC and pH 5.5, while for the enzyme produced by SmF lowered activities were found in 74.1°C of temperature and pH 5.5 This study shows the importance

of studying the enzyme production by fermentation processes because each has specific characteristics that may be advantageous or not

2.3 Enzyme Expression Systems and Engineering

Regarding productivity and production cost, with the steady growth in the use of enzymes in various industrial processes as a sustainable solution (Adrio

et al., 2014), there is the need to create production systems that are compatible with the requirements of industrial processes Due to advances in biotechnology, it was possible to create enzymatic production systems based

on enzyme’s gene cloning in microorganisms with specific characteristics of protein expression, inhibiting resistance to temperature changes and pH (Fernandes et al., 2010) There are many different microorganisms that can be used for this purpose, so it is important to understand the differences between

then.The bacterium Escherichia coli has been widely used for enzyme

production for having the ability to grow to high cell densities in a short time, accumulate about 50% of its dry weight in heterologous enzymes, grow in cheap culture media and for being easy to handle These characteristics make

it a good microorganism for the industrial production of enzymes, however, there are some disadvantages, such as its inability to perform post-translational modifications, which are responsible for correct protein folding and therefore relates to the enzyme activity (Adrio et al., 2014) Because of this, it is

important to evaluate the enzyme amino acid sequence to make sure that E

coli will be able to produce it in the active form According to Morrow,

(2007), an alternative to the use of E coli is the yeast Picchia pastoris, which

also has the ability to produce high levels of heterologous enzymes and may

An Overview on the Main Microbial Enzymes in the Food Industry 9

reach 30 g/L Picchia pastoris is also capable of performing posttranslational

modifications, checking the correct folding of the heterologous enzymes

Other widely used yeast is Saccharomyces cerevisiae, which like P pastoris,

also carries out post-translational modifications and has high levels of enzyme production

Nevertheless, P pastoris offers several advantages over S cerevisiae

such as a responsive promoter that allows methanol tight control of heterologous protein expression and greater capacity to integrate exogenous DNA fragments to the genome, facilitating the construction of stable strains capable of producing the enzymes (Adrio et al., 2014) One example of the

utilization of P pastoris as an industrial enzyme productin system is with the

enzyme laccase Laccases are used in the food industry for wine and beer stabilization and therefore require a suitable production system that is capable

of supplying the industrial needs Kittl et al., (2012) enabled the production of

a laccase from Botrytis aclada by cloning its gene in P pastoris, reaching the

production of 0.495 g/L of the protein in the extra cellular medium which was at the time the highest concentration of this protein obtained from a

heterologous expression system with P pastoris Lipases, enzymes used, for

example, for the manufacture of margarine from fat-oil blends, were produced

with P pastoris under the control of the methanol responsive promoter as high

concentrations of 20 U/mL in the extracellular medium (Shi et al., 2010) Due to advances in genetics and biotechnology, it is now possible not only to improve the production of the enzymes as described above, but it is also possible to improve various characteristics of the enzyme itself such as stability, which is intrinsically related to the amount used in the process, ability to act in a higher pH range without losing activity and greater specificity for certain substrates, among other features Two approaches can be employed in order to improve enzymes features

The first approach is (a) directed evolution and second (b) rational protein design The first is based on a set of techniques that allow the insertion of random mutations in the genome of the host to be selected, aiming the different clones of the microorganism that have obtained the best mutations that somehow have improved the protein that was meant to be improved In this case, a vast knowledge of the enzyme itself is not necessary in order to improve it, as the technique is based on the random insertion of mutations into the sequence of the enzyme and subsequently selected, making this a versatile technique An example of this technique is with glucoamylases, enzymes responsible for starch processing The starch is liquefied at 105°C in the presence of α-amylases but has to be cooled to 60°C so that the glucoamylases

Cristiano José de Andrade, Ana Elizabeth C Fai B de Gusmão et al

10

may be used In order to eliminate this cooling step which is costly for the industry, many researchers applied directed evolution technique in this enzyme to increase its termostability Another example is the application of

the enzyme glucose isomerase derived from Thermotoga neapolitana for

production of sweeteners This enzyme has optimum activity in media with neutral pH and at temperatures around 100°C, however, in the industry, the isomerization of glucose occurs in an alkaline media and at about 50°C With the use of directed evolution, a more thermostable glucose isomerase, rather than the parental one and active at alkaline pH and 50°C, was selected by Sriprapundh et al., (2003)

The second approach used, the rational protein design, was only possible due to advances related to the understanding of protein structures and their functions, which was accompanied by the development of bioinformatics This technique is based on the rational insertion of changes in the protein’s aminoacid sequence To do that, previous bioinformatics analyzes has to be done to indicate which changes could improve the characteristics of that enzyme Unlike the first approach of directed evolution, it is necessary to have thorough knowledge of amino acid sequence of the proteins, the structure and functionality of each part of the enzyme that will be modified (Adrio et al., 2014; Fernandes et al., 2010) An example of this technique was the improvement of other glucose isomerase, when the rational insertion of two point mutations (G138P and G274P) culminated in an enzyme that had a higher specific activity, increased half-life compared to the parental and greater thermostability (Zhu et al., 1999) Although these two techniques that could generate improved enzymes, both are not mutually exclusive and can be used in a complementary manner (Adrio et al., 2014)

Although enzymes are easily found (in animals, plants, microorganisms), there are many advantages of using microbial enzymes instead of animal and plant enzymes such as better stability, higher productivity (shorter generation times), easier scaling up and genetic manipulation, more robust production conditions, less environment impact Also, they are not affected by seasonal fluctuations and can be produced using inexpensive media (Andualema and Gessesse, 2012) In this sense, examples of microorganisms commercially

exploited as enzyme sources are fungi such as Aspergillus, Candida, Mucor,

Penicillium and Saccharomyces, and bacteria such as Bacillus, Klebsiella,

An Overview on the Main Microbial Enzymes in the Food Industry 11

Lactobacilli, and Streptomyces (Ackaah-Gyasi et al., 2015; Pant et al., 2015;

Ray et al., 2012; Abou-Elela et al., 2011) Thus, microbial enzymes that are applied in food industry were classified in 3 groups (carbohydrate-active enzymes, lipases and proteases) and described below

3.1 Carbohydrate-Active Enzymes

Carbohydrates are the main components of food, and they can be found as natural components or as added ingredients Carbohydrates show many different molecular structures, sizes, shapes and exhibit a variety of chemical and physical properties They can be modified by enzymatic process, and these modifications are employed in food industry for improving their properties and extending their uses (BeMiller and Huber, 2007; Park et al., 2008; Sathya and Khan, 2014) The relationship between the major carbohydrates (foods) and carbohydrate-active enzymes (cellulases, hemicellulases, amylases and pectinases) is described below

3.1.1 Cellulases

Cellulases is a collective term referring to a group of enzymes that hydrolyze the β-1,4-glycosidic linkages in cellulose and produce as primary products glucose, cellobiose and cello-oligosaccharides Cellulolytic enzymes consist of three major components: (i) endoglucanases (EC 3.2.1.4), (ii) exoglucanases or cellobiohydrolases (EC 3.2.1.91) and (iii) β-glucosidases (EC 3.2.1.21) (Lynd et al., 2002; Phitsuwan et al., 2013) The synergistic action of these three enzymes is required for the complete hydrolysis of cellulose to glucose Endoglucanase randomly hydrolyses internal β-1,4 linkages of cellulose chains and creates new reducing and nonreducing ends Exoglucanase cleaves disaccharide cellobiose from the nonreducing end and in some cases from the reducing end of the cellulose chain These cellobiose units and short-chain cellodextrins are hydrolyzed by β-glucosidase into individual monomeric units of glucose (Singhania, 2011)

Microbial cellulases are inducible enzymes synthesized by a large diversity of microorganisms during their growth on cellulosic material, but bacteria and fungi appear to be the major sources (Kuhad et al., 2011) Nowadays, the main commercial preparations of cellulases are

obtained from filamentous fungi, such as Aspergillus niger and Trichoderma

reesei Other well-known cellulase-producing microorganisms include

species from Penicillium, Fusarium, Neurosposra, Humicola, Cellulomonas,

Cristiano José de Andrade, Ana Elizabeth C Fai B de Gusmão et al

12

Thermomonospora, Clostridium, Bacillus and Streptomyces (Bhat and Bhat,

1997; Sukumaran et al., 2005; Kuhad et al., 2011; Gupta et al., 2013; Behera and Ray, 2016)

Cellulases have a wide range of potential applications in food industry One of these applications is in the beverage industry for the production of fruit and vegetable juices In this case, cellulases have been used together with hemicellulases and pectinases, collectively known as macerating enzymes, for extraction and clarification of juices (Bhat, 2000; Karmakar and Ray, 2011) The combined use of cellulolytic, hemicellulolytic and pectinolytic enzymes in juices facilitates the complete liquefaction of plant tissues Thus, it is possible

to directly filter the juice from the pulp without any need for pressing and still increase the efficiency of extraction (Lozano, 2006; Fleuri et al., 2015; Molina

et al., 2015) The addition of these enzymes in juices processing have also helped to reduce viscosity, improve cloud stability and aromatic properties of the fruit juices and their pulps during processing, and increase the release of color components from the skins of fruits (Lozano, 2006; Jurutu and Wu, 2014)

Yu and Rupasinghe (2012) investigated the effects of enzymatic mash maceration with commercial cellulases and pectinases as a pre-treatment for making carrot juice The authors observed that the supplementation of pectinolytic enzymes with endoglucanases and β-glucosidases increased about 27% the carrot juice yield when compared with untreated juice The enzymatic pre-treatment reduced the viscosity and turbidity of the carrot juice, and, in addition, increased the total soluble solids content (about 20%) and the recovery of β-carotene (1.6 times)

Cellulases, mainly β-glucosidases, have also been used with hemicelulases (arabinofuranosidase, α-L-rhamnosidase, β-xylosidase) in beverage industry to increase the aroma and improve sensory characteristics in wines, musts and fruit juices (Palmieri and Spagna, 2007; Pgorzelski and Wilkowska, 2007) The aroma in wines and grape juices, for example, are due to the presence of compounds like terpenes, norisoprenoids, aliphatic alcohols and phenolic (Slegers et al., 2015) These compounds are usually present in a free fraction, which contributes directly to must aroma, and in a bound fraction to various sugars, which is non-volatile and flavourless This bound fraction

is quantitatively more significant than the free fraction and represents a significant reservoir of aromatic precursors, which can be released by acid or enzymatic hydrolysis (Romo-Sánchez et al., 2014) The enzymatic hydrolysis using β-glucosidases is considered a promising method for enhancing the aroma because these enzymes can selectively and efficiently release the

An Overview on the Main Microbial Enzymes in the Food Industry 13

glycosidically bound compounds or aroma precursors without modifying or rearranging aglycones (Ferner et al., 2016)

Li et al., (2013) studied the influence of β-glucosidase on volatile profiles

in mango wine, and observed that the addition of this enzyme accelerated the release of volatile substances such as terpenols, acetate esters, benzene derivatives and C13-norisoprenoids These authors related that enzyme-treated wines presented enhanced terpenols by up to ten times and acetate esters by up

to three times Furthermore, enzyme treatment mitigated, by up to five times, the formation of medium-chain fatty acids and ethyl esters to moderate levels Another field of application of cellulases in foods is the bakery industry The use of these enzymes has enabled increased volume and improved quality parameters of bakery products In addition, cellulases can replace the use of chemical bread conditioners (Nadeem et al., 2009; Kuhad et al., 2011) Haros

et al., (2002) investigated the effects of carbohydrases, including cellulases, on fresh wheat bread characteristics, and they observed that the breads treated with cellulases showed an increase in the specific volume and required slightly lower fermentation times to reach the optimum volume compared to the control According to these authors, the cellulase supplementation also modified the texture of breads, reducing the crumb firmness of the fresh bread, and provided an antistaling effect during storage

Cellulases can also be used to improve extraction of oils and other important compounds to food industry from plants According to Sharma et al., (2015), around 24% of the olive oil is not extractable using the existing oil extraction technologies However, when these authors applied one enzymatic treatment with pectinase and cellulase (1:1) at 0.05%, it was possible to increase the oil recovery in 11%, improve sensory quality (appearance and clarity) and enhance total phenols content as compared with nonenzyme treatment In another study, Ribeiro et al., (2012) evaluated the development

of an enzymatic method of extraction of caffeine from the guarana seed

(Paulliniacupana) for use in energetic drinks as an alternative to the traditional

process, based on the extraction with a hydroalcoholic solution Non-alcoholic guarana extracts with low tannin concentration and high caffeine contents were obtained with the use of cellulases in combination with hemicellulase and α-amylase

3.1.2 Hemicellulases

Hemicellulose refers to a group of homo – and heteropolysaccharides consisting of xylose, mannose, glucose and galactose main chains with a number of substituents resulting in a structurally complex polymer (Manju and

Cristiano José de Andrade, Ana Elizabeth C Fai B de Gusmão et al

et al, 2005; Jurutu and Wu, 2013)

Xylans are the most abundant hemicelluloses, and consist of a backbone

of β-1,4-linked xylopyranosyl groups that are further decorated with different side chain residues Xylanases (endoxylanases and β-xylosidase) are central enzymes in the degradation of xylans Endoxylanases randomly cleave the glycosidic linkages in the xylan backbone and β-xylosidase release xylose monomer from cleavage of the nonreducing end of xylo-oligosaccharides and xylobiose (Colins et al., 2005) Another hemicelulose generally found in cereals, like barley, oat, rye, rice, sorghum and wheat gran, are the β-glucans These polysaccharides, that consist of cellotriosyl and cellotetraosyl residues linked by β-1,3 and β-1,4-glucoside linkages, are hydrolyzed by β-glucanases (or lichenases) and also endoglucanases (Grishutin et al., 2006)

One of the main fields of application of hemicellulases in food is the bakery industry Xylanases and β-glucanaseshave been employed in bread making to break down the arabinoxylansand β-glucan present in flours and improve some dough properties and bread quality Kumar and Satyanarayana

(2014) demonstrated that the addition of xylanase produced by Bacillus

halodurans TSEV1 in whole wheat breads provides an increase in volume, a

greater absorption of water, an improvement in reducing sugar and protein soluble contents, an increasing in shelf life and the liberation of xylooligosaccharides In another study, Li et al., (2014) studied the influence

of β-glucanase on the properties of dough and bread from 70% wheat and 30% barley composite flour According to these authors, bread with added β-glucanase (0.04%) showed an increased specific volume (57.5%) and springiness (21%), and reduced crumb firmness (74%) and staling rate In biscuit-making, xylanases allow cream crackers to be lighter and improve some properties of the wafer, like texture, palatability and uniformity (Polizeli,

et al., 2005)

An Overview on the Main Microbial Enzymes in the Food Industry 15

Another important field of application of hemicellulases is manufacturing

of juices, wines and beers (Molina et al., 2015; Amore and Faraco, 2016) During the malting process for brewing, barley undergoes an incomplete natural germination process that involves a series of enzyme degradations of its kernel endosperm As a result of this enzyme degradation, endosperm cell walls are degraded, and starch granules are released from the matrix of the endosperm in which they are embedded (Gupta et al., 2010) Residual polysaccharydes in malted barley, especially β-glucan and arabinoxylan, may cause severe problems such as increased brewer mash viscosity and turbidity Increased viscosity impairs pumping and filtration, causing lower efficiency, reduced yields of extracts, and lowerfiltration rates, as well as the appearance

of gelatinous precipitate sin the finished beer (Celestino et al., 2006; Liu et al., 2007) Microbial hemicellulases, especially xylanase and β-glucanase, have been added to the brewing process to improve the malting process and solve these problems Scheffler and Bamforth (2005) studied the effects of adding exogenous hemicellulases to the malting process and reported that supplementation with xylanase and β-glucanases decreased the viscosity of wort and improved the extract yield In another study, Zhao et al., (2013) also observed that the addition of these enzymes reduced the viscosity and improved the filtration rate of wort

Hemicellulases (xylanases and mannanases) are also used in coffee processing (Polizeli et al., 2005; Chauhan et al., 2012) Mannan is the main polysaccharide component of coffee extract and is responsible for its high viscosity, which in turn negatively affects the technological processing involved in making instant coffee, because of the high cost involved in removing water by freeze-drying (Chauhan et al., 2012) Chauhan et al., (2014) reported a process for reduction in viscosity of coffee extract by enzymatic hydrolysis of mannan It was observed the reduction, about 2 times,

in viscosity of coffee extract polysaccharides using mananase produced by

Bacillus nealsonii PN-11 According the authors, the use of mananase can

simplify industrial production of instant coffee by the reduction in the energy cost of the process

Another recent field of application of microbial hemicellulases in food industry is the production of ingredients for functional food, like low-calorie sweeteners and prebiotics compounds Non-digestible oligosaccharides, like xylooligosaccharides, mannooligosaccharides, are considered non-cariogenic, prebiotics, and have important biological properties These oligosaccharides can be produced from diverse hemicellulosic materials by enzymatic hydrolysis (Carvalho et al., 2013; Ghosh et al., 2013; Samanta et al., 2015)

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Bragato et al., (2013) reported the production of xylooligosaccharides (XOS), showing degrees of polymerization from 2 to 4 xylose units, from sugarcane bagasse pre-treated with hydrogen peroxide and acetic acid (peroxide-HAc) followed by the application of a recombinant xylanase from

Bacillus subtilis After 7 hours of pretreatment with peroxide-HAc and 15

hours of enzymatic hydrolysis a conversion rate of hemicellulose of 91.86% and a yield of 119.5 mg/g were observed Xylitol is a non-cariogenic sweetener and suitable for diabetics, present in some foods, like chewing gums This sweetener can be obtained from conversion of xylose present in hemicellulosic materials after enzymatic hydrolysis of xylans by xylanases (Polizeli et al., 2005; Branco et al., 2011)

3.1.3 Amylases

Amylases are the main group of hydrolyses that can break down the

O-glucosidic linkages in starch Inside the amylase group the glucoamylase and α-amylase are the most important enzymes due to their industrial applications (Chovatya et al., 2014) The amylases are responsible for nearly one quarter of the global enzyme market (Kikani and Singh, 2012) The α-amylases can be obtained through several sources such as plants, animals and microorganisms (Chovatya et al., 2014; Dar et al., 2015; Homaei et al., 2016)

Nevertheless, microbial sources are mostly used for industrial demands, due to their ability to resist the most extremes conditions found in industrial processes such as high and low temperatures, high salinity and for their ability

to catalyze different reactions (Burhan et al., 2003) Amylase is a potential useful enzyme in several industries such as food, pharmaceutical, textiles, detergents and others (Silvaramakrishnan et al., 2006) The main application is

in starch liquefaction and saccharification in textile industry to remove starch sizer, producing glucose, maltose, malto-oligosaccharides and ethanol (Haki and Rakshit, 2003)

Jo et al., (2016) modified sweet potato starch using glycogen branching

enzymes from streptococcus mutans and amylosucrase from Neisseria

polysaccharea The aim of the work was enhancing slowly digestible starch

content of the sweet potato Daeyumi (which has moderate impact to glycemic index being beneficial to reduce the risk of diabetes and cardiovascular diseases), by enzyme modification altering the branch density and chain length The authors obtained a marked increase in slowly digestible starch and resistant starch fractions They both increased from 6.3% to 25-34.8% and from 12.9% to 34.6-41.3%, respectively

An Overview on the Main Microbial Enzymes in the Food Industry 17

Shukla and Singh (2016) immobilized α-amylase from Laceyellasacchari

TSI-2 in six different supports and used the starch hydrolytic and washing characteristics of the immobilized enzyme to evaluate the enzyme potential use in starch processing and detergents industry Among the immobilization methods, DEAE cellulose with 100 µL of glutaraldehyde presented the maximum immobilized efficiency of 68%, and reusability of 6 cycles The starch hydrolytic efficiency of α-amylase was calculated by taking the ratio of the maltose concentration divided by the initial starch concentration The hydrolytic efficiency results were 4.75; 6.65 and 15.55% to gelatinization, liquefaction and scarification, respectively The authors considered these results were useful for gelatinization and scarification processes, being a good choice for a starch processing specially in the maltose syrup preparation For the washing tests the analysis were done evaluating the removal of starch stained contained in a piece of cotton cloth The authors tested water, free enzyme, immobilized enzyme, detergents and a combination of detergents plus immobilized enzyme Better results were found when detergent in combination with immobilized enzyme was used

Hu et al., (2016), used isoamylase from Pseudomonas sp to prepare

high-amylose ginkgo starch The objective was to use response surface methodology to understand which parameters are important to produce high-amylose starch that are employed at packing films, food medical treatments, optical fibers and electronic chips because it is fast retrograded form being classified as a resistant starch The authors analyzed enzyme dosage, pH, temperature and reaction time The maximum conversion efficiency of 74.74% was obtained after 170 minutes; amylose dose was 110 IU/mL at temperature

of 50ºC and pH 5.2 Morphological and physical property changes were observed after enzyme treatment such as irregular surface and porous inner structure, crystallinity changed from C-typeto V-type, higher blue value and water solubility and lower crystallinity, swelling power and pasting enthalpy

3.1.4 Pectinases

Another group of enzymes that are gaining attention globally as biological catalysts in numerous industrial processes are the pectinase enzymes Theseenzymes’action mechanism is through degradation of pectic substances

by depolymerization (hydrolases and lyases) and deesterification (esterases) reactions (Tariq and Latif, 2012)

Pectinase can present three different enzymatic activities: polygalacturonase (PG), pectin lyase (PL) and pectin esterase (PE) depends on their pectic substances breakdown and modifications in plant tissues

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Pectinases can be obtained from plants or from microorganisms (Ahlawat et

al., 2009) The commercial pectinases are produced in majority by Aspergillus

spp and are mixtures of pectinolytic enzymes The pectinase enzymes can used to facilitate clarification processes, releasing more color and flavor compounds contained in the grape skin, facilitating the liberation of phenolic compounds, treatment of pectic waste water, in paper and pulp industry, including retting and degumming of fiber crops, oil extraction, tea and coffee fermentation (Belda et al., 2016; Kohli and Gupta, 2015)

Belda et al., (2016) selected pectinolytic yeasts to improve clarification and phenolic extraction in winemaking The authors isolated and indentified 462 yeast strains from wineries and then tested for several enzymatic activities of recognized interest for the enology industry

Among those, only Aureobasidium pullulans, Metschnikowiapulcherrima and Metschnikowiafructicola showed polygalacturonase activity According to the literature, M pulcherrima reported occurence in wine flavor, being

selected for further analyses by measuring its influence in filterability, turbidity and the increase on color and anthocyanin and polyphenol content of

wines fermented in combination with S cerevisiae The results showed successful numbers in the expected parameters when M pulcherrima NS-EM-

34 was used, in comparison to wines fermented with only with S cerevisiae and combined with other pectinolytic and non-pectinolytic yeasts (A pullulans and Lachancea thermotolerans, respectively), even when compared with commercial enzymes preparations in most parameters In the next step, M

pulcherrima NS-EM-34 was used at a semi-industrial scale combined with

three different S cerevisiae strains, and the results achieved confirmed its

potential application for red wine improvement based on sensorial and technological properties

Combo et al., (2012) evaluated the individual efficiency of six commercial pectinase preparations (Viscozyme L, Pectinase, Endopolygalacturonase M2, Pectinase 62L, Pectinex Ultra SP-Land Macer8 FJ) The authors used high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) analysis of the enzymatic hydrolysates, to evaluate the liberation of pectic oligosaccharides (POS) from polygalacturonic acid that can be employed as functional foods The results showed that all six enzymes generated oligoGalA with different degrees of polymerization The enzyme specificity and the time of enzymatic reaction influenced the quantitative composition of oligoGalA Endopolygalacturonase M2 was the best enzyme preparation for production of oligoGalA, with 18% (wt) of digalacturonic acid and 58% (wt) of trigalacturonic acid after 2 hours of reaction However,

An Overview on the Main Microbial Enzymes in the Food Industry 19

Pectinase 62L was superior to the other enzyme preparations with 47% (wt) after 1 hour of reaction, in galacturonic acid production

3.2 Lipases

Lipases or lipolytic enzymes are generic terms appliedto describe acyl glycerol hydrolases, mainly triacylglycerol acylhydrolases, EC 3.1.1.3, which are the ones considered as “true” lipases However, many authors also consider carboxylesterases, phospholipases and glycolipases in this definition (Hasan et al., 2006; Kotogán et al., 2016; Gerits et al., 2014) The major applications of lipases by food industry are described below: modified lipids, starch, bread, vegetable oil processing, cheese, egg products and tea

3.2.1 Modified Lipids

The sensorial and nutritional values of a triglyceride are influenced by its fatty acids composition (chain length and unsaturation degree) as well as their position in the glycerol backbone (Andualema and Gessesse, 2012; Sharma et al., 2001) Therefore, inter and transesterification are interesting strategies

to obtain high value triglycerides from inexpensive and less desirable ones,

by increasing their nutritional value, creating low calories triglycerides or increasing their oleic acid content (Andualema and Gessesse, 2012; Hasan et al., 2006; Sharma et al., 2001)

Interesterification is often used to control the melting properties of fats, such as melting profile, crystallization and solid fat content (Cowan, 2010), hence it is the method that has been increasingly used for cocoa butter fat substitutes Since cocoa butter fat for chocolate production is often in short supply and usually is subjected to price fluctuation and variability in quality, alternative fats have been developed with lipase-mediated interesterification, being palm oil the most common substrate (Andualema and Gessesse, 2012; Hasan et al., 2006)

With the increasing public awareness of health problems associated with being overweight and consumption of products with high saturated fats, there was a growing interest in the development of new products with lower calories and higher content of polyunsaturated fatty acids (PUFA) (Vieira et al., 2015) Thus, products such as margarine have been manufactured by interesterification not only with the intention of avoiding trans fat, but also to provide higher content of PUFA (Andualema and Gessesse, 2012; Cowan, 2010) and lipase-hydrolyzed triglycerides have been used as emulsifiers in

Cristiano José de Andrade, Ana Elizabeth C Fai B de Gusmão et al

Properties of the final starch ester usually depend on the nature of the substituent and on its degree of substitution (Rajan et al., 2008), nevertheless substitution of OH groups with fatty acids is reported to lead to more thermoplasticity, mechanical properties, thermal stability, emulsion capacity, retrogradation resistance and hydrophobicity (Gao et al., 2014; Rajan et al., 2008; Xin et al., 2012)

3.2.3 Bread

Enzymes have been used for years to improve quality and technological properties of bread substituting traditional volume-improving agents (Moyaedallaie et al., 2009), such as potassium bromate, which was banned in many countries (Joye et al., 2009) Many enzymatic formulations have been developed in the last decades for this purpose, with lipases being a constant in most of them for their synergistic effect (Salmenkallio-Marttila et al., 1999) The use of lipases in bakery products is reported to strengthen dough stability, increase bread volume, improve shelf-life and crumb structure, reduce bread crumb darkness and enhancenon-enzymatic browning and flavor (Castello et al., 1999; Hasan et al., 2006; Moyaedallaie et al., 2009; Paciello et al., 2015) These effects are usually attributed tothe release of emulsifying compounds, among them free polyunsaturated fatty acids, which are also reported to intensify oxidation reactions with protein thiols and carotenoid pigments, strengthening gluten network and increasing bread volume (Castello

et al., 1999)

Lypolitic enzymes have also been used to improve quality of breads with wheat substitution, such as whole quinoa flour (Park et al., 2005), extruded

An Overview on the Main Microbial Enzymes in the Food Industry 21

rice flour (Martínez et al., 2013) or in high fiber wheat breads Marttila et al., 1999)

(Salmenkallio-Lipase can improve biting quality, stickiness, cooking tolerance and color stability in pasta and noodles; besides, it decreases the absorption of oil by instant noodles and could also improve the quality of final products when using poor quality wheat flour The authors hypothesized that products from the lipase hydrolysis can form complexes with amylose, reducing the swelling

of starch granules and leakage of amylose during cooking (Si and Lustenberger, 1999; Halden et al., 2000)

3.2.4 Vegetable Oil Processing

Most edible vegetable oils need to pass through a refining process (that can be either chemical or physical) in order to remove undesirable impurities that affect their taste, smell, appearance and storability The physical refining process usually consists in degumming, dewaxing, bleaching and deacidification/deodorization (Clausen, 2001; Lamas et al., 2014; Yang et al., 2008) Enzymatic degumming using phospholipase A is currently a well-established process during physical refining (Clausen, 2001) and some authors also defend the potential use of lipases during deacidification (Von der Haar et al., 2016)

The aim of degumming is to decrease the concentration of phospholipids and mucilaginous gums to a limit of 10 ppm in order to prevent color fixation during the deodorization process (Roy et al., 2002) The main advantage of lipase mediated degumming is that phospholipases can convert non-hydratable phospholipids into water-soluble lysophospholipids, which can be removed in

a single-step by centrifugation

Other advantages reported were the reduction of some metals and compounds responsible for color and odor in some oils, improvement in the yield and reduction of waste water and operation costs when compared to traditional physical degumming (Cowan et al., 2010; Lamas et al., 2014; Yang

et al., 2008; Yang et al., 2006) The drawbacks of this process can be the necessity of addition of buffer solutions to maintain an optimum pH, resulting

in more retention of water in the final refined oil and higher levels of free fatty acids by hydrolysis when the enzyme also exhibits acyl glycerol lipase activity (Yang et al., 2006)

A process for lipase catalyzed de-acidification of rapeseed degummed oil

by esterification of free fatty acids with a monoacylglycerol was also described and although it is not yet economically feasible, it could be a sustainable alternative in the future (Von der Haar et al., 2016)

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3.2.5 Cheese

One of the first applications of lipases was in flavor generation in milk products since short chain fatty acids are responsible for their “cheesy” flavor (Cowan, 2010) This characteristic is especially important for extra-mature Cheddar and some varieties of Italian cheese, e.g., Parmesan, Romano and Provolone, whereas the release of methyl ketones is important for Blue; hence lipases have been used for decades to accelerate the ripening of these types of cheese (Fox et al., 1996; Fox and Stepaniak, 1993; Kosikowski and Jolly, 1976; Barach and Talbott, 1987) Other dairy products that had their flavor enhanced by the use of lipases are coffee whiteners and butterfat; toffees and caramels also had their texture improved (Hasan et al., 2006)

Enzyme modified cheeses have been used for years as cheese flavor additives, since they are reported to have a flavor more intense than in regular ripened cheeses (Jolly, 2011), sometimes being 15-30 times more intense (Kilcawley et al., 1998) Most common applications are as additives in salad dressings, dips, soups, pasta products, ready-made meals, bakery products and cheese substitutes/imitations, with the advantage of being added in a very low concentration that is capable of giving the desired flavor without much increase in the fat content of the final product (Hasan et al., 2006; Kilcawley et al., 1998)

3.2.6 Egg Products

It is virtually impossible to commercially obtain egg white without contamination with egg yolk, which decreases the foaming ability of the albumen This problem is mainly caused by the triglyceride fraction of the yolk and is usually solved by a treatment with lipases, resulting in glycerol, triglycerides and fatty acids, compounds that have the additional effect of improving the functional properties of egg white (Lomakina and Mikova, 2006; Kobayashi et al., 1980) This process was efficiently demonstrated by using immobilized lipase (Kobayashi et al., 1980) and has been described in a patent in 1959 (Murphy et al., 1959) Phospholipase can also improve functional properties of an egg white composition, as described in the patent (Casagrande and Carstens, 2014), and a combination of lipases and proteases can be used to produce egg white powder with high foaming ability (东北农业

大学, 2007)

Patents have also been deposited to protect processes or products that improve properties of egg yolk Two examples are Saitou et al., (1992) that protected the use of a phospholipase D to improve its emulsifying and heat

An Overview on the Main Microbial Enzymes in the Food Industry 23

gelation properties, and Topinka et al., (2009) that describes its treatment with phospholipase and protease to increase emulsification, stability and flavor in products such as mayonnaise, salad dressings, bread and desserts

3.2.7 Tea

Lipases can be used to increase flavor volatiles in black tea by improving the conversion of unsaturated lipids to aldehydes and alcohols during fermentation (Ramarethinam et al., 2002) Similarly, there are several patents describing the use of enzymes, including lipases, to increase the extraction yield of black tea (Tsai, 1987), to produce enzymatically extracted tea (Lehmberg et al., 1999), as well as to treat green tea leaves to improve their flavor (Yotsumoto, 2009)

3.3 Proteases

Proteases are subdivided into two major groups: exopeptidases and endopeptidases, based on their site of action The exopeptidases act only on the adjacency of polypeptide end chains Depending on their site of action at the N or C terminus, these enzymes are classified as amino and carboxypeptidases Aminopeptidases act at a free N terminus

of the polypeptide chain and release a single amino acid residue, a dipeptide,

or a tripeptide The carboxypeptidases act at C terminals of the polypeptide chain and liberate a single amino acid or a dipeptide In addition, carboxypeptidases can also be classified into three major groups: serine peptidases, metallopeptidases and cysteine peptidases, depending on the nature

of the amino acid residues at the active site of the enzymes Endopeptidases can be categorizedbased on their catalytic mechanism at the inner regions of the polypeptide chain into four subgroups as serine proteases, cysteine proteases, aspartic proteases and metalloproteases The designed names consider the iconic amino acid or metal present in the active site (Souza et al., 2015)

The major applications of proteases by food industry are described below: dairy products, beer production, flavor enhancers, meat extracts, whey and soy, pet food, bakery products and clarification of processed fruit based beverages

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3.3.1 Dairy Products

The use of enzymes in the elaboration of milk products has been practiced for years In early times, calf rennet was applied to cheese because it contains the enzyme chymosin; now there are microbial chymosin or similar proteases available as alternatives (Lemes et al., 2016) Proteases also contribute to precipitate cheese ripening as well as to modify the functional properties of cheese proteins in order to reduce the allergenic potential of some dairy products Proteases have also been used to manufacture infant milk formulas

in order to hydrolysate the milk proteins into peptides and free amino acids, as non-degraded cow’s milk protein can induce sensitization in infants It is interesting to note that only some parts of milk proteins - the epitopes - present

a potential risk for infants, which are eliminated by cutting one or more of their internal peptide bonds Endoproteases with a preference for cleavage peptide bonds in the highly hydrophilic regions of a protein molecule are preferred for this purpose (Souza et al., 2015; Damhus et al., 2013)

Filamentous fungi belonging to the order of Mucorales, as

Rhizomucorsppand Mucorspp have been reported as potential sources of

milk-clotting aspartic proteinases, having unique technological properties The productivity, the structural characteristics and the functional properties of these enzymes on cheese manufacturing and other uses are affected by the producer strain and the mode of cultivation (Yegin et al., 2011)

3.3.2 Beer Production

Yeast fermentation in brewing process needs proteins in order to survive and replicate If the yeast is not provided with sufficient free amino nitrogen, the fermentation will probably be poor, and the beer quality will be devalued

A neutral bacterial protease supplemented at mashing can be used to increase free amino nitrogen and produce consistent, high-quality beer For instance, this strategy is beneficial when working with poorly modified malt (Damhus et al., 2013)

3.3.3 Protein Hydrolysis for Food Processing

In food processing, the enzymatic hydrolysis of animal or vegetable food proteins could be a promising tool for improving functional and nutritional properties (Damhus et al., 2013; Santos et al., 2011) The hydrolysis of proteins of agroindustrial by-products with enzymes is also an attractive strategy to enhance functional properties and to transform industrial food waste into value added products, such as scraps of meat from slaughterhouses (Ryder et al., 2016; Damhus et al., 2013) The industrial transformation of

An Overview on the Main Microbial Enzymes in the Food Industry 25

milk is an example of the addition of protease to create the food itself On the other hand, for the production of functional food ingredients, the structure of protein usually needs to be enzymatically modified in order to obtain soluble protein hydrolysates Thus, food properties as solubility, emulsification, and foaming might be improved on industrialized products Examples of such products are isoelectric soluble soy protein, egg white substitute and emulsifiers from soy protein, soluble and foaming wheat gluten, whey protein hydrolysates, casein hydrolysates, soluble meat proteins and gelatin hydrolysates (Damhuset al., 2013) In this sense, some of the main examples

of the relationship between properties of proteins and their applications in food are emulsification-meats; hydration-doughts; viscosity-beverages; gelation-cheese; foaming-toppings; cohesion binding-textured products; solubility-beverages, etc (Damhuset al., 2013)

The following ingredients and processes are further examples of protein hydrolysis for food processing

3.3.3.1 Flavor Enhancers

In their natural configuration, proteins do not usually participate in food flavoring, although the products resultants of protein hydrolysis (amino acids and peptides) do contribute to the flavor These protein derivatives of low molecular weight react with other components in food developing flavor, including Maillard reactions between amino acids and reducing sugars, thermal degradation caused by Maillard reactions, deamination, decarboxylation, and the degradation of cysteine and glutathione Furthermore, through their interactions at different taste sites on the tongue, peptides might result in flavors which are bitter, sweet, salty, orumami Sourness and astringency have also been associated to peptides isolated from protein hydrolysates or by synthesis Studies have been exploring the effects of flavor

in dairy products, as well as in meat and fish products Flavored products can

be formed using proteases directly on their own or in combination with fermentation processes (Damhus et al., 2013)

3.3.3.2 Meat Extracts

When producing protein hydrolysates from meat, the first phase includes solubilization of the product by endoproteases It is related that meat hydrolysates usually taste bitter when the degree of hydrolysis (DH) exceeds the 10% the necessary for effective solubilization For this reason, the application of exopeptidases is generally pointed as a solution to eliminate the bitterness of high-DH hydrolysates Protein low degree hydrolysates have

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functional characteristics that are appropriate for use as marinade for products such as ham or bacon, improving meat products with respect to flavor, cooking loss and sliceability Meat extracts are also used as flavor enhancers in soups, sauces, snackfood and noodles (Damhus et al., 2013)

Ryder et al., (2016) described five commercially available food-grade microbial protease preparations for their effectiveness to hydrolyse meat myofibrillar and connective tissue from meat industry waste to produce bioactive peptides The results indicate that microbial protease formulations were able to producenon-cytotoxic bioactive peptides which may be stable in the gastrointestinal tract and might consist of novel bioactive peptide sequences

3.3.3.3 Whey and Soy

The alkaline and neutral proteases produced by fungi present significant function in the production of soy sauce and other soy food products Enzymatic process results in soluble hydrolysates with higher solubility and lower bitterness Protein hydrolysates usually obtained from soy, casein and whey are used as components of dietetic and health industrial food, in infant formulations, clinical nutrition supplements, beverages targeted at pregnant/lactating women and individuals allergic to milk proteins, as well as flavoring ingredients (Souza et al., 2015)

3.3.3.4 Pet Food

The most important use of protease in the production of pet food is enhancing its palatability and digestibility Protease is usually coated onto or mixed into dry pet food and it has the ability to hydrolyze meat by-products originated from meats as poultry, pigs, sheep, lambs, etc and consist of parts such as intestines, livers, and lungs, thus liquefying the raw material and producing an acceptable flavor (Vijayaraghavan et al., 2016; Damhus et al., 2013)

3.3.3.5 Bakery Products

A variety of proteases have been used in baking industry for wheat gluten

modification Endo and exoproteinases from Aspergillus oryzae were assayed

to modify wheat gluten in baking processes The addition of proteases diminishes the mixing time of the dough and results in improved loaf volumes (Souza et al., 2015) Stressler et al., (2015) isolated microorganisms from insects and compost samples, which had a preferred enzymatic activity on

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wheat gluten hydrolysis and the best extracellular peptidase for this purpose

was produced by a Bacillus subtilis strain

Microbial protease also may be used to improve the functionality of proteins in non-wheat dough systems Gluten free systems do not possess the viscoelastic network necessary to resist gas production and expansion during baking process Enzymatic treatments of the gluten free flours have been suggested as a strategy to form protein aggregates that mimic gluten functionality as well as to modify protein structures in order to adjust their functionality in these products Each gluten free system entails specific optimization of protease type and levels (Renzettia and Rosell, 2016)

3.3.3.6 Clarification of Processed Fruit Based Beverages

Some proteases are effective against unwanted haze formation and thus are applied in clarification of processed fruit based beverages, including fruit juices and wines (Pinelo et al., 2010) According to Pinelo et al., (2010) studies review addition of a fungal acid protease to kiwifruit juice reduced the immediate turbidity and postponed the appearance of haze in cold storage These authors found that treatment of cherry juice with a preparation

composed by an acid stable protease from Aspergillus niger, a pectinase from

Aspergillus aculeatus, and gallic acid enhanced its clarity and reduced haze

formation throughout cold storage With black currant juice they also observed that treatments with some fungal acid proteases presented substantial juice-clarifying and haze-delaying properties

Theron and Divol (2014), suggest microbial aspartic proteases might be used in the wine industry to prevent the appearance of protein haze while conserving the wines’ organoleptic characteristics In another study, it is

proposed the use of extracellular acid proteases of non-Saccharomyces yeasts genera (e.g., Rhodotorula, Pichia, Candida, Metschnikowia, Kloeckera, and

Hansenula, among others) to fulfill a number of roles in winemaking, such as

improving the available nitrogen sources for the growth of fermentative microbes, affecting the aroma profile of the wine, and potentially decreasing protein haze development (Reid et al., 2012)

3.4 Other Applications – Food Industry Wastewaters Treatment – Lipases, Esterases, Phytases and Xylanases

The food industry has one of biggest consumption of water and consequent effluent generation Due to the usual high content of organic

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matter, nitrogen, phosphor, among others, the improper disposal of food processing wastewater may cause damage to the environment, threatening the local flora and fauna For instance, the dairy effluents have water soluble sugar, proteins, fat and additives The dairy effluents usually have strong odor The extra nutrients will lead to an excessive microbial growth and consequent higher oxygen consume, affecting the microorganisms (natural selection) as well others organisms

In this sense, there is a trend to apply biological methods (enzymes or microorganisms) to withdraw the main contaminants of industrial effluents instead of physical and chemical methods In addition, the direct application of enzymes in effluents results in lower sludge volume (advantageous) compared

to the application of microorganisms (Arun and Sivashanmugam, 2015) The abattoirs and poultry waste processing, among others, are lipid-rich wastewaters, which imply that they need treatment before a safe disposal (Dharmsthiti and Kuhasuntisuk, 1998; Hasan et al., 2006; Arun and Sivashanmugam, 2015) These wastes can form oil films on the surface, which makes harder for aerobic treatment processes such as activated sludge (Dharmsthiti and Kuhasuntisuk, 1998; Facchin et al., 2012) Therefore, the application of lipases and esterases to lipid-rich wastewaters would improve the treatment and also prevent aggregates formed by oil droplets from obstructing water drainage lines (Dharmsthiti and Kuhasuntisuk, 1998) In this sense, Dharmsthiti and Kuhasuntisuk, (1998) described that crude lipases by

P aeruginosa LP602 were added to lipid-rich restaurant wastewater (1:1)

resulting in significant reduction of lipids, 70% in the first 24 hours and after

48 hours lipids were lower than 10 mg/mL (acceptable for discharge of wastewater) (Hasan et al., 2006) Another interesting approach using lipases was described by Waghmare and Rathod, (2016), which studied the hydrolysis

of waste cooking oil by sonication and lipases activity that was produced by

Candida antarctica (Novozyme 435)

Similarly to lipases, phytase can be applied in order to aid solving environmental problems Usually, the animal feed has high content of phosphate resulting in manure with high concentration of this substance (potential polluter) In the animal feed, a significant part of phosphate is complexed with phytate (10 g of phytate/kg of animal feed), which leads to excretion of undigested phosphorus, in particular by poultry and pigs These residues may result in serious environmental problems, for instance, eutrophication In addition, phytate is highly negatively charged, thus phytate trends to chelate (anti-nutritive effects) proteins and cation including calcium, iron, cooper, zinc (parakeratosis), etc resulting in lower bioavailability

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(Bedford and Partridge, 2010; Ndou et al., 2015) Therefore, on one hand the supplementation of minerals, such as dicalcium phosphate, enhances the mineral absorption in animal husbandry, on the other hand, it increases the costs of production and wastewater treatment

Thus, phytase can be added to animal feed for enhancing the animal absorption of minerals resulting in better nutritional efficiency and lower potential polluters by effluents (Bedford and Partridge, 2010) Microbial

phytases are produced by fungi or bacteria, for instance Aspergillusniger and

Escherichia coli, respectively In this context, phytase obtained by A niger

reduced 58% of dietary phytate (corn and soybean meal) in laying hens Another study, but in pigs, demonstrated that approximately 50% of dietary

phytate was degraded by microbial phytase (A niger) (Bedford and Partridge,

2010) In addition, microbial proteases can be applied to animal feed to enhance the nitrogen absorption, resulting in lower excretion of urea (Li et al., 2012; Facchin et al., 2013) According to Li et al., (2012) feed with addition of enzymes phytase, subtilisin, α-galactosidase, glucanase, xylanase, α-amylase and polygalacturonase are current commercially available In 2007 the world market of feed enzymes was estimated ≈ $344 million and a significant increase is expected for the next years (Li et al., 2012)

Cereal co-products can be used as low-cost animal feed, in particular for poultry and swine diets (Bedford and Partridge, 2010) However, cereal co-products have high content of xylans (antinutritional factors) The presence

of xylans directly affect the growth of animals (Ndou et al., 2015) Thus, the use of xylanases such as endo-1,4-β- D-xylanase, β-D-xylosidases, xylo-oligosaccharides and xylobiose (one at a time or as enzymatic pool) for a partial hydroslysis, will improve the bioabsorption (Bedford and Partridge, 2010; Ndou et al., 2015)

Usually, microbial cellulases are used in animal feed, but also paper and wine industries, olive oil and carotene aid extraction and food industry waste treatment (Facchin et al., 2013) Cellulases break down cellulose (recalcitrant compound) to fermentable sugars, which are easily absorbed by animals

Manhar et al., (2016) described a cellulase producer B subtilis AMS6 that also showed a probiotic effects, that is, the presence of B subtilis in the gut may

enhance the animal health and digestibility of feed with high content of cellulose

Biological treatments can be also applied in sewage sludge, in which alkaline protease, lipases, amylases and cellulases act on substrates at 50oC and also reduce the pathogenic microorganisms (Parmar et al., 2001)