Biotechnological Interventions in Beverage Production

BIOTECHNOLOGICAL INTERVENTIONS IN BEVERAGE PRODUCTION

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Santanu Malakar, Sanjib Kr Paul, K.R. Jolvis Pou Department of Agricultural Engineering, School of Technology, Assam University, Silchar, India

1.1 Introduction Beverages can be defined as “any fluid which is consumed by drinking.” It consists of a diverse group of food products, usually liquids, that include the most essential drink “water” in a wide range of commercially available fluids such as fruit beverage, synthetic drinks, alcoholic beverages, milk, dairy beverages, tea, coffee, chocolate drinks, etc. (James et al., 1996; Tamang et al., 2016; Sui et al., 2016). Biotechnology refers to the combination of techniques which involves the application of biological organisms or their components, systems, or processes to manufacture or modify the food products (Timmer, 2003). Presently, beverage industries are continuously focusing on modern biotechnological processes for the production and processing of beverages including overall improvement of quality, nutritional and functional attributes, value addition, aseptic or sterile packaging, and shelf life extension of the products (Shetty et  al., 2006; Balarabe et al., 2017). Industrial application of biotechnological principles contributed in boosting up of efficient fermentation processes, improved starter cultures, recombinant enzymes, genetic engineering, probiotics and prebiotics, safety assessments, biosensors, bio-preservation of beverages, application of nano-biotechnology, etc. Improved fermentation processes have substantially increased the industrial productivity and also enhanced the safety and quality attributes of the fermented beverages (Paul et al., 2014; Paul and Sahu, 2014b; Achi, 2005). Functional starter cultures in beverage industries play a potential role in fulfilling technical and metabolic requirements which accelerate the production of acid, flavor, and bacteriocin (antimicrobial substances) in the fermented beverages to suppress spoilage and Biotechnological Progress and Beverage Consumption. https://doi.org/10.1016/B978-0-12-816678-9.00001-1 © 2020 Elsevier Inc. All rights reserved.

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pathogenic microbes, leading to enhanced preservation and safety. Detection, identification, and elimination of pathogenic bacteria in beverages are extremely essential for ensuring the quality criteria and safety of beverages (Corbo et al., 2014). Molecular typing methodologies involving methods like PCR, RFLP, rRNA, PFGE, etc., as well as techniques of separating large stranded DNA molecules, can be used to characterize and monitor the presence of harmful microbes in beverages (Connor et al., 2005; Casalinuovo et al., 2006; Barthelmebs et al., 2011; Cocolin et al., 2011). With the application of biotechnological principles, biosensors are being functionally improved currently for a wide range of foods and beverages in their processing, analysis, safety, and storage study. Moving ahead of traditional biotechnology, beverage industries are trying to apply modern biotechnological principles including the genetic modification (GM) technique by recombinant DNA and cell fusion-based diagnostic tools leading to the novel concept of GM (genetically modified) beverages (Pan, 2002; Schuller and Casal, 2005). The new concept of nano-biotechnology further opened the scope of application of nanomaterials in quality control and packaging (active and smart) of beverages (Duran and Marcato, 2013; Paul and Sahu, 2014a; Bumbudsanpharoke and Ko, 2015). This chapter is basically aimed at discussing the prospects, potential involvement, and impacts of biotechnology in beverage industries. The critical discussion involved the present-day status of application of biotechnological principles in the production processes of fermented and nonfermented beverages, recombinant enzymes, probiotics and prebiotics, safety assessment, biosensors, bio-preservation of beverages, application of nano-biotechnology, and also the concept of genetic modification/genetically modified (GM) beverages, in order to address the changing consumer trends in the modern-day world.

1.1.1  Biotechnology in Food Biotechnology refers to the techniques which involve the application of biological organisms or their components, systems, or processes in manufacturing and service industries, to make or modify products, to improve plants or animals, or to develop microorganisms for special desired use (Cantarelli, 2012; Balarabe et al., 2017). As a fast developing field, it has already shown its impact on different aspects of day-to-day life such as public health, pharmaceuticals, agriculture, food industry, and bioenergetics. Biotechnology has indeed played a revolutionary role in production, preservation, nutritional enhancement, and value addition of foods. “Traditional” biotechnology refers to the conventional practices which have been applied since a long time for the production and consumption of fermented beverages, cheese, bread, and other food products. “Modern” biotechnology

Chapter 1  Biotechnological Interventions in Beverage Production   3

r­ efers to the techniques applied through the use of GM technology by recombinant DNA and cell fusion-based techniques combined with the recent developments of biotechnological processes in food processing (Tietyen et al., 2000; McCullum et al., 2003; Costa et al., 2017). Biotechnology plays an important role in categorizing the microbes and their enzymes in the food system. It includes the identification of enzymes for use as food ingredients, and the microbial fermentation to manufacture citric acid, glutamic acid, and nucleotides for use as flavor enhancers, bio-preservation, probiotics and enzyme modification of foods are some of the examples where traditional biotechnology has been used (Gašo-Sokač et  al., 2010; Haroon and Ghazanfar, 2016). The most recent advanced biotechnological techniques used by food processing industries are GM, also well known as genetic manipulation or recombinant DNA technology. Biosensors are currently being explored in wide dimensions by food processing industries by the application of biotechnology. Biosensors are being technologically advanced for rapid detection of foodborne microorganisms, toxins, undesirable metabolites, or other compounds (Neethirajan et al., 2015; Stobiecka et  al., 2007; Thakur and Ragavan, 2013; Murugaboopathi et al., 2013). The main purposes of the use of modern biotechnological tools are to increase the trend of production and quality of food products to increase safety and security and protect human health (Bachmann et al., 2015). The prospects and potential of applying biotechnology in food and beverage production and to enhance the quality improvement in food systems with the objective of addressing food security and responding to changing consumer trends are increasing day by day.

1.1.2  Beverage Production Technology Beverages are a diverse group of essential drinks (fermented and nonfermented) which are liquids made for consumption. Alcoholic drinks are broadly classified into five classes, starting from beers, wines, hard liquors, liqueurs, and others. Some major alcoholic beverages are listed in Table 1.1. Similarly, nonalcoholic drinks are classified into carbonated drinks, noncarbonated drinks, and hot beverages. These include juices, energy drinks, carbonated drinks, tea, coffee, and bottled water. Different beverage industries have experienced rapid growth in recent years with the application of various modern technologies. While age-old traditional beverages and drinks have sustained among consumers, at the same time, production of new value-added juices, multifeatured fermented beverages, alcoholic and nonalcoholic beverages containing microencapsulated nutrients, nutraceutical and herbal-based value-added drinks produced through modern biotechnological approaches has also created a business

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Table 1.1  Major Types of Alcoholic Beverages Beverage

Source

Technique

Brandy Whisky Rum Vodka Wines (Port, Sherry, Champagne, etc.) Beer Gin Liqueurs

Fruit juices Cereal grains Molasses/sugarcane Grains Grapes (also other fruits) Barley malt, wheat, rice Malt, other grains Fruits, herbs, or flowers

Fermentation

Alcohol Content (%) 40–50 40–55 40–55 40–45 10–22 4–8 36–50 15–30

Source: Shahidi, F., Alasalvar, C., 2016. Handbook of Functional Beverages and Human Health. CRC Press.

Beverage

Nonalcoholic

Noncarbonated

Fruit juices Fruit drinks

Alcoholic

Carbonated

Fruit nectars

Soda water flavoured drinks

Tea

Tonic water

Coffee

Grain

Fruit/sap

Nondistilled

Distilled

Nondistilled

Distilled

Beer

Wishky

Wine

Brandy

Fig. 1.1  Classification of beverages. Source: FAO, 2010. Current status and options for biotechnologies in food processing and in food safety in developing countries, in: FAO International Conference, Guadalajara, Mexico.

f­ocus in the beverage sector for their commercial growth. The beverages may be classified as shown in Fig. 1.1. Wide ranges of materials are used in the production of different types of beverages. The production of beverages can be categorized depending on the raw materials used and the method by which the materials are processed. Alcoholic beverages are produced by the fermentation and distillation process. Raw materials which contained sugars are converted into alcohol (ethanol) and carbon dioxide by

Chapter 1  Biotechnological Interventions in Beverage Production   5

microorganisms, which also impart characteristic flavors and aromas to the beverage. Food fermentations have not only facilitated the production of highly palatable products but also increased their nutritive value (Ogunshe et  al., 2006; Paul et  al., 2014; Paul and Sahu, 2014b; Haroon and Ghazanfar, 2016; Balarabe et al., 2017). In this direction, modern biotechnological techniques are extensively applied for the production and use of microbial starter culture to enhance the quality attributes (flavor, taste, color, and food additives) of value-added products across the spectrum of beverage industries (Okonko et  al., 2006; FAO, 2010). A nonalcoholic beverage refers to those drinks which do not contain any liquor percentage or, in other words, yeast or any fermenting microbe is not used to convert sugar into alcoholic compounds during the production process (Bothast and Schlicher, 2005; Holzapfel, 2002).

1.2  Biotechnology in Beverage Processing There are several potential sectors in food and beverage industries where conventional and modern biotechnological tools are being used during processing for the overall improvement of quality, safety, and health promoting potentials of the produced food and beverages. Impact and interventions of biotechnological techniques used in beverage industries also focus on the energy drinks for the all-round enhancement of the nutritional qualities. The recent trend on the biotechnological approach and its potential areas in beverage technology is shown in Fig. 1.2.

1.2.1 Fermentation Fermentation is a slow decomposition process of organic substances induced by microorganisms or enzymes that essentially convert carbohydrates to alcohols or organic acids including gaseous Starter cultures technology

Diagnostic tests for food safety Recombinant enzymes

Fermentation

Probiotics, prebiotics Biosensors

Biotechnological applications in beverage production

Others

Genetically modified foods Biopreservation of beverage

Fig. 1.2  Application of biotechnology used in food processing.

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by-products (Demir et al., 2006; Swain et al., 2014). Fermented foods such as bread, beer, wine, vinegar, sauerkraut, pickles, dahi, and lassi are conventionally processed by the application of traditional biotechnological approaches (Haroon and Ghazanfar, 2016). Genetic manipulation of microorganisms used as microbial cultures in food processing is also being carried out through molecular and biotechnological approaches for the improvement of quality characteristics of traditional and modern fermented beverages and also enhanced enzymatic activity and flavor development. For preparation of fermented beverage, culture is required of desirable microorganisms that are intentionally added to the base material to initiate and accomplish the desired fermentation in raw material under controlled conditions. Lactic acid (LA) fermentation of foods such as milk, vegetables, and fruits is a common practice to preserve and improve the nutritional and sensory features in the food and beverage industries (Demir et al., 2006; Parvez et  al., 2006; Di Cagno et  al., 2013; Sathe and Mandal, 2016; Costa et al., 2017). Most of the lactic acid bacteria (LAB) were isolated from various traditionally fermented foods and observed as predominant microflora of fermented beverages (McCullum et  al., 2003; Anandharaj et al., 2014). In beverage processing, carbohydrates are converted to alcohol, organic acids, and/or carbon dioxide by the microorganisms such as bacteria, yeast, etc. (Tamang et  al., 2012). Biotechnology plays a vital role in the processing of fermented beverages that undergo microbial or enzymatic activities to produce significant modifications by biochemical changes in the substrate during beverage production. The main fermentation types applied in beverage production are: lactic acid fermentation, alcoholic fermentation, solid state fermentation, submerged fermentation, etc. (Soni and Sandhu, 1990). Fermented beverages can be classified in many different ways, based on the kind of microorganisms involved, fermentation process, raw materials or substrate, and also on the function of the food. Major fermented beverages produced and consumed around the world, and some fermented food and beverages produced in India, are presented in Tables 1.2 and 1.3, respectively.

1.2.1.1  Lactic acid fermentation Lactic acid is now considered as one of the most useful substances for the beverage industry as a preservative, and in the chemical industry as a raw material for the production of lactate ester, propylene glycol, 2,3-pentanedione, propionic acid, acrylic acid, acetaldehyde, and dilactide (Wee et al., 2006). Lactic acid fermentation (e.g., fermented milks and cereals) is mainly of anaerobic respiration carried out by bacteria (Lactobacillus and others) to convert the 3‑carbon pyruvate to the 3‑carbon lactic acid (C3H6O3) and regenerate NAD+ in the process,

Chapter 1  Biotechnological Interventions in Beverage Production   7

Table 1.2  Major Fermented Beverages From Different Parts of the World Source

Name of the Fermented Beverage

Barley Rye Corn Wheat Rice Juice of fruits, other than apples or pears Juice of apples Juice of pears Juice of sugar cane, or molasses Juice of agave Juice of plums Pomace Honey Potato and/or grain Milk

Beer, ale Rye beer Corn beer Wheat beer Sake sonti Wine (most commonly thought of from grapes) Hard cider Perry, or pear cider Basi, betsa-betsa (regional) Pulque Plum wine Pomace wine Mead Potato beer Kumis

Name of the Distilled Beverage Scotch whisky Rye whisky Bourbon whiskey Wheat whisky, korn (Germany) Shochu (Japan), soja (Korea) Brandy, cognac (France), branntwein (Germany), pisco (Peru/Chile) Apple jack, apple brand, calvados Pear brandy Rum, cachaça, aguardiente, guaro Tequila, mezcal Slivovitz, tzuica, palinca Grappa (Italy), trester (Germany), marc (France) Distilled mead (mead brandy or honey brandy) Vodka(potato mostly used in Ukraine, otherwise grain) Araka

Source: Marshall, E., Mejia, D., 2011. Traditional fermented food and beverages for improved livelihoods. FAO Diversification Booklet 21. FAO, FIAT PANIS.

allowing glycolysis to continue to make ATP in low-oxygen conditions (Mckay and Baldwin, 1990). C3 H3 O3 ( pyruvate ) + NADH → C3 H6 O3 ( lactic acid ) + NAD+ Biotechnological processes for the production of lactic acid usually include lactic acid fermentation and biochemical changes. In the lactic acid fermentation process, sugar molecules are converted into lactic acid with the help of organisms such as Leuconostoc, Streptococcus, and Lactobacillus bacteria (Ghaffar et al., 2014). Recently, strains used in the commercial production of lactic acid have become almost proprietary, and it is believed that most of the LAB used belongs to the genus Lactobacillus (Hofvendahl and Hahn-Hägerdal, 2000). LAB can increase levels of vitamins in food, especially vitamin B (Di Cagno et al., 2013).

Table 1.3  Traditional Fermented Food and Beverages of India, Involved Microorganisms and Specific Region of Production Food Source

Fermented Food/Beverage

Dairy based

Yogurt Dahi Lassi Buttermilk Mistidahi Chhu/sheden Kefir Chhurpi

Cereallegume based

Haria

Microorganism

Area/Region

Lactobacillus bulgaricus and Streptococcus thermophilus Lactococcus lactis L. acidophilus and S. thermophilus Lactobacillus lactis Lactobacillus plantarum, Bacillus subtilis L. lactis ssp. lactis, L. lactis ssp. diacetylactis, Lueconostoc dextranicum ssp. Citrovorum Lb. caucasicus, Strep. thermophilus, Lb. bulgaricus, Lb. plantarum, Lb. casei, Lb. brevis Lb. farciminis, Lb. casei, Lb. biofermentans, W. confuses

Not specific

Simaltarulkojaanr Gahoonkojaanr Faaparkojaanr Opo Pona Pachwai

Saccharomyces cerevisiae, Lactobacillus fermentum, Bifidobacterium sp. S. cerevisiae, Hanseniaspora sp., Kloeckera sp., Pischia sp., and Candida sp. Yeast, LAB S. cerevisiae Not properly studied LAB Bacillus pumilus, Bacillus firmus B. laterosporus, B. pumilus, B. firmus; D. hansenii, Sacch. cerevisiae Yeasts, LAB Not properly studied Not properly studied Saccharomycopsis fibuligera, Ped. pentosaceus, Lb. bifermentans Yeast, molds, Pediococcus Not properly studied Not properly studied Yeast Molds, yeast, LAB Lactobacillus coriniformis, L. lactis, L. fructosus

Sujen Buza Atingba

Not properly studied S. cerevisiae Mucor sp., Rhizopus sp., S. cerevisiae

Apong Ennog Jou Aara Aliha Gahoonkojaanr Handia Jhara Dekuijao Zutho/zhuchu Mingari/lohpani

Throughout India Throughout India Not specific West Bengal, Odisha Darjeeling, Sikkim Not specific Sikkim, Darjeeling, Arunachal Pradesh West Bengal, Odisha, and Jharkhand Arunachal Pradesh Arunachal Pradesh Nagaland, Assam Arunachal Pradesh, Sikkim – Sikkim Bihar, Jharkhand, Odisha, Madhya Pradesh West Bengal Nagaland Nagaland Arunachal Pradesh Darjeeling, Sikkim Darjeeling, Sikkim Darjeeling, Sikkim Arunachal Pradesh Arunachal Pradesh West Bengal and Northern India West Bengal and Assam Ladakh Manipur

Table 1.3  Traditional Fermented Food and Beverages of India, Involved Microorganisms and Specific Region of Production—cont’d Food Source

Fermented Food/Beverage

Cereal and pulse based

Judima Zutho Bhaatijaanr

Ahom Zu Bhang-chyang Kiad Thiat Daru Dekuijao Juhning Juharo Duizou Judima Makaikojaanr

Fruits and vegetable based

Nduijao Nchiangne Angoori/kinnauri Chulli Ghanti Kanji Kodokojaanr Ark/ara Rak Soor Toddy or tari

Microorganism

Area/Region

Pediococcus pentosaceus, Bacillus circulans, Bacillus catarosporous S. cerevisiae, Rhizopus sp. Mu. circinelloides, Rhiz. chinensis, Sm. fibuligera, Pic. anomala, Sacch. cerevisiae, Cand. glabrata, Ped. pentosaceus, Lb. bifermentans lactic acid bacteria, Pediococcus and L. bifermentous Not properly studied Yeasts, molds LAB, yeasts Molds, yeasts, LAB Yeasts, LAB Not properly studied Not properly studied Lb. bifermentans, Pic. anomala, Sacch. cerevisiae, Cand. glabrata, Ped. pentosaceus D. hansenii, Sacch. cerevisiae Ped. pentosaceous, B. circulans, B. laterosporus, B. pumilus, B. firmus Sm. fibuligera, P. anomala, Sacch. cerevisiae, Cand. glabrata, Ped. pentosaceus, Lb. bifermentans LAB, yeasts Not properly studied Lactobacillus plantarum, Bacillus subtilis Bacillus cereus, B. subtilis, Staphylococcus aureus, Enterococcus faecium, Candida sp. LAB Debaryomyces hansenii, S. cerevisiae Pediococcusas Lactobacillus fermentum, L. plantarum, L. casei Not properly studied Lactobacillus, Leuconostoc Lactobacillus fermentum, L. brevis and L. plantarum Pseudoplantarum and Pediococcus pentosaceus

Assam Nagaland Darjeeling, Sikkim, and Northeast states Assam Assam Arunachal Pradesh Meghalaya Meghalaya Himachal Pradesh Nagaland Assam Assam Nagaland Assam Darjeeling, Sikkim Nagaland Nagaland Himachal Pradesh Himachal Pradesh Himachal Pradesh South India Sikkim Himachal Pradesh Himachal Pradesh Himalayan region – Continued

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Table 1.3  Traditional Fermented Food and Beverages of India, Involved Microorganisms and Specific Region of Production—cont’d Food Source

Fermented Food/Beverage

Bamboo based

Mesu Soibum

Bastanga Tuaithur Lung-siej Ekung

Microorganism

Area/Region

Lactobacillus plantarum, L. brevis and L. pentosaceus Enterococcus durans, Streptococcus lactis, B. subtilis, B. licheniformis, B. coagulans, Candida spp., Saccharomyces spp., Torulopsis spp. LAB Lb. plantarum, Lb. brevis, Ped. pentosaceou, Lc. lactis, Bacillus circulans, B. firmus, B. sphaericus, B. subtilis Lb. brevis, Lb. plantarum, Lb. curvatus, Ped. pentosaceus, Leuc. mesenteroides Lb. plantarum, Lb. brevis, Lb. casei, Tor. halophilus

Darjeeling and Sikkim Manipur

Nagaland Assam Meghalaya Arunachal Pradesh

Source: Tamang, J.P., Thapa, N., Tamang, B., Rai, A., Chettri, R., 2015. Microorganisms in fermented foods and beverages, in: Health Benefits of Fermented Foods and Beverages. CRC Press, Taylor & Francis Group, New York, pp. 1–110; Tamang, J.P., Watanabe, K., Holzapfel, W.H., 2016. Diversity of microorganisms in global fermented foods and beverages. Front. Microbiol. 8, 7–16, Tamang, J.P., 2016. Ethnic Fermented Foods and Alcoholic Beverages of Asia. Springer; Hugenholtz, J., 2013. Traditional biotechnology for new foods and beverages. Curr. Opin. Biotechnol. 24, 155–159, Marshall, E., Mejia, D., 2011. Traditional fermented food and beverages for improved livelihoods. FAO Diversification Booklet 21. FAO, FIAT PANIS; Wilson, T., Dahl, R., Temple, N., 2016. Beverage trends affect future nutritional health impact, in: Beverage Impacts on Health and Nutrition. Springer; Kumari, A., Pandey, A., Ann, A., Raj, A., Gupta, A., Chauhan, A., Sharma, A., Das, A.J., Kumar, A., Attri, B., Neopany, B., 2016. Indigenous Alcoholic Beverages of South Asia. CRC Press, New York, 503–596.

1.2.1.2  Alcoholic Fermentation Alcoholic fermentation is a biotechnological process accomplished by yeast, some kinds of bacteria, or a few other microorganisms to convert sugars into ethyl alcohol and carbon dioxide. In this fermentation process, yeast is mostly used as a bio-culture and aqueous solution of monosaccharide (raw materials) as the culture media for the production of beverages. In the alcoholic fermentation process, yeast generally carries out the aerobic fermentation process, but it may also ferment the raw materials under anaerobic conditions. In the absence of oxygen, alcoholic fermentation occurs in the cytosol of yeast (Sablayrolles, 2009; Stanbury et  al., 2013). Alcoholic fermentation begins with the breakdown of sugars by yeasts to form pyruvate molecules, which is also known as glycolysis. Glycolysis of a glucose molecule produces two molecules of pyruvic acid. The two molecules of pyruvic acid are then reduced to two molecules of ethanol and 2CO2 (Huang et al., 2015). Under anaerobic conditions, the pyruvate can be transformed to ethanol, where it first converts into a midway molecule called

Chapter 1  Biotechnological Interventions in Beverage Production   11

a­ cetaldehyde, which further releases carbon dioxide, and acetaldehyde is converted into ethanol. In alcoholic fermentation, the electron acceptor called NAD+ is reduced to form NADH. The exchange of electrons that occurs in the process helps to build ATP.

1.2.1.3  Solid-State Fermentation Solid-state fermentation (SSF) is defined as a fermentation process in which microorganisms grow on solid materials in the absence of free liquid. SSF is mostly used for food processing and production of enzymes using filamentous microorganisms like fungi. In the SSF technique, microorganisms are grown on and inside the humidified solid substrate (Prabhakar et al., 2005). It has been defined as the fermentation process which involves a solid matrix and is carried out in the absence or near absence of free water; however, the substrate must possess enough moisture to support the growth and metabolism of the microorganism (Couto and Sanromán, 2006; Singhania et al., 2009). Several applications of solidstate fermentation through biotechnological intervention are studied for the production of beverages. Agro-industrial residues have been employed for the production of microorganisms to produce enzymes for the application in food and beverage processing. The production of enzymes through SSF, such as α-amylases and fructosyl transferase, was extensively employed in the processed food industry such as baking, brewing, preparation of digestive aids, production of vinegar, fruit juices, starch syrups, etc. (Couto and Sanromán, 2006; Gašo-Sokač et al., 2010). Maximum enzyme production was obtained on wheat bran supplemented as a substrate for the production of beverage in industries (Brijwani et al., 2010; Teixeira and Vicente, 2013). Lipases are widely used nowadays on an industrial scale through incorporation in beverages for the improvement of flavor, palatability, and further value addition. Pectinases are widely used in the beverage industry to clarify fruit juices and wine, to improve oil extraction, to remove the peel from citrus fruit, and to increase the firmness of several fruits. Other enzymes such as phytase, inulinase, cellulase, protease, α-galactosidase, tannase, chitinase, and l-glutaminase are employed through SSF for different desired applications in food and beverage processing (Cavalcanti et al., 2005; Anisha et al., 2008; Singhania et al., 2009; Chandrasekaran et al., 2013).

1.2.1.4  Submerged Fermentation Submerged fermentation is the technique where microorganisms are grown in the liquid medium which is vigorously aerated and mostly agitated in contrast to the solid media. It uses free flowing substrates like molasses and broths to achieve a quite rapid fermentation

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process. This fermentation process is suitable for microorganisms that require high moisture content to grow during the fermentation process (Saqib et al., 2010). Substrates selection is extremely important as different organism reacts in a different way to each substrate and so does affects productivity. The most common substrates used in submerged fermentation are molasses, soluble sugars, fruit and vegetable juices, broth, and sewage/waste water (Paul et  al., 2014; Paul and Sahu, 2014b). During the fermentation process various medium ingredients and different submerged culture conditions, such as temperature, pH, oxygen supply, incubation period, and inoculation rate, have an effect on the production of fermented beverages (Tang et al., 2011; Shah et al., 2014; Paul et al., 2014; Paul and Sahu, 2014b). In this fermentation process, organisms growing in a vigorously aerated and agitated liquid may be either batch or continuous type fermentation. Batch Fermentation In this fermentation process, the organism is grown in a known amount of culture medium and biochemical synthesis is allowed for a defined period of time. During the fermentation process, a modified and enhanced mode of conventional closed fermenter is cleaned, re-sterilized, and another batch of fermentation, called fed-batch fermentation, is started (Tang et al., 2009). Due to the metabolism and synthesis of cells, the composition of the culture medium and cell concentration will change constantly from time to time (Negi and Banerjee, 2009; Hashemi et al., 2011). Neither the inoculum nor the nutrient solution is added but oxygen in the form of air and an antifoaming agent may be added to control foam and pH during the batch fermentation process (Vidyalakshmi et al., 2009; Speight and Harmon, 2010; Paul et al., 2014; Paul and Sahu, 2014b). Continuous Fermentation Continuous fermentations are those in which fresh nutrient medium is added continuously or intermittently to the fermentation vessel, accompanied by a corresponding continuous or intermittent withdrawal of a portion of the medium for recovery of cells or fermentation products (Bayramoğlu et al., 2004; Saqib et al., 2010).

1.2.2 Starter Cultures Technology Starter cultures are preparations of live microorganisms which have an effect on the processing of beverages through biotechnological approaches containing large numbers of specific or variable microorganisms, which may be added to facilitate control over the initial phase of a fermentation process (Holzapfel, 2002). Starter cultures

Chapter 1  Biotechnological Interventions in Beverage Production   13

comprising various LAB, such as lactobacilli, lactococci, propionic, and pediococci bacteria, are mainly being used for the production of cultured dairy and food products including dairy-based beverages such as yogurt, dahi, lassi, buttermilk, etc. Most of the traditional fermented foods are prepared by processes of solid substrate fermentation, in which the substrate is allowed to ferment either naturally or by adding starter cultures (Marsh et al., 2014). Microorganisms normally break down carbohydrates, proteins, and lipids present in the raw materials by releasing enzymes into the medium (Sathe and Mandal, 2016). Moreover, the breakdown products such as peptides and amino acids can be further converted into smaller volatile molecules that are odoriferous and hence improve the flavor characteristics of the fermented foods (Champagne, 2006). Microbes present in the raw materials and the processing condition function as inoculants in spontaneous fermentations, while the high concentrations of live microorganisms are used as starter cultures to increase the efficiency of the fermentation process (FAO, 2010; Sablayrolles, 2009; Costa et al., 2017). Appropriate starter cultures are usually produced using a back slopping method that makes use of the previous batch’s sample as inoculants for the production of fermented products. Defined starter cultures consist of single or mixed strains of pure microorganisms for use in the production of dairy and other food products such as kefir, yogurt, dahi, cheeses, alcoholic beverages, etc. (Holzapfel, 2002; Tamang et  al., 2012; Hugenholtz, 2013). In spontaneous fermentation, the multifunctional starter cultures fulfill the desired requirements which accelerate the production of acids, flavor, and bacteriocin during fermentation of the beverage to suppress spoilage, pathogenic bacteria, and also contribute in developing additional health promoting functions (Marsh et al., 2014; Corbo et al., 2014). Hence, multifunctional starter cultures in food processing play a potential role in food preservation and safety. LAB produces organic acids (lactic acid, acetic acid, formic acid, phenyllactic acid, caproic acid, carbon dioxide, hydrogen peroxide, diacetyl, ethanol, bacteriocins, and acetic acid) which contribute to the aroma and prevent mold spoilage and microbial contamination (De Vuyst, 2000; Gálvez et al., 2007). Moreover, they also improve the aroma and flavor characteristics of the products and the product quality. Recent developments in the field of metabolic manipulation techniques, genomics, and bioinformatics are expected to contribute in the future improvement of starter cultures to gain commercial profits by food and beverage processing industries. Exploration of wild strains present in traditional beverages and fermented dairy products, etc. will permit a thorough screening of promising strains for the development of GM starter cultures through biotechnological approaches.

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1.2.2.1  Appropriate Starter Cultures Appropriate starter cultures are used in back slopping process by taking out the sample from a previously prepared appropriate product as inoculum in the fermentation (Holzapfel, 2002). The use of appropriate starter cultures is generally preferred in alcoholic beverage production and suggested as the appropriate approach to alleviate the problems of variations in organoleptic quality and microbiological stability of traditional products (N'Guessan et al., 2016)

1.2.2.2  Defined Starter Cultures Defined starter cultures are bacterial or fungal strains, pure or mixed, which are used to initiate a fermentation process. This type of starter culture has been developed to use as an inoculum for commercial fermentation processes in beverage industries. This pure or mixed culture may be incorporated to produce adjunct culture preparations to achieve its specific additional abilities, such as inhibition of pathogenic or spoilage organisms by the production of hydrogen peroxides, organic acids, bacteriocins, diacetyl, etc. (Hutkins, 2006). These cultures are also generally imported by most of the developing countries for their use in commercial production of dairy products and for alcoholic beverages (FAO, 2010).

1.2.2.3  Genetically Modified (GM) Starter Cultures GM starter cultures are produced through a biotechnological process by genetic engineering with the use of molecular mechanisms. The first GM wine yeast, ML01, was released in 2005 by Springer Oenologie (a division of Lesaffre Yeast Corporation). This was modified by inserting two foreign genes, one from the pombe yeast and another from the bacteria Oenococcus oeni, for the production of an alcoholic beverage. There are two commercially available GM yeasts widely used in beverage industries. One such yeast has been genetically manipulated to better degrade urea during the wine-making process (Saccharomyces cerevisiae strain ECMo01). The second GM yeast (ML01) has been designed to allow malolactic fermentation with more effectiveness in beverage industries. GM wine bacteria also play a vital role in secondary beverage fermentation, known as malolactic fermentation. Oenococcus oeni is, an LAB, is used commercially in the wine industry for development of tools for the GM of lactic acid bacterial strains (Bhatia, 2017).

1.2.3 Recombinant Enzymes Production Enzymes are important biocatalysts that demonstrate a broad range of industrial applications in food and beverage industries to facilitate and speed up biochemical reactions in and around the living organisms and/or raw materials (James et al., 1996). Enzymes can

Chapter 1  Biotechnological Interventions in Beverage Production   15

be obtained from plant [β-amylase, papain, bromelain, urease, ficin, polyphenol oxidase (tyrosinase), lipoxygenase, etc.] and microbial (α-amylase, penicillin acylase, protease, invertase, lactase, dextranase, pectinase, pullulanase, etc.) sources (Chi et  al., 2011). Several microbial strains have been selected and genetically modified to increase the efficiency in producing enzymes to accelerate the fermentation process. The industrial manufacture of enzymes from different microbes is carried out in large tanks that involves culturing and straining the microorganisms where enzymes are secreted into the fermentation/culture media as primary or secondary metabolites. These enzymes as a processing aid show beneficial effect on increasing the efficiency and productivity of fermentation processes (Tietyen et al., 2000; Mackey, 2002; Senker and Mangematin, 2006). The techniques used for the production of enzymes through biotechnological approaches are shown in Fig. 1.3. The majority of the enzymes are used, for example, in cheese making (lipases, proteases), wine and juice production (pectinases), and lactose reduction (lactase). Pectinases and cellulases are used to break down cell walls of fruit and vegetables, resulting in improved extraction and an increase in yield. They can also be used to decrease the viscosity of purees or nectars, and to provide ‘cloud stability’ and texture in juices (Renge and Khedkar, 2012; Fernandes, 2010; Tapre and Jain, 2014). Enzymes are an important biotechnological tool whose activity and rate of reaction can be monitored and controlled in the substrate or raw materials to produce high-quality products. Enzymes require catalyzing conditions for food production, for example, optimum pH, time, temperature, and oxygen concentration (Tapre and Jain, 2014). Enzymes were isolated traditionally, mainly from plant and animal sources, for the production of food and beverage, but nowadays different modern biotechnology can produce a variety of enzymes through DNA modification, genetic engineering, and protein engineering

Filtration Removal of nucleic acids

Cell disruption Inoculum

Inoculum development

Enzyme powder

Inoculation tank

Freeze drying

Fermenter

Filtration and purification

Cold storage

Fig. 1.3  Production of enzymes through a biotechnological approach.

Salt treatment

16  Chapter 1  Biotechnological Interventions in Beverage Production

(Patel et  al., 2016; Costa et  al., 2017). Modern techniques are being used to produce novel enzymes with modified structures that produce unique desired properties. Moreover, modified enzymes with improved efficiency have also mainly been made possible by the GM of the microorganisms. These techniques involve producing large numbers of modified enzyme by random GM and consequently screening them to categorize the improved variants during industrial enzyme production (Baniasad and Amoozgar, 2015). The production of novel and modified enzymes by use of recombinant DNA technique through modification of known enzymes has been made possible by the application of modern methods of protein engineering (Adrio and Demain, 2014; Bruins et al., 2014). As a result, several important food-processing enzymes such as amylases and lipases were developed, introducing the change in their properties through recombinant DNA technology. Another important achievement is the improvement of microbial strains recently developed for enzyme production to increase enzyme yield by removing native genes encoding extracellular proteases (Whitehurst and Van Oort, 2009). To date, numbers of enzymes used in food and beverage industries are developed using recombinant microorganisms. At present, these modified enzymes can be produced in large quantities for their desired subsequent applications, including the use in beverage industries through modern biotechnological approaches (Patel et al., 2016; Tamang et al., 2016). Some of the enzymes used for the industrial production of alcoholic and nonalcoholic beverages are listed in Tables 1.4 and 1.5, respectively.

Table 1.4  Enzymes and Their Uses in the Production of Alcoholic Beverages Enzyme

Beverage

Application

Lichenase Acetolactate decarboxylase Pectinase β-Glucanase Pectinases, cellulases, hemicellulases Amylases Glucose oxidase Terpene glycosidase

Beer Beer Wine Wine Distilled spirit Beer/spirits Beer/wine Wine

Hydrolysis of lichenan Decomposition of acetolactate Improvement of aromatic profile Influence on the autolysis of yeast, improved filtering ability Hydrolysis of bound monoterpenes Starch breakdown Oxygen scavenger in bottled beverages Improve aroma

Source: Whitehurst, R.J., Van Oort, M., 2009. Enzymes in Food Technology. John Wiley & Sons; Kumar, S., 2015. Role of enzymes in fruit juice processing and its quality enhancement. Health 6, 114–124.

Chapter 1  Biotechnological Interventions in Beverage Production   17

Table 1.5  Enzymes and Their Uses in the Production of Nonalcoholic Beverages Enzyme

Beverage

Functions

Pectinases, Cellulases, Hemicellulases Pectin lyase

Fruit juice

They hydrolyze pectin and soluble components of the cell wall, reduce viscosity, and maintain fruit juice texture It cleaves pectin into oligosaccharides without the action of esterase It removes methyl esters and releases methanol, enabling polygalacturonase to digest the pectin It randomly digests pectin and hydrolyzes polygalacturonan in the cell walls They hydrolyze cell wall polysaccharides They hydrolyze xylan and arabinoxylan during fruit juice clarification Liquefication of fruit and accelerate color removal during fruit processing Starch breakdown in early season fruit Debittering and enhancement of juice aromas by the enzymatic hydrolysis Modify the polyphenolic composition of the orange juice Aroma development Color and flavor development Aroma development

Cider

Pectin methyl esterase Polygalacturonase

Apple juice

Hemicellulases Xylanases

Fruit/grape juice

Cellulases

Apple juice

Amyloglucosidases α-l-Rhamnosidase

Apple juice Citrus fruit juices

Tannases Esterase Peroxidase Lipoxygenase

Orange juice Apple juice Tea Apple juice, tea

Source: Whitehurst, R.J., Van Oort, M., 2009. Enzymes in Food Technology. John Wiley & Sons; Kumar, S., 2015. Role of enzymes in fruit juice processing and its quality enhancement. Health 6, 114–124; Chandrasekaran, M., Basheer, S.M., Chellappan, S., Krishna, J.G., Beena, P. 2013. Enzymes in food and beverage production: an overview. Enzymes Food Beverage Process, 117.

The main applications of the abovementioned enzyme groups in juice processing industries involve the extraction, clarification, and concentration stages. There are mainly two groups of enzymes which are used in the fruit juice industry, that is, amylases and pectinases. Amylases are biotechnologically very important with applications in the beverage industry such as the production of glucose syrups, apple juice, orange juice, and maltose syrup and reduction of viscosity and turbidity to produce clarified fruit juice for longer shelf life (Achi, 2005; Gašo-Sokač et al., 2010). α-Amylases are extracellular enzymes which catalyze the hydrolytic breakdown of α-1,4-glycosidic linkages in starch to increase the clarification of fruit juice. Pectinases were the first broadly used enzymes and their biotechnological application was first studied for the preparation of wines and

18  Chapter 1  Biotechnological Interventions in Beverage Production

fruit juices in the beverage industries. This group of enzymes is one of the most important groups since they help in obtaining well clarified and stable juices with higher yields and maintaining the viscosity and structure of the juice (Kumar, 2015; Costa et al., 2017).

1.2.4  Bio-Preservation of Food and Beverages Bio-preservation is the process used for food preservation by using microbial metabolites and microbiota to increase the shelf life of food and beverages (Giraffa et al., 2010; Bigliardi and Galati, 2013). In order to harmonize consumer demands with the necessary safety standards, traditional means of controlling microbial spoilage and safety hazards in foods are being replaced by alternative food preservation technologies such as bio-preservation to extend the shelf life and assure safety and quality (Lacroix, 2010).

1.2.4.1  LAB as Bio-Preservatives The most common and traditionally used bio-preservation of food products through fermentation is LAB like lactobacilli, lactococci, streptococci, leuconostocs, pediococci, etc. These bacteria are being widely used as starter cultures for the production of dairy, fruits, and vegetable products including beverages (Papagianni, 2012). The preservative effect of LAB is due to the production of active metabolites, such as organic acids (lactic, acetic, formic, propionic, and butyric acids), that intensify their action by reducing the pH of the media, and other substances, such as ethanol, fatty acids, acetone, hydrogen peroxide, diacetyl, and antifungal compounds (propionate, phenyllactate, hydroxyphenyl-lactate, cyclic dipeptides, and 3-hydroxy fatty acids) (Crowley et al., 2013; Awojobi et al., 2016; Cheong et al., 2014; Cousin et  al., 2017). Inhibitory effects of lactic and acetic acid from LAB against different fungal strains of Aspergillus flavus in the processing of juice were also reported (Awojobi et al., 2016). These bacteria enhance the nutritional values as well as inhibit the spoilage causing and pathogenic microbes due to the production of hydrogen peroxide, organic acids, and bacteriocins. LAB can be used as protective cultures to restrict the growth of undesired organisms, such as certain spoilage and pathogenic bacteria, with the subsequent benefits in terms of food safety (Crowley et al., 2013; Fatima and Fernanda, 2015). LAB are essential as natural bio-preservatives which produce effective metabolites that possess antibacterial and antifungal properties against harmful or undesirable microbes. LAB is used in the fermentation process to produce these metabolite components which act as a promising preservative for food and beverages (Ameen and Caruso, 2017; Rani et al., 2016; Tomadoni et al., 2016). The antagonistic and inhibitory properties of LAB are due to the competition for

Chapter 1  Biotechnological Interventions in Beverage Production   19

nutrients and the production of one or more active metabolites such as organic acids (lactic and acetic acid), hydrogen peroxide, and antimicrobial peptides (Rani et al., 2016; Awojobi et al., 2016).

1.2.4.2  Bacteriocins as Bio-Preservatives Bacteriocins play a very important role as novel food preservatives and have received greater attention in beverage industries for enhancing safety and extending the shelf life of the product (Rani et al., 2016). Bacteriocins like nisin, reuterin, reutericyclin, pediocin, lacticin, and enterocin are used as food and beverage preservatives (Cousin et al., 2017; Rocha et  al., 2017). A large number of bacteriocins produced by LAB have been identified, although their potential application as bio-preservatives is yet to be studied thoroughly (Bali et  al., 2016). Antimicrobial properties of various bacteriocins have been extensively studied by different food researchers (Balciunas et  al., 2013; Marie et al., 2012). Lactococcus lactis subsp. lactis BZ or its bacteriocin, which has a wide inhibitory spectrum, has the potential for use as a bio-preservative in food products. In recent years several bacteriocins are produced and some among them have been patented for their applications in foods. To date, the only commercially produced bacteriocins are those belonging to the group of nisins produced by Lactoccocus lactis, and pediocin PA-1 produced by Pediococcus acidilactici (Balciunas et al., 2013; Fatima and Fernanda, 2015). Though the mode of action of bacteriocin is not yet clearly understood, it is assumed that they are involved in the inhibition of cell wall synthesis or create depolarization of the cell membrane (Bali et  al., 2016; Kantachote et al., 2017).

1.2.5  Probiotic and Prebiotic Functional Beverages Probiotics are the preparations containing single or mixed cultures of live microorganisms, which, when administered to humans or animals in appropriate amounts, have a beneficial effect on their health (Marsh et al., 2014). There are a wide variety of traditional nondairy fermented beverages produced around the world, and many of them are nonalcoholic beverages manufactured through encapsulation of probiotic microorganisms and prebiotics (Parvez et al., 2006; Saad et al., 2013). Probiotics and prebiotics are combinedly called synbiotics. Major microorganisms used as probiotic foods and beverages are—Lactobacillus casei, Lb. acidophilus, Lb. brevis, Lb. lactis, Lb. plantarum, Lb. fermentum, Lb. delbrueckii var. bulgaricus—Bifidobacterium breve, Bf animalis, Bf. lactis, Bf. bifidum, Bf. longum, Bf. Adolescentis—other organisms (Lactococcus lactis, Enterococcus faecium, Enterococcus faecalis, Pediococcus acidolactici, Streptococcus sali var. thermophilus,

20  Chapter 1  Biotechnological Interventions in Beverage Production

Saccharomyces boulardi) (Corbo et al., 2014; Sekhon and Jairath, 2010; Shori, 2016; Yerlikaya, 2014; Pereira et al., 2011). Different sources of potential probiotics are currently in use. Lactobacillus, Enterococcus, and Bifidobacterium are some species extensively used as probiotics in food formulations and encapsulated supplements through biotechnical applications (Dündar, 2016). The advances in applications of biotechnological techniques in food processing are also leading towards the rapid developments of the strains of probiotic microorganisms by recombinant DNA technology, genetic engineering, and sequence modification of common probiotic strains. The cutting edge researches in this direction are contributing in the development of novel and modified probiotics with enriched nutritional and functional efficacy for safety of human health and new product development (Prado et al., 2008; Champagne, 2009; Panesar et al., 2013; Singla and Chakkaravarthi, 2017). In addition to these, by using the modern tools it is also being tried to develop probiotic bacteria which can survive during the harsh processing conditions used during the production process (Cantarelli, 2012; Panesar et al., 2013). With microencapsulation technologies, probiotics can also become an important ingredient in the functional foods, expanding the probiotic application outside the pharmaceutical and supplement industries. Mainly fermented dairy beverages are recognized as an excellent carrier of probiotic microbes those already evidenced their health benefits among the consumers. Probiotic LAB has the ability to resist acidic conditions and bile salts, and additionally it produces bacteriocins that are active against food pathogens and spoilage microorganisms, which may have potential applications for improving the safety of food products (Parvez et al., 2006; Sekhon and Jairath, 2010). The probiotic LAB can be present in the spontaneous fermentation of different foods and beverages, and this group is generally recognized as safe, having GRAS status. However, the therapeutic value of LAB was one of the major reasons behind the popularity in dairy and beverage industries for exploring probiotics as a possible bio-therapeutic against intestinal disorders and lactose intolerance, altered vitamin content of milk, antagonism against various pathogenic organisms, and anti-mutagenic and anticarcinogenic activities (Bali et al., 2016; Shori, 2016; El-Baily, 2016; Shahidi and Alasalvar, 2016; Fleet and Rahman, 2017).

1.2.6 Genetically Modified Food and Beverage GM can be defined as a process involving the alteration of the genetic orientation or makeup of the living organism for a certain intended purpose. The purpose of GM is to isolate specific genes of known functions from one organism and transfer its copies to a new

Chapter 1  Biotechnological Interventions in Beverage Production   21

host organism for introducing target characteristics (Marushkina, 2009; Gjermansen et al., 2001). GM food refers to products developed through numerous processes and applications of biotechnology where the organisms are produced by specific changes introduced into their DNA by genetic engineering techniques. Advancement in this field has already started to bring various GM foods as well as microbes and raw materials for food and beverage processing. GM strains of yeast have been used in fermented beverage industries to benefit the beverage production by enhancing fermentation conditions, raising yeast ethanol tolerance, nitrogen assimilation, and sugar utilization, and also altering the sensory aspects (Schuller and Casal, 2005). Some of the GM yeast strains developed to enhance wine fermentation can also concurrently carry out malolactic fermentation along with the alcoholic fermentation due to the presence of the malate permease gene introduced from malolactic wild strains. This is beneficial because it permits microbial stability in beverages while decreasing the acidity and also significantly reduces the time required for the overall fermentation process. The technology also helps to convert malate to lactic acid which does not produce the same expected flavor attributes with LAB (Akada, 2002; Schuller and Casal, 2005; Varela et al., 2012). GM yeast strain is also used to decrease the production of hydrogen sulfides (H2S) in wine fermentation because H2S has a negative impact on wine flavor and aroma, and other factors during alcoholic beverages production (Biyela et al., 2016; Varela et al., 2012). Modifications to microbial strains including yeast do not change basic characteristics of the native strain in the fermentation but slightly transform their metabolic processes (Chambers and Pretorius, 2010). The products of general genetic crossing and breeding are not recommended as a GMO, because genetic engineering tools need to be used to produce GM products. If the recombinant DNA technology is used to transfer genes or to alter specific genes in a new strain and the product is identical with the wild type, it would still be recognized as a GMO in several countries due to the use of recombinant DNA tools. Production and development of GM food through genetic engineering are governed by strict rules and regulations throughout the globe. The strict regulations are due to the fact that the probable benefits expected from modern GM organisms, food, or beverages (improved nutritional value, functional foods) may lead to new issues (metabolic changes, major impact on nutritional status and genetic as well as subnuclear orientations) which may impose new challenges in terms of safety assessments in food including beverage industries (Marden, 2002). Concerns about the impact of applications of this modern biotechnology on ecological balance due to its growing

22  Chapter 1  Biotechnological Interventions in Beverage Production

exposure towards GM foods, microbes, and beverages are also increasing. Many countries control the use of GMO with almost the same objectives but sometimes with slight differences. In many countries, regulations of GMO apply to all such products produced by the use of recombinant DNA techniques, but in many countries the definition of GMO is restricted to transgenic products with exemption for self-cloned organisms (Pretorius and Høj, 2005). The applications of GM organisms in food and beverages are continuing to be a matter of intense controversy, and hence there are many challenges ahead for governments, especially in the areas of safety testing, regulation, international policy, and labeling in food and beverage industries. However, the potential of the modern biotechnologies is enormous for developing countries for the production of food and beverages to ensure food and nutritional security.

1.2.7 Diagnosis of Safety and Quality Assurance Biotechnology is helping in many ways to enhance food safety and quality assurance. It is providing many tools to detect microorganisms and the toxins they produce. Monoclonal antibody tests, biosensors as well as PCR and DNA probes are being developed to determine the presence of harmful bacteria such as Listeria, Clostridium, Escherichia coli 0157:H7, and many more (Mandal et  al., 2011). Biotechnologybased detection methods have been developed now to detect toxins like aflatoxin. Modern biotechnological methods need less time in identification of pathogenic microbes and other substances. It is also more specific, sensitive, and faster than normal conventional methods. PCR-based methods are also used for the detection of ingredients in food product and allergens in diverse foods and beverages. Nucleic acid-based identification and diagnostic systems can significantly improve the sensitivity, specificity, and speed of microbial assays. Molecular typing methodologies like PCR, restriction fragment length polymorphism (RFLP), analysis of ribosomal ribonucleic acids (rRNA), and pulsed field gel electrophoresis (PFGE), etc. can be used for typing microbial strains. These techniques are also used to detect and monitor the existence of spoilage microbes in food and beverages as well as their further characterization (Tauxe, 2002; Valderrama et al., 2016). PCR can detect a single copy of a target DNA sequence and thus can be used to detect a single pathogenic bacterium in food. So PCR is a very widely used technique in the detection of pathogens in food, forming the basis for detection systems utilizing nucleic acid. A real-time PCR-based assay is also developed for the rapid detection of bacterial species during the processing and preservation of food at an industrial level (Gammon et  al., 2007; Elsisi, 2015). RFLP

Chapter 1  Biotechnological Interventions in Beverage Production   23

in combination with PCR has been used for the accurate detection of Staphylococcus at the species level (Mandal et al., 2011). PFGE is a DNA-based subtyping method that generates DNA banding patterns after DNA is cut into fragments with rare cutter restriction enzymes and used to study the characteristics of Staphylococcus aureus isolates (Lukinmaa et al., 2004). Of the many bacterial pathogens which cause contamination of different foods and hence illnesses, the major ones include Salmonella, Escherichia coli O157, Campylobacter, Listeria monocytogenes, Clostridium perfringens, Staphylococcus, Shigella, and Bacillus. The emergence of lab-based technologies, such as PCR, HPLC (High Performance Liquid Chromatography), ELISA (Enzyme Linked Immunosorbent Assay), flow cytometry, and biosensors, has made it easier to identify and quantify the pathogens. Progress has also been made in determining the presence of toxins produced by various bacteria in foods and beverages (Murugaboopathi et al., 2013). With the advancements in biotechnology, various advanced molecular techniques have been applied recently in developed countries like the United States and Europe for enhanced food safety and quality in food and beverage sectors. This is providing many tools to detect microorganisms and the toxins they produce. Monoclonal antibody tests, biosensors as well as PCR and DNA probes are being developed to determine the presence of harmful bacteria such as Listeria and Clostridium. Biotechnology-based detection method rapid assay molecular techniques are also used which are more reliable and enable quick detection of pathogens present in food.

1.2.8 Biosensors Biosensors represent an important area of biotechnological applications in product quality monitoring for food and beverage industries. A biosensor is an analytical device which converts a biological response into an electrical signal. It consists of two main components: a bio-receptor or bio-recognition element, which recognizes the target analyte, and a transducer, for converting the recognized event into a measurable electrical signal (Singh et al., 2017). The biological indicative material may be antibodies, enzymes, proteins, cell organelles, DNA, microbial cells, or plant/animal tissues. A receptor is generally immobilized within a cellulose, polyacrylamide, acetyl cellulose, or suitable gel membrane next to the transducer. Biosensors representing a group of instrumental devices based on biological interactions/ catalysis coupled with physical transducers and electronics are emerging as a revolutionary analytical technique having diverse applications in the area of dairy, food, and beverage processing and ensuring quality as well as safety (Mutlu, 2016).

24  Chapter 1  Biotechnological Interventions in Beverage Production

There are several types of biosensors developed through the application of biotechnology which could be classified as: enzyme-based biosensors, electrode-based biosensors, whole cell-based biosensors, antibody and receptor-based biosensors, tissue/whole organism- based biosensors, amperometric biosensors, immuno-sensors, acoustic biosensors, potentiometric biosensors, electrochemical biosensors, calorimetric biosensors, and optical biosensors. Among various biosensors, enzyme-based biosensors have been most frequently reported for dairy, food, and beverage applications, for example, determination of lactose, lactulose, lactic acid, amino acids, pesticide residues, antibiotic residues, including determination of triglycerides, cholesterol, starch, etc. in food products (Murugaboopathi et al., 2013). A rapid assay-based biosensor has been developed that needs minimum culture enrichment and employs immuno-based biosensors for quick detection and identification of harmful microorganisms in food systems. Acoustic wave biosensors, immuno-based biosensors, and radio frequency identification (RFID) sensors are recently developed tools used in dairy and other food industries to prominently improve food safety and quality (Thakur and Ragavan, 2013).

1.3  Other Applications of Biotechnology Biotechnology has a potential role in the beverage processing industry and hence can help in meeting the nutritional requirements and food security effectively. Other functions of biotechnological approaches of diverse areas in food and beverage industries like food ingredients, functional beverages, flavor production, and packaging of beverages are discussed below.

1.3.1  Biotechnology for Food Ingredients and Nutritional Enhancement Biotechnology has a vital role to play in the beverage industry and in the application of biotechnological techniques focused on the major value-added and energy-providing foods and their ingredients, such as alcohol, fermented beverages, yogurt, cider, vinegar, etc. and also on encapsulation of bioactive ingredients. Fermentation processes enhance the flavor and nutritional value of food or beverages by the biosynthesis of vitamins, proteins, and essential amino acids. It also causes improvement in protein and fiber digestibility, enhances micronutrient bioavailability, and degrades antinutritional factors (Lindley Consulting, 2009). Novel ingredients can also be produced by fermentation. There has been a long trend to replace natural ingredients with a broad range of flavors produced by

Chapter 1  Biotechnological Interventions in Beverage Production   25

fermentation. Recombinant DNA can also be used to increase the production of scarce enzymes from microbial sources (Corbo et al., 2014). Food ingredients are substances used to increase nutritional value, change consistency, and enhance flavor. The compounds inxanthan gum and guar gum are produced by microbes and are usually of plant or microbial origin to develop flavor and texture and increase nutritional value in different foods and beverages. Many of the amino acid supplements, flavors, flavor enhancers, and vitamins added to beverages are produced by microbial fermentation (Shahidi and Alasalvar, 2016; Kantachote et al., 2017). Functional and nutraceutical beverages are currently attracting attention across the globe because of their tremendous health benefit potentials and commercial value. Applications of biotechnology in food processing are reaching towards far more advanced stages in production of easily preparable and consumable foods and beverages, like RTE or RTS. The attention further focused in recent years due to the shift in consumer trends towards the same direction (Fleet and Rahman, 2017). The functional food production through modern biotechnological approaches has emerged to meet the challenge of bioactive ingredients through encapsulation and fortification of functional and health promoting foods. These bioactive ingredients include bioactive peptides, probiotics/prebiotics, bio-therapeutic proteins, omega-3, omega-6, low calorie sugars, flavones, etc., which can be encapsulated to enrich their functionality in protecting the consumers from different diseases such as strokes, gastrointestinal illnesses, CVD, hypertension, diabetes, cancers, etc. (Wilson et al., 2016). These approaches have already contributed in making the fruit and dairy-based microencapsulated prebiotic and probiotic beverages as an interregnal part of the diet of peoples living in higher and middle income countries (Varnam and Sutherland, 1994).

1.3.2  Biotechnology in Flavor Enhancement Flavors consist of attributes including both the perception in the mouth (sweetness, acidity, or bitterness) and the aroma (produced by several volatile compounds). Aroma profile is very important in wine and other beverages as it contributes to the quality of the final product. In wine, it is due to the collective effects of major volatile compounds, mainly aldehydes, alcohols, monoterpenes, esters, acids, and other minor compounds that are present in the grapes which cause enhanced flavor during the fermentation and maturation process of wine production (Verzera et al., 2008). The aroma profile radically increases during fermentation due to the synthesis of several volatile compounds and the release of varietal aroma precursors. The amount and nature of the volatile compounds synthesized during the fermentation

26  Chapter 1  Biotechnological Interventions in Beverage Production

process depend on various factors, such as pH, nitrogen content, fermentation temperature, and the yeast strain (Swiegers et  al., 2005). The volatile compounds produced by wine yeast include higher alcohols (floral aromas, fusel, and marzipan), medium and long-chain volatile compounds (sweaty aromas, fatty, and cheesy), ethyl esters and acetate esters (floral and fruity aromas) and aldehydes (nutty, buttery, and fruity aromas), and several others. The volatile fatty acids also contribute to the aroma during the fermentation of beverages (Lindley Consulting, 2009). The flavor of fermented food products is greatly influenced by acid fermentation as it produces lactic acid, thus resulting in a lowering of the pH causing sourness in food. During fermentation, metabolism of sugar produces acid or alcohol, thus decreasing sweetness. LAB converts carbohydrates to organic acids and produces other flavoring compounds, such as diacetyl, acetaldehyde, and ethanol, which contribute to the desired taste and flavor of the food. Mango wines fermented with yeast stains also produce various volatile compounds like fatty acids, alcohols and esters, and other volatile compounds including terpenoids, esters, alcohols, acids, aldehydes, and ketones due to fermentation of mango wine with Saccharomyces cerevisiae and Williopsis yeast strains (Li et al., 2012). Due to the impact of different strain during fermentation of mango and grape wine, the chemical profile concluded that macerated wine contained more terpenes, terpenols, higher alcohols, and fruity acetate esters than the nonmacerated wine, which improved the flavor characteristics during fermentation through biotechnological approaches (Li et  al., 2011; Shahidi and Alasalvar, 2016). Nowadays, the consumer preference for natural food additives has led to an increasing demand for natural flavors which also include those flavors obtained from living cells, food-grade microorganisms, and their enzymes that determine their quality and value. Strains like M522 of Basidiomycetes is developed to make it suitable for producing improved aroma such as esters and fatty acids to contribute towards improving the fruity intensity in desired food and beverages. Therefore, Basidiomycetes, which served as a biological aroma factory, offers an alternative to natural plant sources. The use of the unique characteristics of Basidiomycetes represents a potent and promising alternative to industrially produced foods and beverages with chemical as well as plant-based flavors (Carrau et al., 2008).

1.3.3  Biotechnology in Beverage Packaging Biotechnology also has tremendous potential to play a vital role in packaging of beverages at the industrial level. Traditionally, beverages have been packaged in containers made of plastic, glass, paper, or

Chapter 1  Biotechnological Interventions in Beverage Production   27

other substance that is simply designed to be an active or passive barrier between the beverage product and the environment. The packaging of beverages, both carbonated and noncarbonated, is a complex technological branch in the packaging industry. The current trend is to improve or replace the conventional containers, bottle or can, through different bio-based active and intelligent or smart packaging technologies. These technologies can be used to extend the shelf life of the products by quality monitoring, safety indications, greater consumer convenience, and ultimately to produce economic packages. Modern techniques like nano-biotechnology are intended to produce bio-based packaging films that can adapt with the packaging stuff, with enhanced barrier characteristics, improved mechanical and heat resistance, effective antimicrobial exteriors, and demonstrate higher biodegradability. Improving intelligent packaging to elevate the nutritional validity and shelf life via nano-biotechnology became the ambition of many corporations (Paul and Sahu, 2014a; Helmy, 2016). Nanotechnology applications for food packaging or food contact materials (FCM) are presently occupying the largest market share in terms of the applications of nanomaterials in the food processing sector. Edible nano-coatings or films can prevent the food products from various invasive microorganisms leading to increase in shelf life and food safety (Duncan, 2011; Paul and Sahu, 2014a; Castillo et al., 2017). Nanotechnology is rapidly bringing a revolution in the food industry by designing nutrient delivery systems, developing nano-formulated packaging materials, enriching nutritional values, production of novel products through bioactive encapsulations, and development of nanosensor-based intelligent monitoring and alert systems to detect and control the food-spoilage organisms. Inorganic nanomaterials of some metals and metal oxides such as silver, iron, titanium dioxide, zinc oxides, magnesium oxide as well as silicon dioxide and carbon nanoparticles have been used as antimicrobial agents in food packaging and in some cases as nutritional ingredients of food. Due to the excellent antimicrobial efficacy, nano‑silver-based active FCMs have been developed which are found to be highly effective to preserve the food products and beverages by inhibiting the growth of microorganisms (Paul and Sahu, 2014a). Further, the embedded nano sensors in a packaging system can detect food-spoilage organisms (Luo et al., 2016; Samanta et al., 2016; Helmy, 2016).

1.4  Impact of Biotechnological Intervention in Food and Beverage Industries As the awareness increases in the consumption of industrially processed food and beverages, the role of the food industry becomes

28  Chapter 1  Biotechnological Interventions in Beverage Production

extremely pertinent in producing high-quality nutritious and wholesome products which are cost-effective and safe to cater to the needs of its vast global consumers. The intentions are to increase the range and quality of products, consumer safety, reduce their price, and keep the environment safe. The application of biotechnology has even now made a strong commercial influence on the food, dairy, and beverage industries by improving and expanding the range of flavors, shelf life extension, enrichment in labeled nutritional quality and safety with lots of value additions including expected health benefits. The implementation of new innovations through modern biotechnology such as transgenics, rDNA technology, tissue culture, animal/plant cloning, and other bio-based improved interdisciplinary tools in the food industry has already started getting immense commercial benefits. The same is not only improving the external quality of the processed food products but also contributing in the variety of product diversification by manufacturing novel foods and beverages customized for specific consumers. The practice of modern biotechnological applications (including recombinant DNA technology) to produce foodstuffs with improved features for the consumers is also considered as a matter of concern with respect to the long-term effect on consumers’ health and environmental safety (Balarabe et al., 2017; Panesar et al., 2013).

1.5  Safety Issues With the Intervention of Biotechnological Tools Based on the published evidences and broad representation of scientific committees (also comprising industry experts in many cases) and governmental organizations, there is no acknowledged significant food safety concern in consuming food products produced through biotechnological interventions. Different globally powerful food and health organizations such as the Institute of Food Technologists and American Medical Association recognize and support the application of food biotechnology in food and beverage production. However, use of the products of biotechnology may get transferred to other food through different ways unintentionally and may lead to problems in consumers and may lead to allergic reactions with the food when they consume the same contaminated with the gene or modified bacteria (FAO, 2010). In Europe, a process-based regulatory system, triggered by the use of GM, exists for the release of GMOs to the environment. In the USA, a product-based approach has been adopted which evaluates the risks according to the final product and whether genetic material from plant pathogens has been used in the process. The possible adverse effects of eating GM foods may bring new allergens being formed through the inclusion of novel proteins which trigger allergic reactions

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at some stage. Antibiotic resistant microbes are also being transferred into the community of gut microbes through GM foods and intensifying problems of antibiotic-resistance (Bawa and Anilakumar, 2013). Hence, it is clear that the potential of biotechnological intervention in food and beverage industries is already recognized and continuously receiving a rising response of concern from producers and consumers (though commercial organizations are putting their best efforts to portray the matter in a attractive and modified way). It is also expected by different global agencies that, to meet the challenges of global food and nutritional security and safety, modern biotechnological intervention in food and beverage industries is one of the best alternatives. However, looking into some long-term possibility of health and environmental hazards, global regulatory authorities should carry out a thorough and reliable study neutrally (in the absence of any influence from the commercial producers) to approve or implement the norms and guidelines for the application of biotechnological tools in food and beverage processing.

1.6 Conclusion Biotechnology in the beverage processing sector targets the selection, production, and improvement of useful microorganisms and their products, as well as their technical application in product quality. Nowadays, the application of modern biotechnology in beverage industries is upgrading the traditional processing in the fermentation and other production processes. These modern technologies improve various bacteria used in fermentation which produce compounds that kill other food poisoning and spoilage bacteria leading to the enhanced nutritional and flavor profile of the product. In the old biotechnology, it was prudent to include directed control of the physical and chemical environments of the fermentation process, but new biotechnological methods, such as modified starter culture, recombinant DNA techniques, etc., have overcome this problem. Therefore, these technologies are bringing tremendous contributions in producing super strains of microbes that could enable acceleration of fermentation processes. Moreover, several of the modified enzymes used in beverage processing industries are manufactured using recombinant microorganisms through a biotechnological approach. Biotechnological intervention is also continuously contributing in the production of functional beverages through encapsulation of micronutrient and other bioactive compounds for providing new prospects for improving safety and human nutrition. Bio-preservation also plays a key role in enhancing the shelf life and safety standards, leading to reduced incidence of product spoilage. LAB and bacteriocins are the major bio-preservative used in beverage industries which can be isolated

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or produced through biotechnological processes. Improved strains of microorganisms can be produced by a variety of techniques like GM by mutagenesis, gene transfer mediated conjugation by using plasmid DNA, or by genetic recombination by hybridization with better yielding microorganisms for better production of desired functional products. Biotechnology-based detection tools are being effectively utilized in various food and beverage industries to detect various undesirable microorganisms and the toxins produced by them. Monoclonal antibody tests, biosensors as well as PCR and DNA probes are being developed to determine the presence of harmful bacteria such as Listeria and Clostridium, Escherichia coli 0157:H7, and also to detect toxins like aflatoxin. The PCR-based method is also used for the detection of ingredients and foodborne pathogens in food and beverage products. Nano-biotechnology is also applied in bio-based packaging systems to enhance the quality and shelf life of beverage products. Biotechnology has the potential to solve many health and nutritional concerns among the people throughout the globe. Although, developments in innovative modern biotechnologies implemented in food and beverage processing and preservation but stringent food-safety standards for biotechnological applications are need to be adopted by various regulatory bodies in view of the predicted long term effect on human health and environment. The need for further neutral studies is still felt to establish more concrete regulations to eliminate any chance of a long-term impact on consumers’ health and environmental hazards.

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Further Reading Kumari, A., Pandey, A., Ann, A., Raj, A., Gupta, A., Chauhan, A., Sharma, A., Das, A.J., Kumar, A., Attri, B., Neopany, B., 2016. Indigenous Alcoholic Beverages of South Asia. CRC Press, New York, pp. 503–596. Marshall, E., Mejia, D., 2011. Traditional fermented food and beverages for improved livelihoods. In: FAO Diversification Booklet 21. FAO. FIAT PANIS. Tamang, J.P., 2016. Ethnic Fermented Foods and Alcoholic Beverages of Asia. Springer. Tamang, J.P., Thapa, N., Tamang, B., Rai, A., Chettri, R., 2015. Microorganisms in Fermented Foods and Beverages. In: Health Benefits of Fermented Foods and Beverages. CRC Press, Taylor & Francis Group, New York, pp. 1–110.