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This review presents an overview of probiotic food development, various matrices for probiotic foods, and processing and packaging of probiotic foods.
Nutrafoods (2012) 11:117-130 DOI 10.1007/s13749-012-0055-6

Review

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Technological aspects of probiotic functional food development a review Shalini Mishra, H.N. Mishra Correspondence to: Keywords: Shalini Mishra functional foods [email protected] fermentation

probiotics food matrix processing and packaging

Received: 15 May / Accepted: 3 October 2012 © Springer Healthcare – CEC Editore 2012

Abstract

Introduction

Fermented food containing live cultures called probiotics are considered as fermented functional foods. Probiotics, when consumed in adequate numbers, provide a health benefit directly or indirectly by production of metabolites to the host. The development of probiotic products is a research priority and challenge for both industry and science sectors due to increasing consumer awareness of their ability to maintain good health along with the supply of essential nutrition. This review presents an overview of probiotic food development, various matrices for probiotic foods, and processing and packaging of probiotic foods.

Due to increased consumer awareness of the health benefits associated with specific foods, development of functional food has gained special attention from food scientists and industries. Functional food has the ability to maintain good health along with providing essential nutrition [1]. There has been a growing awareness of the role of gut microbes in human health, in terms of intestinal development, innate immunity or digestion of food and protection of the host against diseases [2]. Therefore, development of functional food has focused on dietary supplements that may favourably influence gut microbial composition and activities. Fermented foods with live cultures of beneficial microbes are considered as functional foods with probiotic benefits. Probiotics are commonly consumed as part of fermented foods such as fermented dairy products like yogurt, kefir, kumis cheese and fermented non-dairy products like soy yogurt, yosa, ogi, fruit, vegetable and malt beverages [3]. The global market for probiotic ingredients, supplements and foods was worth US$16 billion in 2008 and reached $21.6 billion in 2010, with an estimated target of $31.1 billion in sales in 2015, with a compound annual growth rate (CAGR) of 7.6% [4]. The potential health benefits of fermented functional foods are expressed either directly through

Abbreviations LAB Lactic acid bacteria Lb. Lactobacillus Lc. Leuconostoc Bf. Bifidobacterium GIT Gastrointestinal tract

Shalini Mishra (•), H.N. Mishra Department of Agricultural and Food Engineering Indian Institute of Technology Kharagpur Kharagpur, West Bengal 721302, India tel +91 3222 283130, fax +91 3222 283130 [email protected]

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Lactic acid bacteria

Non-lactic acid bacteria

Lactobacillus spp.

Bifidobacterium spp.

Other lactic acid bacteria spp.

Lb. acidophilus Lb. amylovorus Lb. casei Lb. crispatus Lb. delbrueckii Subspp. bulgaricus Lb. gallinarum Lb. fermentum Lb. gasseri Lb. johnsonii Lb. paracasei Lb. plantarum Lb. reuteri Lb. rhamnosus Lb. shirota Lb. sporogenes

Bf. adolescentis Bf. animalis Bf. bifidum Bf. breve Bf. longum Bf. lactis Bf. infantis

Enterococcus faecalis Enterococcus faecium Lactococcus lactis Leuconostoc mesenteroides Pediococcus acidolactici Streptococcus oralis Streptococcus uberis Streptococcus rattus

Bacillus cereus var. toyoi Escherichia coli (Nissle, 1917) Propionibacterium freudenreichii Sporolactobacillus inulinus Saccharomyces cerevisiae Saccharomyces boulardii

Sources: Refs. [96, 97] Table 1

Lactic and non-lactic acid-producing microorganisms considered as probiotics

the interaction of ingested live microorganisms with the host (probiotic effect) or indirectly as a result of ingestion of microbial metabolites like vitamins, proteins, peptides, oligosaccharides and organic acids, produced during the fermentation process (biogenic effect) [5]. Probiotics are live microorganisms thought to be beneficial to the host organism. According to the definition adopted by the Food and Agriculture Organization of the United Nations (FAO) and World Health Organization (WHO) [6], probiotics are: “Live microorganisms which when administered in adequate amounts confer a health benefit on the host”. The use of probiotic cultures stimulates the growth of preferred microorganisms and elimination of harmful bacteria, and enhances the body’s natural defence mechanisms [7, 8]. Lactobacillus spp. and Bifidobacterium spp. are the most common probiotics [9] and other potential probiotics are yeast [10], bacilli [11], Escherichia coli [12] and enterococci [13] (Table 1). Numerous health benefits of probiotic bacteria have been reported, including enhancement of immunity; prevention of colon cancer, hypercholesterolaemia, type 2 diabetes and upper gastrointestinal tract (GIT) diseases; reduction of osteoporosis and

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obesity; improvement in lactose utilisation; and stabilisation of the gut mucosal barrier [14, 15]. Many researchers have reported that activities of probiotics include cell-mediated immune responses, activation of reticuloendothelial system, augmentation of cytokine pathways and stimulation of pro-inflammatory pathways such as interleukin regulation [16, 17]. Probiotics have been proposed to exert therapeutic and health effects via several mechanisms [18–20].

Food matrices for formulation of probiotic functional food Probiotics are normally added to foods as a part of the fermentation process [21]. In the production of probiotic food one of the important factors is the matrix of food substrate. It acts as a medium to achieve the growth of microbes to at least 9 log cfu/g or ml [22], which is considered necessary to confer health benefits to the host [23]. Characterisation of specific probiotic strains, food matrix and dietary content interaction with the probiotics are the new research areas for food technologists and industrialists [24]. Composition of food substrate such as fat content, type of proteins, carbohydrates and pH can

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affect probiotic growth and survival. Charalampopoulos et al. [25] suggested that the nature of food matrix could affect the stability of the probiotic microorganisms during gastrointestinal transit. Dairy and non-dairy substrates are considered as a vehicle for delivering probiotic bacteria to the human GIT and base for the development of probiotic foods. Dairy matrix Dairy matrices are an extremely promising source for the development of probiotic foods [21]. They are associated with the strain, bioactive substance or substrate specificity, which provide evidence of their efficacy, safety and organoleptic quality [26]. Various food products have been developed as carriers for probiotics, mainly of dairy origin because consumers commonly associate them with fermented dairy products and perceive health benefits in the presence of probiotic cultures [27]. The base for the production of dairy fermented products is milk, which has a typical composition of 87.4% water, 4.7% lactose, 3.8% fat, 3.3% protein (80% casein and 20% whey protein), 0.2% citrate and 0.6% minerals, with pH in the range 6.5–6.7 [28]. Most probiotics proliferated well in a dairy-based matrix due to the lactose-hydrolysing enzyme and proteolytic system involved in casein utilisation, which provides probiotic cells with a carbon source and essential amino acids for growth. Metabolism of these nutrients produced organic compounds that are essential for the development of flavour, preservation and appearance of the products [21]. Some additives like prebiotics, pulses and cereal flours are used to speed up the acidification process and survivability of probiotics, as some lactobacilli are unable to consume lactose as a carbon source. According to Rogelj [29], dairy-fermented products such as yogurt, probiotic beverages and cheese-containing lactic acid bacteria and their constituents such as omega-3 fatty acid, phytosterols, isoflavones, conjugated linoleic acid (CLA), minerals and vitamins have a prominent position in the development of functional foods. Casein glycomacropeptide, amino sugars and

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oligosaccharides promote the growth of bifidobacteria and lactobacilli, which led to development of probiotic foods [30]. In some cases, fermented milk products are fermented by monocultures of probiotic bacteria, but usually supporting cultures are applied to speed up the acidification process and provide the desired texture and flavour [31]. Many lactobacilli and bifidobacteria survive in fermented milk products for 4–8 weeks in refrigerated storage. Probiotic dairy products, which contain healthpromoting lactic acid bacteria (LAB) in addition to traditionally used starter LAB, are good examples of successful fermented functional foods. Today, numerous commercial dairy-based beverages incorporate various strains of probiotic bacteria that are available for human consumption (Table 2). Non-dairy food matrices The food industry is focusing on the development of new non-dairy foods containing probiotics that can overcome the limitations (lactose intolerance and cholesterol content) imposed by milk-based products, which prevent their consumption by certain segments of the population [32]. Increasing demand for new foods and tastes initiated development of non-dairy probiotic products that are part of the day-to-day normal diet to maintain the minimum therapeutic level [33]. Technological advances have made it possible to change some structural and physico-chemical characteristics of plant matrices [33–36] by modifying food components in a controlled way such as pH modification and fortification of culture media [37, 38]. Food grains Cereals The application of probiotic microbial strains for fermentation of cereals and legumes is a rational approach for the development of functional foods. Cereals contain high levels of carbohydrates, which act as a source of carbon and energy for microbes during fermentation. Most of the carbohydrates in cereals are present as starch and only available for microbes

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Product name

Substrate

Starter culture/probiotic microorganism

Lb. acidophilus Lb. delbueckii subsp. bulgaricus Lc. lactis subsp. lactis, Lb. delbrueckii subsp. bulgaricus, Lb. plantarum, Streptococcus lactis, S. thermophilus, S. cremoris Buffalo’s/cow’s S. lactis subsp. diacetylactis, S. cremoris milk skim milk Cow’s, buffalo’s, Lc. lactis subsp. lactis, Lc. lactis subsp. goat’s milk, cremoris, Lc. lactis subsp. diacetylactis, sheep milk S. thermophilus, Lb. delbueckii subsp. bulgaricus Priopionibacterium shermanii, Penicillium roqueforti, etc. Mare’s, camel’s Lb. acidophilus, Lb. bulgaricus, or ass’s milk Saccharomyces, Micrococci Sheep’s, cow’s, S. lactis, Leuconostoc sp., goat’s or mixed Saccharomyces kefir, Torula kefir, milk Micrococci Goats, sheep’s S. lactis, S. thermophilus, Lb. bulgaricus, milk lactose-fermenting yeast Cow’s milk/ S. thermophilus, Lb. bulgaricusan buffalo’s milk Various combinations of probiotics

Acdiophilus milk Cow’s milk Bulgarian butter milk Cow’s milk Curd Buffalo’s or cow’s milk Cultured butter milk Cheese

Kumiss Kefir Leben Yoghurt

Commercial products Acidophilin

Acidophilus buttermilk Acidophilus-yeast milk

Buffalo’s or cow’s milk or skim milk or mixture Buffalo’s or cow’s milk or skim milk or mixture Whole or skimmed milk

Lb. acidophilus, Lc. lactis subsp. lactis, kefir yeasts Lb. acidophilus, Lc. lactis subsp. lactis, subsp. cremoris, subsp. lactis biovar. diacetylactis

Product name

Substrate

Starter culture/probiotic microorganism

Bifighurt®

Buffalo’s or cow’s milk or skim milk or mixture Cow’s milk Buffalo’s or cow’s milk or skim milk or mixture Whole or skimmed milk Buffalo’s or cow’s milk or skim milk or mixture Buffalo’s or cow’s milk or skim milk or mixture of milk Whole or skimmed milk

Bf. longum (CKL 1969)

Bifidus milk Cultura® or A/B milk Chamyto Diphilus milk

Nu-Trish A/B

Onaka He GG, Gefi lus (Valio Ltd) Procult drink bulgaricus Vitagen Yakult Yakult Miru-Miru

Whole or skimmed milk Whole or skimmed milk Skim milk or skim milk powder Skim milk or skim milk powder

Bf. bifidum or Bf. longum Lb. acidophilus, Bf. bifidum, Bf. longum (DSM 2054) Lb. helveticus, Lb. johnsonii Lb. acidophilus, Bf. bifidum

Lb. acidophilus, Bf. spp.

Streptococcus thermophilus, Lb. delbrueckii subsp. bulgaricus, Lb. rhmnosus GG Lb. delbrueckii subsp. B longum BB536, Streptococcus thermophilus Lb. acidophilus Lb. paracasei subsp. paracasei Shirota Lb. paracasei subsp. paracasei, Bf. bifidum or Bf. bereve, Lb. acidophilus A-38 fermented milk, Lb. acidophilus, mesophilic lactic cultures

Lb acidophilus, Saccharomyces lactis

Sources: Refs. [98, 101] Table 2

Fermented dairy products with different strains of probiotics

after amylolytic hydrolysis. Endogenous cereal enzymes, malt or selected enzymes can be used to break down the starch to simple fermentable sugars (i.e., maltose and glucose), which can be utilised by probiotics as a carbon source [39]. Pediococcus spp. VA403 [40], Lb. manihotivorans [41] and Lb. plantarum [42] are known as LAB, which have the ability to break down the starch and utilise it as a carbon source to produce lactic acid. Cereal-based products’ ability to support the growth of probiotics is mainly due to their high concentration of fibres such as xylooligosaccharides, xylan and arabinoxylan, which may act as a growth substrate for probiotics. Besides carbohydrates, cereals also contain relatively high levels of minerals, vitamins, sterols, and other growth factors, which support the growth of

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microbes, including the LAB. Whole grains are also a source of many phytochemicals, including phytoestrogens, phenolic compounds, antioxidants and phytic acid [43], which provide additional functionality to probiotic foods. The nutritional quality of grains is sometimes inferior to that of milk because of its lower protein content, deficiency of certain essential amino acids, low starch availability, antinutrients (phytic acid and tannins) and the coarse nature of the grains [44]. Fermentation has been postulated to decrease the level of starch as well as some non-digestible poly- and oligosaccharides, improve protein quality and increase the level of amino acids and group B vitamins. Fermentation also provides optimum pH conditions for enzymatic degradation of phytate

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and release minerals such as manganese (which is an important growth factor of probiotic), iron, zinc and calcium [44]. Strains of Lactobacillus have been recognised as complex microorganisms that require fermentable carbohydrates, amino acids, vitamin B, nucleic acids and minerals to grow [45]. Charalapompoulos et al. [46] conducted experiments with different cereals to determine the main parameters required for the growth of probiotic microorganisms, such as composition and processing of cereal grains, substrate formulation, growth capability and productivity of the starter culture, stability of the probiotic strain during storage, organoleptic properties and nutritional value of the final product. Different cereals provide different conditions to support the growth of probiotics [25]. It has been reported that Lb. reuteri, Lb. acidophilus and Bf. bifidum grow well in oat-based substrates [47]. Yosa, a new oat-based fermented food similar to flavoured yogurt or porridge, is considered as a health food due to its oat fibre β-glucan, lactobacilli and bifidobacteria [44]. Helland et al. [48] studied the growth ability of probiotics in a corn-based fermented substrate and observed that maize fermentation induces fruity flavours in traditional Mexican foods, which could have good worldwide acceptance. Nyanzi et al. [49] evaluated the sensory attributes of a maize beverage fermented by four species of probiotics and reported that the beverages fermented by L. acidophilus or L. rhamnosus were well accepted by trained and untrained panels. The possible applications of cereals or cereal constituents in fermented functional food can be summarised as [46]: • Fermentable substrates for growth of probiotics, especially lactobacilli and bifidobacteria • Prebiotics due to their content of specific nondigestible carbohydrates • Encapsulated materials for probiotics in order to enhance their stability Legumes (soy) Soy is an excellent raw material for the development of non-dairy probiotic foods to overcome the limi-

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tations associated with dairy products. The benefits of soy have drawn much attention recently and numerous soy products have been evaluated as possible probiotic vehicles. Experiments revealed that soy milk is a good food matrix for probiotics such as Lactobacillus spp., Lb. casei [50], Lb. helveticus [51], Lb. fermenti [52], Lb. fermentum [53], Lb. reuteri [54] and Lb. acidophilus [55]. Soy-based fermented foods may provide additional benefits for the consumer due to their various functional properties: they are hypolipidaemic, anticholesterolaemic and antiatherogenic and have reduced allergenicity [56]. According to Champagne et al. [57], development of a fermented soy product containing probiotics requires strain selection for the ability to grow in the substrate, as well as the ability to compete or even establish a synergy between strains. Donkor et al. [58] reported that the protein in fermented soy milk could encourage the growth of many probiotic strains such as Lb. acidophilus, Lb. casei and St. thermophilus. Scientific research has shown that probiotic-containing soy-fermented beverages have good sensory acceptance for potential consumers [59]. Hauly et al. [60] reported that soy yoghurt supplemented with fructooligosaccharide had an acceptance index above 70%. The texture [61] and taste of soy yoghurt are essential attributes for product acceptability [62]. Gel formation of soy milk proteins is a key process step in the manufacture of a non-dairy fermented product like yoghurt. The rheological properties of set gels determine the texture, organoleptic properties and shelf life of the product [63, 64]. Soy milk has a low acidification rate and slow growth of probiotic bacteria, which take longer to complete fermentation and produce undesirable changes in the product that are not acceptable to the consumer [62]. Addition of certain additives like prebiotics (inulin and fructooligosaccharide) and whey protein concentrate improved the textural and sensory characteristics of fermented soy yoghurt [60, 62]. Farnworth et al. [65] inoculated cows with a mixture of two starters (S. thermophilus and Lb. delbrueckii

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subsp. bulgaricus) and soy milk with probiotic bacteria (Lb. rhamnosus, Lb. johnsonii and Bifidobacteria) and reported better growth with the soy substrate due to the availability of various sugars (stachyose, raffinose, sucrose and glucose). In addition to supporting the growth of probiotics, soy-based products also protected probiotic cells during transit from the harsh conditions of the GIT [66]. Soy is the most studied matrix for the formulation of probiotic food, but other substrates like peanut have also been explored for the development of probiotic food [67]. Table 3 represents various developed probiotic cereals and legume food products.

Fruits and vegetables Fruits and vegetables are a rich source of minerals, vitamins, dietary fibres and antioxidants [68]. Therefore, there has been increasing interest in the application of vegetable and fruit juices as alternative carriers of probiotics. A number of studies found that probiotic strains have the capability to grow in fruit and vegetable matrices [69]. Researchers also observed significant differences in the acid resistance of lactobacilli and bifidobacteria in orange, pineapple, cranberry, bitter gourd, carrot and other juices. According to Sheehan et al. [70], Lb. casei, Lb. rhamnosus and Lb. paracasei survived for longer

Remarks

References

Product name

Substrate

Starter culture/probiotic microorganism

Boza

Substrate Maize, wheat, rye, millet and other cereals

Lb plantarum, Lb. acidophilus, Lb. fermentum, Traditional fermented product native to Turkey, Romania, Albania and Bulgaria Lb. aprophilus, Lc. raffi nolactis, Lc. mesenteroides, Lb. brevis, Saccharomyces cerevisiae, Candida tropicalis, Candida glabrata, Geotrichum penicillatum and Geotrichum candidum

44, 102

Bushera

Sorghum, or millet flour

Lactobacilli, Lactococcus, Leuconostoc, Enterococcus and Streptococcus, Lb. brevis

Traditional beverage prepared in the Western highlands of Uganda

103

Lb. plantarum A6

The level of probiotic is 2.3×109 cfu/ml

104

Mahewu

Corn meal

Lactococcus lactis subsp. lactis

Sour beverage consumed in Africa and some Arabian Gulf countries

44, 105

Fermented soybean beverage and yoghurt

Soybean

Bifidobacterium spp., Lactobacilli spp.

Higher protein level in soybean increases the buffering capacity and growth of probiotic strains

58, 106, 107

Ogi

Maize, sorghum or millet

Lb. plantarum, Lb. fermentum, Leuc. mesenteroides and Sacch. cerevisiae

Popular breakfast and complementary food for young children in several West African countries

44

Probiotic oats drink

Oats

Lb. plantarum B28

The number of probiotic bacteria is 7×1010 cfu/ml

108

Rice yoghurt

Rice

Lb acidophilus and Lb. casei subsp. rhamnosus

Rice-based yoghurt with strawberry contains 109 3.05% protein, 2.67% fat, 24.4% glucose, 1.5% sucrose, 47 mg/100 g of calcium, 0.86% acidity as lactic acid, pH 3.48 and probiotic count of 107 cfu/g. Storage period was more than 20 days at 4°C

Fermented cassava flour Cassava flour

Commercial products Grainfields Whole grain probiotic liquid

Grains, beans and seeds such Lb. acidophilus, Lb. delbrueckii, as pearl barley, linseed, maize, Saccharomyces boulardii, Saccharomyces rice, alfalfa seed, mung beans, cerevisiae rye grains, wheat and millet

ProvivaR

Malted barley, oatmeal gruel

Table 3

Lb. plantarum 299v

Probiotic fermented cereal and legume products

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Grainfields B.E Wholegrain Liquid is considered as gluten free

110

Commercialised by Skane Dairy (Sweden)

111

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in orange and pineapple juice than in cranberry juice. They survived at levels above 7.0 and 6.0 log cfu/ml in orange juice and pineapple juice for at least 12 weeks at refrigerated storage temperature. Sheehan et al. [70] reported that fruit juices appear as a more complex system for the development of probiotic foods, due to the more acidic pH of the products. Thus, the selection of probiotic strains that are more resistant to acidic environments is crucial in the development of a probiotic juice [21]. Microencapsulation has been shown to provide protection to acid-sensitive probiotics. Ding and Shah [71] studied the effect of microencapsulation on the viability of probiotic bacteria in orange and apple juices and reported that encapsulated probiotic bacteria was found to survive over 6 weeks of cold storage with counts of more than 105 cfl/ml or g, while free probiotic cells lost their viability within 5 weeks. The addition of prebiotics can also improve the viability and stability of the probiotics [72].

Kyung et al. [73] developed a probiotic red beet beverage using Lb. acidophilus and Lb. plantarum and reported that both strains reduced the pH of the juice from an initial value of 6.3 to less than 4.5 after 48 h of fermentation, due to their ability to produce a greater amount of lactic acid. The juice supported a significant number of beneficial LAB (109 cfu/ml) but Lb. acidophilus was considerably less stable in the fermented juice than Lb. plantarum, Lb. casei and Lb. delbrueckii during cold storage at 4°C. Table 4 represents the developed probiotic fruit and vegetable products and their fermentable substrates.

Processing, packaging and storage of probiotic functional food Processing of probiotic food The incorporation and viability of probiotic bacteria during storage is a constant challenge for the food industry and requires the understanding of

Product name

Substrate

Starter culture/probiotic microorganism

Remarks

References

Probiotic cabbage juice

Cabbage

Lb. plantarum C3, Lb. casei A4 and Lb. delbrueckii D7

The fermented cabbage juice could serve as a healthy beverage for vegetarians and lactose-allergic consumers

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Probiotic fermented beet juice

Beet

Lb. acidophilus LA39, Lb. plantarum C3, Lb. casei A4, Lb. delbrueckii D7

Probiotic tomato juice

Tomato

Lb. plantarum C3, Lb. casei A4, Lb. delbrueckii D7

Fermentation was achieved with 24-h-old 115 culture at 30°C. After 48 h fermentation, the counts of Lb. plantarum C3, Lb. casei A4 and Lb. delbrueckii D7 reached 109 cfu/ml

Probiotic fermented Carrot and beetroot carrot and beetroot juice

Lb. acidophilus, yeast

Yeast autolysate stimulated the growth of Lb. acidophilus. Amino acids, vitamins, minerals and antioxidants reduced the fermentation time

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Probiotic noni juice

Bf. longum and Lb. plantarum

Noni juice fermented with Bf. longum and Lb. plantarum had a high antioxidant capacity.

66

Commercialised by Superfoods (USA)

117

113, 114

Commercial products Vita BiosaR

Aromatic herbs and other plants Lactic acid baceria

BioprofitR

Fruit juice-based product

Lb. rhamnosus GG, P. freudenreichii subsp. shermanii JS

Commercialised by Valio Ltd (Finland)

118, 119

Rela

Fruit juice

Lb. reuteri MM53

A popular beverage of Biogaia, Sweden

120

BiolaR

Fruit juice

Lb. rhamnosus GG

Commercialised by Tine BA (Norway

121

Table 4

Probiotic fermented fruit and vegetable products

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all intrinsic and extrinsic factors associated with processing [74]. From a technological perspective, it would be advantageous if microbial cultures were capable of growing in substrate media, survived during processing and maintained their efficiency throughout the storage [75]. In addition, probiotic strains should be suitable for large-scale industrial production and must have good stable properties so that they can be cultured and incorporated into a range of food matrices without losing viability and functionality and creating unpleasant flavours or textures in the product [75, 76]. Selection criteria for probiotic bacteria include an ability to survive the transition through the GIT, including acid and bile resistance, attachment abilities to intestinal epithelial cells, human intestinal colonisation, antimicrobial substance production and conveyance of beneficial effects on human health [77, 78]. It should be of human origin and certified as GRAS (generally recognised as safe) status [78, 79]. Probiotics must grow well in simple media to sufficiently high cell concentrations and survive during various processes like centrifugation, freeze drying and freezing [80]. Processing of microbial systems for functional foods is dependent on the composition and processing history of the raw material used as a substrate, the viability and productivity of the starter cultures applied, and processing and storage conditions of the final food products [81]. Pre-treatment for the development of a fermentable substrate for dairy probiotic food involves heat treatment (pasteurisation), homogenisation, ultrafiltration, stirring (incorporation of air) and addition of additives. Soaking, amylolytic treatment, grinding, sieving/ultrafiltration and pasteurisation/steaming are basic steps for the development of fermentable substrate for probiotic non-dairy foods. Processing conditions (i.e., heating, homogenisation and packaging) effect the starter culture growth, fermentation time and subsequent handling. Most commonly, substrate fortification, pH adjustment, competitive microbial flora, thermal processing or aseptic packaging are employed during

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the manufacture of fermented functional foods. Bacteria must be added at a suitable concentration to remain greater than 109 cfu/g or ml for the shelf life of the product, which is the requirement for the probiotic claim of any products [21]. A number of factors, such as incubation temperature, incubation time, acidity, hydrogen peroxide produced by bacteria, concentration of lactic and acetic acid, and antagonistic and synergistic interaction of the probiotic species and starters can affect the survival of probiotic bacteria in dairy and non-dairy fermented foods [82, 83]. Probiotic fermentation of raw substrate allows the bacteria to multiply and impart distinctive flavours and organoleptic changes to the food [76]. The quality of the final products depends upon selection of probiotic strains, type and amount of acids and other metabolites produced by it. Table 5 presents a list of starter cultures and probiotic microorganisms and their physiological conditions used to develop functional foods. Hetero- and homo-fermentative lactobacilli imparts different flavours during fermentation. Homo-fermentatives produce an “acidic sour taste” from lactic acid and hetero-fermentatives yield a “sharp acidic taste” by the production of acetic acid [84]. Taste, flavour, appearance and composition will determine the acceptability of products among consumers. The various steps involved in the development of fermented functional foods can be summarised as: • Formulation of fermentable substrate for probiotic bacteria • Inoculation of probiotic bacteria • Incubation at the optimum temperature • Ceasing the fermentation by maintaining low temperature • Sensory analysis • Packaging and storage • Evaluation of nutritional and health claims Packaging and storage “Packaging is often described as a silent salesman and is defined as a device to contain what it sells or

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Micro-organism

Growth temperature (°C)*

Fermentation type

Acidity

Final pH

Lb. delbrueckii subsp. bulgaricus Lb. delbrueckii subsp. lactis Lb. helveticus Lb. acidophilus Lb. kefir Lb. brevis Lb. casei subsp. casei Lb. plantarum S. thermophilus Lc. lactis subsp. lactis Lc. lactis subsp. cremoris Lc. lactis subsp. lactis biovar. diacetylactis L. casei shirota Lc. mesenteroides subsp. cremoris Ln. mesenteroides subsp. dextranicum Bifidobacterium (bifidum, infantis, etc.)

22–52 (45) 18–50 (40) 22–54 (42) 27–48 (37) 8–43 (32) 8–42 (30) (30) 15–45 22–52 (40) 8–40 (30) 8–37 (22) 8–40 (22–28) 5–45 (35) 4–37 (20–28) 4–37 (20–28) 22–48 (37)

Homo fermentative Homo fermentative Homo fermentative Homo fermentative Hetero fermentative Hetero fermentative Homo fermentative Homo/hetero fermentative Homo fermentative Homo fermentative Homo fermentative Homo fermentative Homo fermentative Hetero fermentative Hetero fermentative Hetero fermentative

1.5–1.8 1.5–1.8 1.5–2.2 0.3–1.9 1.2–1.5 1.2–1.5 1.2–1.5

3.8 3.8 3.8 4.2 – – –

0.6–0.8 0.5–0.7 0.5–0.7 0.5–0.7 – 0.1–0.2 0.1–0.2 0.1–1.4

4.5 4.6 4.6 4.6 6.5 5.6 5.6 4.5

*Optimum temperature in parens. Source: Ref. [122] Table 5

Starter culture and probiotic microorganism used to develop fermented functional foods

a device which sells what it contains” [85]. Careful considerations should be given to the packaging of probiotic fermented products to provide suitable environmental conditions to maintain the viable numbers. Mattila-Sandholm et al. [86] reported that the packaging material and the storage conditions are important factors for the quality of fermented functional foods. The oxygen content in the product and oxygen permeation through the package are considered the most significant factors affecting the viability of probiotics [87]. The use of oxygen-impermeable packaging, microencapsulation of nutrients [88] and selection of stress-resistant strains [89] are applied to solve these problems. In this respect, the use of oxygen-scavenging plastics as chemical barriers to permeation should provide products equivalent to canned foods. Plastic materials having low oxygen permeability dominate food packaging materials in the dairy and non-dairy sector. Fermented foods may become contaminated with components or degraded products due to product package interactions. In fermented products where lactic acid is produced, it could penetrate into the structure of the plastic polymer packaging and form stubborn bacterial films on polymer surfaces [90]. Tetra Pak, and probiotic tubes and films are the

latest invention in the field of probiotic beverage packaging. Unistraw’s unique system stores the probiotics as dry, stable UniBeads in the straw, where they are kept in position by filters located at both ends of the straw. The UniBeads dissolve in the liquid as it passes through the straw when sipping it and shelf life of this straw is 12 months [91]. There is some innovative packaging such as active packaging and antimicrobial packaging being used for the preservation of fermented functional foods. Active packaging is an exciting area of food technology that can confer many preservative benefits of fermented food products. The objectives of this technology are to maintain sensory quality and the shelf life extension of foods, while maintaining nutritional quality and ensuring microbial safety [92, 93] Antimicrobial packaging systems are particularly important in fermented foods to inhibit growth of spoilage and pathogenic microorganisms, contribute to the improvement of food safety and extend the shelf life of packaged food. Many factors need to be considered in designing an antimicrobial packaging system; however, most factors are closely related to the characteristics of antimicrobial agents, packaged foods and target microorganisms [94]. Packaging of food in a modified atmosphere can of-

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fer extended shelf life and improved product presentation in a convenient pack, making the product more attractive to the retail consumer [95]. Storage temperature is a critical parameter to maintain the viability of probiotic foods and reduce undesirable changes in fermented food products. It must be kept low to prevent further fermentation once optimum acidity is achieved. Freeze-drying or refrigerated storage are generally applied storage/distribution routes.

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Conclusions Development of fermented food is a multistage process that is affected by many factors, such as sensory acceptance, physical and microbial stability, and price. Probiotic food is one of the largest functional food markets and has growth potential in the food industry among dairy and non-dairy probiotic products; those made with milk and plant sources have been reported to have numerous health benefits. The future of probiotic foods will undoubtedly involve a continuation of the labelling of health claims and safety debates. As consumers become more health conscious, the demand and market value for health-promoting foods and food components is expected to grow. Before the full market potential can be realised, however, consumers need to be assured of the safety and efficacy of probiotic foods. There is a need to test bioactive ingredients, explore more options of fermentable substrates that have not yet been industrially utilised, and optimise products and processes for the development of fermented foods. Culture viability during storage, inoculum size and inoculum strength of probiotics are other major issues related to product development, which have to be studied.

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