Fermented Milk and Dairy Products 84 FERMENTED MILK AND DAIRY PRODUCTS 4.6 Application of Starter Cultures 99 4.7 Conclusion 105 References 106 4.1 Introduction Starter cultures are used in the manufacturing of different types of fermented milk and dairy products including yogurt, dahi, cultured buttermilk, sour cream, quarg, kefir, koumiss, and cheese. The use of these cultures for the preparation of products has been practiced since time immemorial. Traditionally, the common method was to use the previous-day’s product (i.e., milk, dahi, whey, buttermilk, etc.) as an inoculum to produce the fresh batches of fermented product. Such methods were not reliable and often resulted in off- flavor, inconsistent products, and product failure due to undesir- able fermentation. These drawbacks were mainly due to the lack of scientific knowledge of starter culture technology. The ideal of pure culture for making good-quality fermented milk became more visible in the mid-nineteenth century, where starters were widely studied and their metabolisms were well established. This leads to manufacturers handling large volumes of milk started selection of appropriate starters to obtain uniform quality product. This made the selection of appropriate starters even for manufacturers han- dling large volumes of milk to obtain uniform quality of product. Thereafter, companies started producing pure cultures for commer- cial application. Starter cultures may be defined as the carefully selected group of microorganisms that are deliberately added to milk and milk prod- ucts to bring desirable fermentative changes. These have multifunc- tional role in dairy fermentation; the primary one is to produce lactic acid, hence, popularly called as lactic acid bacteria (LAB). Besides production of lactic acid, certain cultures perform secondary func- tions such as the production of acetic, propionic, and folic acids, CO2, H2O2, ethanol, bacteriocins, exopolysaccharides (EPS), and so on (Cintas et al. 2001; Padalino et al. 2012; Yang et al. 2012). This chapter reviews the characteristics of different starters, their types, scaling up, problems associated with starters, and their application. TYPES AND APPLICATION OF LACTIC StARTERS 85 4.1.1 Functions of Starter Cultures Different functions of starter cultures are to • Produce lactic acid and other metabolites (i.e., alcohol, CO2, propionic acid, acetic acid, etc.). • Produce aromatic compounds like diacetyl, acetaldehyde, and acetoin. • Control the growth of pathogens and spoilage causing microorganisms. • Produce certain vitamins (i.e., folic acid, vitamin B12, niacin, etc.). • Bring proteolytic and lipolytic activities. • Improve body and texture of certain products by producing EPS. • Assist in overall acceptability of the final product. 4.2 Characteristics of Dairy Starter Cultures Starter cultures form a large group of microorganisms that include bacteria, yeasts, and molds (Table 4.1) for particular fermented milk products. Although starter cultures are genetically diverse, the common characteristics of these groups include Gram-positive, non-spore Table 4.1 Microorganisms Used as Starter Cultures MICROORGANISM(S) MAJOR CHARACTERISTICS A. BACTERIA Lc. lactis subsp. lactis, Lc. lactis subsp. Cocci shaped, production of lactic acid, cremoris, Lc. lactis subsp. lactis biovar. exopolysaccharides, riboflavin, bacteriocins, diacetylactis diacetyl, etc. Leuconostoc mesenteroides subsp. Cocci shaped, production of lactic acid, dextranicum, Leu. mesenteroides subsp. exopolysaccharides (homopolysaccharides, mesenteroides, Leu. cremoris dextran), diacetyl, CO2, etc. Streptococcus thermophilus Cocci shaped, production of lactic acid, exopolysaccharides (homo- or heteropolysaccharides), bacteriocins, folate, etc. Lactobacillus delbrueckii subsp. bulgaricus, Rod shaped, production of lactic acid, Lb. reuteri, Lb. casei, Lb. fermentum, exopolysaccharides (homo- or Lb. plantarum, Lb. helveticus, Lb. heteropolysaccharides), riboflavin, bacteriocins, acidophilus, Lb. paracasei, Lb. rhamnosus etc. Few species are used as probiotic. (Continued) 86 FERMENTED MILK AND DAIRY PRODUCTS Table 4.1 (Continued ) Microorganisms Used as Starter Cultures MICROORGANISM(S) MAJOR CHARACTERISTICS P. acidilactici Cocci shaped, forms tetrads, production of lactic acid, bacteriocins, etc. Bifidobacterium adolescentis, B. brevis, Anaerobic heterofermentative, non-spore forming B. bifidum, B. infantis, B. lactis, B. longum rods, production of two molecules of lactate and three molecules of acetate. Species are also used as probiotic. Brevibacterium linens, B. casei Rods, pleomorphic, obligate aerobes, impart reddish-orange color in cheeses. Propionibacterium freudenreichii subsp. Rods, pleomorphic, production of propionic acid, freudenreichii, P. freudenreichii subsp. acetic acid, and CO2. shermanii Enterococcus faecium, E. faecalis, E. durans Cocci shaped, production of lactic acid, bacteriocins. B. YEASTS Candida kefir Short ovoid to long ovoid, budding yeast-like cells or blastoconidia, production of ethanol and CO2. Kluyveromyces marxianus Formation of pseudomycelium, fermentation of lactose and inulin, produces aroma compounds such as fruit esters, carboxylic acids, ketones, furans, alcohols, monoterpene alcohols, and isoamyl acetate. Saccharomyces cerevisiae Forms blastoconidia (cell buds), produces ascospores, production of alcohol and CO2. Torulospora delbrueckii Forms buds, produces ethanol and CO2. C. MOLDS Penicillium roquefortii, P. camemberti Production of asexual spores in phialides with a distinctive brush-shaped configuration, production of mycotoxins like roquefortine and PR toxin. Geotrichum candidum Produce chains of hyaline, smooth, one-celled, subglobose to cylindrical, slimy arthroconidia (ameroconidia) by the holoarthric fragmentation of undifferentiated hyphae. Septate hyphae that disarticulate into arthroconidia and do not form budding yeast cells. Contributes to an aroma. Aspergillus oryzae Filamentous fungus, highly aerobic and are found in almost all oxygen-rich environments. Mucor rasmusen Spores or sporangiospores can be simple or branched and form apical, globular sporangia that are supported and elevated by a column-shaped columella. TYPES AND APPLICATION OF LACTIC StARTERS 87 forming, non-pigmented, and unable to produce catalase and cytochrome, growing anaerobically but are aero-tolerant and obligatorily ferment sugar with lactic acid as the major end product. The nutritional require- ment of these cultures varies from species to species. Most of the cul- tures are nutritionally fastidious, often requiring specific amino acids, vitamin B, and other growth factors, while unable to use complex carbohydrates. 4.2.1 Bacteria Lactococci have been widely used for manufacturing a variety of fer- mented milk products. So far, five species are recognized but only Lactococcus lactis is used as starter culture that has practical significance in dairy fermentations. There are two subspecies,Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris, and one variant, Lc. lactis subsp. lactis biovar. diacetylactis, which are commonly used as single or in mixed cultures. Lactococci are homofermentative and mesophilic, and when grown in milk, more than 95% of their end product is lactic acid, l(+) isomer. However, being weakly proteolytic, they can use milk proteins and grow at 10°C but not at 45°C. Lc. lactis subsp. lactis is more heat and salt tolerant than other subspecies. It ferments maltose, grows at 40°C, and in pH 9.5, produces ammonia from arginine, whereas Lc. lactis subsp. cremoris did not show these characteristics. Lc. lactis biovar. diacetylac- tis shows a close relationship with Lc. lactis subsp. lactis but differs by exhibiting citrate positive ability and does not produce as much lactic acid in milk as the latter. Nisin and diplococin are produced by Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris, respectively, while bacteriocins produced by Lc. lactis subsp. lactis biovar. diacetylactis are not named. Some lactococci can produce EPS and improve textural properties of cultured dairy products (Cerning 1990; Behare et al. 2009a). Leuconostoc ssp. occur in pairs and chains of cocci and are often ellipsoidal. It is difficult to differentiateLeuconostoc from lactococci. Both are catalase negative and form chains of coccal to oval shaped cells. However, a useful method by which one can fairly distinguish these two is by growing them in litmus milk. Lactococci reduce litmus before coagulation, whereas leuconostocs do not. Fundamentally, leu- conostocs are heterofermentative and produce d-lactate, and with the exception of Leuconostoc lactis, these show no change in litmus milk 88 FERMENTED MILK AND DAIRY PRODUCTS (Garvie 1960). These also do not hydrolyze arginine and require vari- ous B vitamins for growth. Leuconostoc ssp. grows at 10°C but not at 40°C, and can ferment lactose, galactose, fructose, and ribose. The end product produced includes diacetyl, carbon dioxide, and acetoin from citrate. The species that are widely used as dairy starters includeLeu. mesenteroides subsp. cremoris (previously referred as Leu. cremoris or Leu. citrovorum), Leu. mesenteroides subsp. mesenteroides, Leu. mesen- teroides subsp. dextranicum, and Leu. lactis. They are primarily used as flavor producers in butter, cheeses, and flavored milks. These cultures are often used in combination with other fast growing lactic cultures. Streptococcus thermophilus used as a starter is fairly close with Str. salivarius, a common inhabitant