Microorganisms used to manufacture fermented milk primarily include those that can ferment lactose to lactic acid and may be either of the mesophilic or thermo-philic type. Nomenclature for these organisms has evolved over the years as a greater understanding of their genetics has been acquired. Current nomenclature of selected microorganisms used to manufacture fermented milks is in Table 2. Pure strains of these organisms are readily available from commercial suppliers, but it is not uncommon, especially for small-scale manufacturers, to use product from a previous batch as culture for the next batch. In such instances, there is a potential for quality of the endproduct to vary from batch to batch because of changes in culture characteristics that may occur over repeated transfers. This is especially evident in products that normally require a combination of organisms in a specific ratio, such as rods and cocci in a 1:1 ratio for yogurt. Over repeated transfers as a mixed culture, one of the species is likely to dominate and hence alter the characteristics of the fermentation and consequently flavor and texture qualities of the product.
In addition to lactic acid producers, other types of organisms may also be employed to impart desired flavor or therapeutic properties to fermented products. Examples include organisms that produce diacetyl or acetaldehyde for flavor or small amounts of alcohol in products such as kefir. Organisms such as Bifido-
Table 2 Nomenclature of Microorganisms Used in the Manufacture of Fermented Milks
Previous name* Lactobacillus bulgaricus
Leuconostoc cremoris and Leuconostoc citrovorum Leuconostoc dextranicum
Streptococcus cremoris Streptococcus lactis subsp. diacetilactis and Streptococcus diacetilactis Streptococcus lactis subsp. lactis Streptococcus salivarius subsp. thermophi-lus
Lactobacillus delbrueckii subsp. bulgari-cus
Leuconostoc mesenteroides subsp. crem-oris
Leuconostoc mesenteroides subsp. dex-tranicum
Lactococcus lactis subsp. cremoris
Lactococcus lactis subsp. lactis (biovar. diacetylactis)
Streptococcus thermophilus a The names here reflect the most current previous names. Historically, various names have been used for these organisms. For example, Leuconostoc dextranicum was previously known as Streptococcus paracitrovorus. Such nomenclature can be found in Hammer (1928) and Swithinbank and Newman (1903). Wood and Holzapfel (1995) discuss in detail the nomenclature of lactic acid bacteria. Source: Kosikowski and Mistry (1997)
bacterium spp. and Lactobacillus acidophilus are added for therapeutic purposes. Leuconostocs are used in products such as cultured buttermilk to produce diacetyl via citrate fermentation (Vedamuthu, 1994). Some functions of organisms for specific applications in fermented milks are given in Table 3, and metabolic pathways are discussed in Chapter 7.
Legislation in some countries and codex regulations require the presence of viable organisms in yogurt. In the United States, the National Yogurt Association requires the presence of at least 10 million yogurt bacteria per gram at the time of consumption if manufacturers wish to display the ''Live and Active Cultures'' symbol on yogurt packages (Kosikowski and Mistry, 1997). Furthermore, many fermented milk products possess therapeutic properties largely because of the presence of selected viable organisms. These organisms have to be present in specified numbers to impart such therapeutic properties. Therefore, the use of proper enumeration procedures is vital. Such procedures have been developed (Dave and Shah, 1996; Frank et al., 1992; International Dairy Federation, 1997a,
Table 3 Functions and Applications of Microorganisms in Fermented Milks
Lactobacillus delbrueckii subsp. bulgaricus Acid and flavor
Lactobacillus acidophilus Lactobacillus kefir Streptococcus thermophilus Lactococcus lactis subsp. lactis (biovar. diace-tylactis)
Lactococcus lactis subsp. lactis & Lactococ-
cus lactis subsp. cremoris Leuconostoc lactis & Leuconostoc mesentero-ides subsp. dextranicum
Bifidobacterium longum Bifidobacterium bifidum Bifidobacterium breve
Acid Acid Acid
Acid and flavor
Acid and flavor
Bulgarian buttermilk, yogurt, kefir Acidophilus milk Kefir Yogurt
Sour cream, cultured buttermilk Cultured buttermilk, sour cream Cultured buttermilk, sour cream, ripened cream butter Yogurt
Source: Kosikowski and Mistry, 1997.
1997b; Lee et al., 1974; Matalon and Sandine, 1986). Lactic agar is used to enumerate lactic acid bacteria, whereas deMan, Rogosa, and Sharp (MRS) and lactobacillus agars are suitable for lactobacilli. Special consideration is given to products that are made with a combination of cultures. An example is yogurt that is manufactured with rods and cocci and sometimes also with bifidobacteria. It is important not only to enumerate but also differentiate these types of organisms. Enumeration procedures such as those that use yogurt lactic agar are recommended for differentiating between rods and cocci. On this agar, Streptococcus thermophilus colonies are small and white and Lb. delbrueckii subsp. bulgaricus colonies are large and white and have a white cloudy zone.
A critical issue in enumeration of bacteria in cultured products is the occurrence of acid injury to cells, especially during storage of the product. The pH in most fermented milk products drops to below 4.6 and causes sublethal injury to surviving lactic acid bacterial cells. Such sublethally injured cells are not able to multiply in media used in routine counting procedures but require an enriched medium which will help repair the injured cells (Andrew and Russell, 1984; Ray, 1993). Pariente et al. (1987) demonstrated that counts of heat-injured Lb. casei were underestimated when Lactobacillus selection (LBS) and Rogosa media were used for enumeration. Application of soya trypticase broth to recover injured lactobacilli has been recommended (Briceno-Graciela et al., 1995). Standard Methods for the Examination of Dairy Products (Frank et al., 1992) suggests the use of standard methods agar (SMA) for enumerating injured cells, but this agar is not selective. It can be made more selective by adding 0.02% sodium azide, which does not inhibit lactic acid bacteria but does inhibit others such as enteric bacilli. In a direct epifluorescent filter technique for differential determination of sublethally injured bacterial cells, the RNA of viable cells is stained orange by acridine orange, whereas inactive cells and DNA are stained green (Sto et al., 1986). This characteristic also is applicable to Lb. delbrueckii subsp. bulgaricus, S. thermophilus, and Lb. acidophilus. Working with cells injured by freeze drying, de Valdez et al., (1985) demonstrated that highest recovery was obtained on LAPTg agar for various lactobacilli and lactococci.
Development of adequate flavor and texture in fermented milk products requires optimal growth of culture organisms. This is readily attained with proper manufacturing conditions and handling of cultures. If a batch starter is used daily, facilities for aseptic culture transfer and maintenance of cultures should be available (Kosikowski and Mistry, 1997). Presence of substances in milk such as phages and sanitizers can inhibit cultures. Antibiotics have a static effect on bacteria, but yogurt bacteria, S. thermophilus and Lb. delbrueckii subsp. bulgaricus, are particularly sensitive (Reinbold and Reddy, 1974). Penicillin at 0.01 IU/mL of milk will inhibit these organisms, whereas mesophilic lactococci are not as sensitive. It is important, therefore, to test every batch of milk for antibiotics.
Bacteriophages of organisms used to manufacture fermented milks have been identified. Phages of mesophilic lactic acid bacteria are well known to cheese makers but have also been found in buttermilk production facilities. Moineau et al. (1996) isolated 27 different phages from 27 buttermilk plants in the United States. Although not as common as phages of mesophiles, those of thermophiles, such as yogurt bacteria, have also been reported (Kilic et al., 1996) and can arrest the fermentation. Phage control systems have been described (Kos-ikowski and Mistry, 1997) and involve culture rotation, the use of phage-inhibi-tory media (Vedamuthu, 1992), and, most important, proper sanitation at the plant. Phage-inhibitory media are usually rich in phosphates to chelate calcium. Some strains of S. thermophilus do not grow well in high-phosphate media. Chlorine as a sanitizer is very effective against phages. Sanitizers must be used with caution, however, because residual sanitizers in fermentation vats, piping, or packaging cups will also inhibit starter organisms. The latter is particularly applicable for products that are fermented in consumer cups. Sanitizers such as quaternary ammonium compounds in particular can be a problem. If a residual film of such sanitizers is left on equipment surfaces, the sanitizers are released slowly over time and inhibit culture organisms that come in contact with them (Guirguis and Hickey, 1987a; Miller and Elliker, 1951; Pearce, 1978; Valladao and San-
dine, 1994). Sensitivity is strain dependent but thermophiles are generally more sensitive than mesophilic lactococci (Guirguis and Hickey, 1987a).
Another mode of inhibition in milk is by the naturally present lactoperoxi-dase system. This system has to be activated for inhibition to occur and requires the presence of the lactoperoxidase enzyme, H2O2, and thiocyanate. Some starter bacteria used to produce fermented products, such as Lb. acidophilus and Lb. delbrueckii subsp. bulgaricus, produce H2O2 during fermentation and consequently activate the lactoperoxidase system. Guirguis and Hickey (1987b) concluded that inhibition by this system was strain dependent and that strains most affected were those that produced H2O2. S. thermophilus was not inhibited by the lactoperoxidase system.
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