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III. MILK DERIVATIVES
7 Fermented Milks Past, Present, and Future M. Kroger, J. A. Kurmann, and I. L. Rasic Milk is the most important foodstuff for a mammal and has always been the first food of the newborn. One could argue that the deliberate souring or fermentation of milk was one of the key achievements that nurtured mankind to grow and develop into a productive and preeminent species. Had fermented milk been considered spoiled and inedible and thus not have entered the human diet in the thousands of years to come, human development would have taken an entirely different course. Although there is no perfect food, milk is the most nearly perfect food known. At some stage in the course of human evolution it was recognized that the milk of other mammals was equally satisfying in meeting physiological demands for moisture, energy, and nutrients. Milk from eight species of domesticated mammals (cow, buffalo, sheep, goat, horse, camel, yak, and zebu) has been used to make traditional fermented milk products throughout the world. From a biological standpoint, fermented milks are characterized by the accumulation of microbial metabolic products. It was realized very early that such microbial metabolites as lactic acid, ethyl alcohol, and dozens of other chemicals collectively called flavor substances, were not altogether unpleasant and even contributed to overall preservative action. CLASSIFICATIONS Despite the long historical record and worldwide distribution of fermented milks, few people know more than five or 10 of the several hundred specific products that could be described. Even current food science and dairy technology textbooks fail to do the subject justice. 6
62 FERMENTED FOODS For example, the latest (fourth) edition of Food Microbiology (1) covers fermented dairy products in only two pages. The textbook used in the Pennsylvania State University dairy technology course is The Science of Providing Milk for Man (2). Cultured and acidified milk products occupy 10 pages, and cultured buttermilk, sour cream, yogurt, acidophilus milk, and ymer and lactofil are given only subchapter status. Koumiss and kefir are merely mentioned as being popular in Eastern Europe. Cheese and Fermented Milk Foods (3) is somewhat more comprehensive, but it deals mainly with practical concerns and primarily with cheese. By far the best compilations on fermented milks have been and are being published as documents of the International Dairy Federation (4,51. One chapter of the latter lists some 80 fermented milks, including both traditional and nontraditional products. A soon-to-be-published encyclopedia of fermented fresh milk products (6) describes some 200 traditional fermented milks and several hundred nontraditional ones. Traditional and Nontraditional The most fundamental division of fermented milk products is into traditional and nontraditional types. Traditional fermented milk prod- ucts have a long history and are known and made all over the world whenever milk animals were kept. Their production was a crude art. It was not until the days of Pasteur about 100 years ago that the microbiology underlying fermentations was revealed. In contrast, nontraditional fermented milk products are recently developed. They are based on known scientific principles; their microbial cultures are known; and their quality can be optimized. This is not the case with traditional products made with ill-defined, empirical cultures where you have to take what you get out of the fermentation. Yogurt is both a traditional and a nontraditional product the latter being represented by ever-changing varieties. Medium and Procedure Classification by technology differentiates between fermented milks and fermented products not based directly on milk. It is obvious that products other than fresh milk can serve as the fermentation medium or substrate, such as cream, whey, buttermilk, and dry milk solids. It is also possible to further manipulate or change the curd recovered after coagulation.
FERMENTED MILKS 63 Further Processing Neither law nor taboo forbids experimentation with fermented milks. Numerous products are known that are mixtures of milk and other foodstuffs and that have been subjected to fermentation. These include fermented milk-vegetable products, fermented milk-meat extract mix- tures, and fermented milk-fishmeal hydrolyzate mixtures. Conse- quently, we find societies that have utilized specific plants, meat extracts, or fishmeal hydrolyzates to enhance their nutritional status and the flavor and variety of their cuisine. Pharmaceutical preparations are unique in that they emphasize microorganisms only instead of milk nutrients or product flavor. The subject of probiotics (a word coined in 1974) will undoubtedly emerge as a major field of study. We see it in animal science now where some work is being done to get specific bacteria implanted or colonized in the gastrointestinal tract of animals, obviously in the interest of animal health and improvement of farm animal food production. So-called health food stores make available preparations that provide people with specific doses of bacteria, such as Lactobacillus acidophilus, commonly found in some fermented milk products. The subjects of health and probiotics, as well as myth and faddism, are beyond the scope of this paper. End Uses Traditionally, fermented milk products have been consumed as beverages, as meal components, or as ingredients in cookery. As social patterns have changed, however, meal eaters have become snackers and grazers. Furthermore, food technologists and food innovators have created a multitude of new products for the shelves of modern supermarkets. Most of the developments have been in the dessert and confectionery category. Microbial Actions Homemade fermented milk products, especially in nomadic or village environments, are still occasionally made by spontaneous fermentation, but most likely they are made by the use of an empirical culture. In other words, the inoculum is obtained from a previous production and its microbial identity is unknown. The bacteria utilized are either mesophiles or thermophiles, terms
64 FERMENTED FOODS indicating optimum bacterial growth temperatures, roughly 70° and 100°F (22° and 38°C), respectively. More specific and important is the bacterial species present. A fermented milk is mainly characterized by its sensory properties, and the sensory properties, such as taste, odor, and viscosity, are the direct results of specific bacterial action. The current names of microorganisms recognized in fermented milks are listed in Table 1. TABLE 1 Current Names of Microorganisms in Fermented Milks Current Name Number of Former Designations and Synonyms Genus Lactobacillus L. delbrueckii L. delbrueckii subsp. Iactis L. delbrueckii subsp. bulgaricus L. acidophilus L. helveticus L. cased L. brevis L. fermentum L. kefir Genus Leuconostoc L. mesenteroides L. mesenteroides subsp. dextranicum L. mesenteroides subsp. cremoris L. Iactis Genus Pediococcus P. pentosaceous P. acidilactici Genus Propionibacterium P. freudenreichii subsp. shermanii P. freudenreichii subsp. freudenreichii Genus Streptococcus S. Iactis S. Iactis subsp. diacetylactis S. Iactis subsp. cremoris S. thermophilus Genus Bifidobacterium B. bifidum B. Iongum' B. infantis B. breve Genus Acetobacter A. aceti Yeasts Torulaspora delbrueckii Kluyveromyces marxianus subsp. marxianus Kluyveromyces marxianus subsp. bulgaricus Candida kefyr Saccharomyces cerevisiae 8 10 8 2 7 6 29 10 4 2 6 3 1 3 1 31 13 1 3 2 11 3 1 3 o ~In an earlier edition of Bergey's Manual, B. Iongum was listed as having two subspecies: B. Iongum subsp. Iongum and B. Iongum subsp. animalist The latter was translocated in the new Bergey's into two species: B. animalis and B. pseudolongum.
FERMENTED MILKS 65 With regard to bacterial species, a number of products have evolved that are now characterized by the presence of specific organisms. Modern yogurt is now defined by the regulations of many governments to be made from and to contain only Lactobacillus bulgaricus and Streptococcus thermophilus. But there are no hard-and-fast rules, and, theoretically, any combination of organisms could be utilized to make a fermented milk product. The ultimate test is palatability. Frankly, there is still much confusion over the microbial identity of most of the known traditional fermented milk products in the world. Some have never been studied in depth. Some are very variable from batch to batch. Only yogurt has been given a proper definition by regulatory authorities in some countries. All other products are only loosely defined. RESEARCH Milk has always turned sour, but at some point in human history artisans deliberately caused milk to coagulate. However, the scientific principles behind the phenomenon of milk fermentation have remained unrevealed until recent decades. We had to wait for the pioneers in microbiology to lead the way. Louis Pasteur (1822-1895) studied alcohol fermentation; Heinrich Anton DeBary (1831-1888) studied the infection of plants by fungi; and Robert Koch (1843-1910) studied human disease caused by bacteria. It was Elie Metchnikoff (1845-1916) who, while working at the Pasteur Institute in Paris, moved milk fermentations and the unheard-of subject of probiotics into the limelight. In 1908 he shared the Nobel Prize in Physiology and Medicine. Metchnikoff developed a theory that lactic acid bacteria in the digestive tract could, by preventing putrefaction, prolong life. His book, The Prolongation of Life (7), was translated into English in 1907 (reviewed in Harper's Weekly, February 8, 1908) and received much exposure worldwide. In a way it made Metchnikoff the godfather to everyone who, to this day, believes in the therapeutic value of fermented milk. World War I put a damper on this type of human diet/health preoccupation. In the United States, it was 1921 before an American figure emerged who should be given much more credit, Leo Frederick Rettger. Rettger was a professor of bacteriology at Yale for most of his career. Two of his publications are A Treatise on the Transformation of the Intestinal Flora with Special Reference to the Implantation of Bacillus Acidophilus (8) and Lactobacillus Acidophilus and Its Therapeutic Application (91. On the practical front at that time, A. D. Burke, head of the Dairy
66 FERMENTED FOODS Department of Alabama Polytechnic Institute, published Practical Manufacture of Cultured Milks and Kindred Products (101. Burke's book is, according to the subtitle, "a complete and practical treatise on the manufacture of commercial cultured buttermilks of all types- lactic, Bulgarian, acidophilus, kefir, kumiss, yogurt." It is also a practical treatise on commercial casein, cottage cheese, cream cheese, and commercial sour cream, with information on dried, condensed, and fruit-flavored buttermilk. Then came World War II, and until about 1950 very little research and development was seen on fermented milks. Since then increasing attention has been paid to fermented milk products worldwide. The American Cultured Dairy Products Institute was created in the United States in 1965. Several good books have been published, and scientific publications on the subject are proliferating. Manufacturers, research- ers, and the public are experimenting with cultured dairy products in North America and not only with yogurt but with other products as well. Kefir has been available in Los Angeles for more than a decade. In 1985 a New Jersey corporation began producing kefir for the East Coast, and in 1987 several major grocery chains began selling leben. The future of fermented milk in North America and elsewhere will undoubtedly be exciting and complex. REFERENCES 1. Frazier, W. C., and D. C. Westhoff. 1987. Food Microbiology. 4th ed. New York: McGraw-Hill Book Co. 2. Campbell, J. R., and R. T. Marshall. 1975. The Science of Providing Milk for Man. New York: McGraw-Hill Book Co. 3. Kosikowski, F. V. 1977. Cheese and Fermented Milk Foods, 2nd ed. Ann Arbor, Mich.: Edwards Brothers, Inc. 4. International Dairy Federation. 1984. Fermented Milks. Docu- ment 179, International Dairy Federation, Brussels, Belgium. 5. International Dairy Federation. 1989. Monograph on Fermented Milks: Science and Technology. International Dairy Federation, Brus- sels, Belgium. 6. Kurmann, J. A., J. L. Rasic, and M. Kroger. 1990. Encyclopedia of Fermented Fresh Milk Products. New York: Van Nostrand Reinhold. 7. Metchnikoff, E. 1906. The Prolongation of Life. New York: G. P. Putnam and Sons. 8. Rettger, L. F., and H. A. Cheplin. 1921. A Treatise on the Transformation of the Intestinal Flora with Special Reference to the Implantation of Bacillus Acidophilus. New Haven, Conn.: Yale University Press.
FERMENTED MILKS 67 9. Rettger, L. F., M. N. Levy, L. Weinstein, and J. E. Weiss. 1935. Lactobacillus Acidophilus and Its Therapeutic Application. New Haven, Conn.: Yale University Press. 10. Burke, A. D. 1938. Practical Manufacture of Cultured Milks and Kindred Products. Milwaukee, Wis.: The Olsen Publishing Co.
8 Lactobacillus GG Fermented Whey and Human Health Seppo SaIminen and Karl Salminen Traditionally, whey has been a troublesome waste product at cheese factories. New uses have now been developed for cheese whey to utilize the whey nutrients, including protein and carbohydrates. Fermented milk products have been reported to have an important role in the treatment of infant diarrhea in malnourished children (1,21. More recently, Isolauri and co-workers (3) have shown in a double- blind controlled trial that Lactobacillus GG bacteria promote recovery from acute diarrhea in children. These results suggest that whey-based products may be used in this application. A process for manufacturing a fermented flavored whey drink has been developed that combines the nutritional properties of whey and the health benefits of Lactobacillus strain GG. The objective has been to improve the utilization of whey through use of a scientifically selected Lactobacillus strain with proven health benefits. For this purpose, demineralized lactose-hydrolyzed whey concentrate has been fermented with Lactobacillus GG. Whey and lactic acid bacteria have thus been combined to provide a wholesome and nutritious beverage. WHEY HYDROLYSIS PROCESS Important steps in whey processing are the hydrolysis of lactose and demineralization to remove excess salt. A continuous whey hydrolysis process has been developed using immobilized 13-galactosidase enzyme. This process is more economical than batch hydrolysis. Lactose hydrolysis is important for lactose-intolerant populations and for malnourished children. Malnourished children may experience worsen- ing of acute diarrhea when lactose is given during treatment (1J. Salt removal can be completed using an ion exchange process. After 68
FERMENTED WHEY 69 concentration to 60 percent dry matter, a hydrolyzed demineralized whey syrup is obtained that has a good shelf life and a pleasant rich taste. LACTOBACILLUS GG La ctoba cillus cased strain GG (La ctoba cillus GG) is a new La ctoba- cillus strain that is of human origin and has been shown to colonize the intestinal tract (41. This strain was originally isolated from a healthy human volunteer based on its ability to tolerate acid and bile, to produce an antimicrobial substance, and to adhere to human intestinal cells (5,6~. It is among the first strains with clinically proven health benefits in various intestinal disorders in adults, children, and infants. The most important evidence of its health benefits comes from studies of infant diarrhea. Isolauri and co-workers (3) published the first study on infant rotavirus diarrhea in which the duration of diarrhea was reduced by 50 percent through the use of either freeze-dried Lactobacil- lus GG or Lactobacillus GG fermented milk products. LACTOBACILLUS GG FERMENTED WHEY DRINK A new fermented flavored whey drink has been manufactured from demineralized lactose-hydrolyzed whey concentrate using Lactobacil- lus GG. It is a low-lactose product that contains no fat and is lightly sweetened with fructose. It has special sensory characteristics- smooth texture, mild acidity, and the rich taste from whey. Fruit juices or fruit flavoring have been used to modify the Havor to appeal to different people. Fermentation of whey may also influence lactose content when suitable bacteria are used. Additionally, whey proteins may undergo slight changes to ease their digestibility. The end product may offer alternatives for people not currently attracted to fermented milks. CONCLUSIONS This development in whey processing offers new alternatives for utilizing cheese by-products and applies new technologies to nutri- tionally important products. Combining whey processing with lactoba- cilli that have been obtained using new selection methods may prove to be beneficial to human health in many intestinal imbalances. It may also offer possibilities in utilizing new technologies in food production
70 FERMENTED FOODS in different cultures and in providing nutritionally attractive foods from low-value by-products. REFERENCES 1. Bhan, M. K., S. Sazawal, S. Bhatnagar, B. L. Jailkhani, and N. Arora. 1989. Efficacy of yoghurt in comparison to milk in malnourished children with acute diarrhea. Pp. 229-232 in: Les tails fermenter: Actualite de la recherche. U.K. John Libbey Eurotext Ltd. 2. Boudraa, G., M. Touhami, P. Pochard, R. Soltana, J. Y. Mary, and J. F. Desjeux. 1990. Effect of feeding yogurt versus milk in children with persistent diarrhea. Journal of Pediatric Gastroenterology and Nutrition 11:409-512. 3. Isolauri, E., M. Juntunen, T. Rautanen, P. Sillanaukee, and T. Koivula. 1991. A human Lactobacillus strain (Lactobacillus cased strain GG) promotes recovery from acute diarrhea in children. Pediat- rics 88:90-97. 4. Saxelin, M., S. Elo, S. Salminen, and H. Vapaatalo. 1990. Dose response colonisation of faeces after oral administration of Lactobacillus cased strain GG. Microbial Ecology 4:209-214. 5. Silva, M., N. Jacobus, C. F. Deneke, and S. Gorbach. 1987. Antimicrobial substance from a human Lactobacillus strain. Antimicro- bial Agents and Chemotherapy 31:1231-1233. 6. Elo, S., M. Saxelin, and S. Salminen. 1991. Attachment of Lactobacillus cased strain GG to human colon carcinoma cell line Caco-2: Comparison with other dairy strains. Letters on Applied Microbiology 13: 15~156. 7. Siitonen, S., H. Vapaatalo, S. Salminen, A. Gordin, M. Saxelin, R. Wikberg, and A.M. Kirkkola. 1990. Effect of Lactobacillus GG yoghurt in prevention of antibiotic associated diarrhoea. Annales Medicinae 22:57-60.
9 The Microbiology of Ethiopian Ayib Mogessie Ashenafi In Ethiopia, smallholder milk processing is based on sour milk resulting from high ambient temperatures, while meeting consumers' preferences and improving keeping quality (1~. Ayib, a traditional Ethiopian cottage cheese, is a popular milk product consumed by the various ethnic groups of the country. It is made from sour milk after the butter is removed by churning. Traditional ayib making has been described by O'Mahony (11. Milk for churning is accumulated in a clay pot over several days. This is kept in a warm place (about 30°C) for 24 to 48 hours to sour spontaneously. Churning of the sour milk is carried out by slowly shaking the contents of the pot until the butter is separated. The butter is then removed from the churn and kneaded with water. The casein and some of the unrecovered fat in skim milk can be heat precipitated to a cottage cheese known as ayib. The defatted milk is heated to about 50°C until a distinct curd forms. It is then allowed to cool gradually, and the curd is ladled out or filtered through a muslin cloth. Temperature can be varied between 40° and 70°C without markedly affecting product composition and yield. Heat treatment does not appear to affect yield but gives the product a cooked flavor. AYIB CHARACTERISTICS Ayib comprises about 79 percent water, 15 percent protein, 2 percent fat, 1 percent ash, and 3 percent soluble milk constituents. The yield should be about 1 kilograms of ayib from 8 liters of milk (1~. The safety of cheese with respect to food-borne diseases is of great concern around the world. This is especially true in developing countries, where production of milk and various dairy products often takes place under unsanitary conditions. Since there was no published 71
72 FERMENTED FOODS information on the microbiology of milk and milk products in Ethiopia, a study was carried out in our laboratory to evaluate the microbiological quality of ayib as available to the consumer (21. One hundred samples of ayib were purchased at the Awassa market over 10 weeks. Since Awassa is an open-air market, ayib was generally handled at ambient temperatures (about 25° to 27°C during the study period). Samples were microbiologically analyzed within two hours of purchase. Standard microbiological procedures were followed to determine the counts of aerobic mesophilic microorganisms, psychrotrophs, yeasts and molds, coliforms, bacterial spores, enterococci, Bacillus cereus, Listeria monocytogenes, staphylococci, and lactic acid bacteria. The pH of the samples was also measured. Ayib samples showed high numbers of mesophilic bacteria, entero- cocci, and yeasts (Table 1~. More than 90 percent of the samples had aerobic mesophilic counts of 108 cfu/g (colony forming units) or higher; more than 75 percent of the samples had yeast counts of 107 cfu/g or higher, and over 85 percent contained enterococci in numbers of 107 cfu/g or higher. The majority of the samples had mold and lactic acid bacteria counts of 105 cfu/g or higher, spore-formers of about 104, and psychrotrophs of about 106 cfu/g (Table 14. Over 32 percent had coliform counts of more than 109/g, and about 27 percent contained fecal coliform loads of more than 102/g. Listeria spp. were not detected from the samples. B. cereus and S. aureus were isolated in 63 percent and 23 percent of the samples, respectively, but at very low numbers (102 to 103 cfu/g). About 40 percent of the ayib samples had pH values of less than 3.7, and 60 percent had values of 3.7to4.6. Most of the production of milk and various milk products in Ethiopia is generally a household process that usually takes place under unsanitary conditions. However, despite its high moisture content, the low pH of ayib may prevent the further proliferation of various microorganisms. Yeasts, which can grow at lower pH values, may affect the Havor and keeping quality of ayib. In another study (3), proteolytic yeasts made up 47 percent of the total yeast isolates and all isolates showed lipolytic activities. Since traditional ayib making involves removal of fat from the sour milk, ayib contains only about TABLE 1 Frequency Distribution (Percent) of Aerobic Mesophiiic Organisms, Yeasts, and Enterococci in Ayib Samples Microorganisms <107 107 108 >108 Aerobic mesophils Yeasts Enterococci 8 25 70 13 45 65 27 5 37 aColony forming units
ETHIOPIAN A YIB 73 1 percent fat, and thus the lipolytic isolates may not play an important role in affecting the havor or keeping quality of ayib. Although proteolytic yeasts are important in cheese types that require ripening, their presence in a fresh product such as ayib is undesirable. The findings in the previous studies indicated that ayib purchased from local markets was highly contaminated with various microorgan- isms. It was not known, however, whether these microorganisms were survivors of the heat treatment process or were postheating contaminants. MODIFIED PROCESS Another study was therefore conducted to determine the effect of cooking temperatures used in various parts of Ethiopia on the microbiological quality of the finished product and to recommend cooking temperatures that can decrease or destroy most microorgan- isms (41. Ayib was made in the laboratory using traditional methods. Pooled raw milk was allowed to sour naturally at room temperature. After removal of the fat by churning, the casein in the sour skimmed milk was heat precipitated at 40°, 50°, 60°, and 70°C in a water bath, and the curd was recovered by filtering through sterile cheese cloth. Microbial analysis of raw milk, sour milk, and ayib indicated that heat treatment of the curd was effective at higher temperatures (Table 2~. At these temperatures the time required for casein precipitation was also low. Heating the curd at 70°C for 55 minutes at pH 4 destroyed most of the microorganisms. The low pH also inhibited the proliferation of most surviving microorganisms. The high degree of contamination of market ayib could be due to either low curd cooking temperatures or addition of various plant materials to the finished product to give it desirable flavor, the packaging of ayib with Musa leaves, or other unhygienic handling practices. Thus, heat treatment of curd at 70°C and an appropriate handling of the product could result in a less contaminated and safer ayib. TABLE 2 Frequency Distribution (Percent) of Lactic Acid Bacteria, Bacterial Spores, Molds, and Psychrotrophs in Ayib Samples acfu/g Microorganisms 103 104 105 1o6 Lactic acid bacteria 14 33 13 40 Bacterial spores 4 68 24 4 Molds 12 25 35 23 Psychrotrophs 13 20 67 aColony forming units
74 REFERENCES FERMENTED FOODS 1. O'Mahony, F. 1988. Rural Dairy Technology Experiences in Ethiopia. ILCA Manual No. 4, International Livestock Center for Africa, Addis Ababa. 2. Ashenafi, M. 1990. Microbiological quality of ayib, a traditional Ethiopian cottage cheese. International Journal of Food Microbiology 10:263-268. 3. Ashenafi, M. i989. Proteolytic, lipolytic and fermentative proper- ties of yeasts isolate from ayib, a traditional Ethiopian cottage cheese. SINET: Ethiopian Journal of Science 12:131-139. 4. Ashenafi, M. 1990. Effect of curd-cooking temperatures on the microbiological quality of ayib, a traditional Ethiopian cottage cheese. World Journal of Microbiology and Biotechnology 6:159-162.
10 Moroccan Traditional Fermented Dairy Proclucts Abed Hamama In Morocco 20 to 30 percent of all milk produced is still processed by private individuals. These dairy shops and farmers manufacture traditional Moroccan dairy products such as Iben and raib (fermented milks), zabda (farm butter), and jben (fresh cheese). These products are made from raw milk, and their physical properties are similar to those of commercially produced buttermilk, yogurt, butter, and fresh cheese. Although they are usually made from cow's milk, milk from sheep, goats, and camels also can be used. These products are very popular in Morocco mainly because of their refreshing qualities. Basically, all these traditional dairy products are prepared by simply allowing the raw milk to ferment spontaneously at room temperature (15° to 25°C) for 1 to 3 days depending on the season. The coagulated milk is called raib. It can be consumed as such or churned in a clay jar to separate the liquid phase (Iben) from fat (zabda). Jben is prepared by placing the coagulated milk in a cloth at room temperature and draining the whey. Salt is added to jben made in northern Morocco. COMPOSITION AND MICROBIOLOGICAL CHARACTERISTICS The Moroccan traditional fermented dairy products have been investigated (1-4) for their composition (Table 1) and their microbiology (Table 2~. Data in these tables are average results only. In fact, a high level of variability for all the parameters was seen among samples of the same product. This heterogeneity is a consequence of the lack of standard procedures for preparation of these products. Despite the acidic nature of these products (pH 4.0 to 4.5), they showed high counts of indicator microorganisms (e.g., coliforms, enterococci). This probably reflects poor hygienic conditions in the 7s
76 FERMENTED FOODS TABLE 1 Average Physical-Chemical Composition of Moroccan Traditional Dairy Products Composition Lben(4) Jben(3) Raib(3) Zabda(1 ) pH 4.25 4.1 4.2 4.5 % lactic acid 0.81 1.04 0.62 0.77 % total solids 6.5 37.5 10.7 76.7 % fat 0.9 16.47 2.22 73.7 % protein 2.5 15.8 3.1 1.8 % lactose 2.7 4.1 4.2 1.2 % chlorides 0.17 0.5 0.17 ND % ash ND 1.26 0.54 ND ND, not determined. preparation of these products and/or poor bacteriological quality of the raw milk used for their manufacture. In addition to the indicator microorganisms, pathogens such as Salmonella sp., Yersinia enterocoli- tica, Listeria monocytogenes, and enterotoxigenic Staphylococcus aureus have been recovered mainly from samples of Iben and jben. Although there are no epidemiological reports of outbreaks linking Moroccan traditional dairy products with diseases caused by these pathogens, their presence in these products indicates potential health hazards for consumers. Therefore, there is need to implement correc- tive procedures to eliminate or reduce this risk. This can be achieved by the use of heat-treated milk instead of raw milk and through the use of selected starter cultures for preparation of these products. OBJECTIVES The application of modern technology to Moroccan traditional dairy products aims to assure the following: ~ Large-scale production of these products year-round by replacing raw milk with dry milk and butter oil. This will solve the problem of seasonality in Moroccan milk production. · Production of dairy products with standardized chemical and microbiological composition so that their quality can be more easily controlled and standards for each product can be established. Streptococci Lactobaciiii Leuconostocs T. conforms F. coliforms Enterococci Fungi Total flora TABLE 2 Average Microbiological Counts of Moroccan Traditional Dairy Products (cfu/g or ml) _ . . . Microorganism Lben(4) Jben(3) Raib(3) Zabda(2) 5.0x 106 2.4x 105 1.8x 104 6.5 x 1 04 2.1 x 104 8.6x 104 ND 4.6x 107 7.6x 108 1.0x 103 1.7x 105 5.0x 104 1.0x 103 1.0x 105 8.5x 102 2.9x 109 5.1 x 108 3.2x 108 2.6x 108 4.3 x 105 2.7x 104 2.4x 105 2.3x 106 8.2x 108 1.4x 108 2.6x 106 2.8x 106 1.7x 105 4.2x 103 2.2x 104 2.3x 104 3.5x 108 ND: not determined.
MOROCCAN DAIRY PRODUCTS 77 · Elimination of massive contamination of these products and reduction of health hazards associated with these contaminations by using heat-treated milk and improving the sanitation and fermentation conditions. · Adoption of simple and standardized processes for the preparation of these products that could be easily applied in the dairy industry. PRELIMINARY STUDY Preparation of traditional dairy products using improved technologi- cal processes requires, for each type of product, determination of the characteristics that constitute an excellent-quality product. For this purpose, samples of each product were evaluated by a gustatory panel. The best products were then analyzed to determine their physical characteristics, chemical composition, and microbiological profiles. The objective of the study was to assess the censorial and compositional parameters (e.g., acidity, total solids) that the improved product should have to be acceptable to consumers. Selection of Starters Microbiological analysis of the different traditional fermented dairy products showed that an important proportion of their microflora was represented by lactic acid bacteria. Lactic streptococci were predominant in Iben, raid, and zabda, while streptococci, lactobacilli, and leuconostocs were found in jben at almost the same average levels (108 cfu/g or ml) (colony forming units). From each product isolates from the predominant lactic flora were identified using biochemical tests. The principal species found in Iben, raib, and zabda were Streptococcus lactis, and S. diacetylactis, while 5. Iactis, Lactobacillus cased casei, and Leuconostoc lactis were the main species recovered ~ r In Joen. Owing to the nature of traditional Moroccan dairy products (fresh fermented products), the major criterion considered for selection of lactic starters was their acid production ability at different incubation temperatures. Production by lactic strains of certain substances contrib- uting to the overall aroma of these products also was taken into account. Thus, several lactic strains were retained to be used for preparating improved products. Manufacture of Traditional Dairy Products from Heat-Treated Milk To prepare each type of product, a simple and economically feasible technology, which industrial dairy plants could easily adopt, was used.
78 FERMENTED FOODS The improved processes proposed for use with raib (fermented milk) and jben (fresh cheese) are as follows: · Manufacture of raib: Reconstitution of dry milk to 90 percent water and 10 percent solids. Pasteurization at 63°C for 30 minutes. Addition of calcium chloride and storage at 7°C for 10 hours. Addition of fresh pasteurized milk (60 percent of the total volume). Inoculation (S. Iactis, S. diacetylactis @ 3.0 percent). Distribution into plastic containers and incubation at 30°C for 3 to 4 hours. Refrigeration at 4° to 6°C. · Manufacture of jben: Reconstitution and pasteurization of powdered milk. Addition of calcium chloride and storage at 7°C for 10 hours. Addition of fresh pasteurized milk (60 percent of the total volume). Inoculation (S. Iactis, S. diacetylactis, L. cased cased (it? 3.0 percent). Storage of inoculated milk at 20° to 25°C until 0.25 percent lactic acid is formed. Addition of rennet (5 to 10 milliliters/100 liters). Fermentation at 20° to 25°C until 0.60 percent lactic acid is formed. Curd cutting and whey draining. Unmolding when titratable acidity reaches 0.9 percent lactic acid and total solids content reaches 28 to 30 percent. Cutting of cheese into suitable pieces (150 grams/piece). Surface dry salting, if desired (1 percent salt) and wrapping. RESULTS This study is still in progress. The final results regarding censorial quality, chemical composition, and microbiological quality of tradi- tional dairy products made with the improved technology are not yet available. Nonetheless, preliminary data obtained for raib and jben are very encouraging: ~ Sensorial quality: Laboratory samples of improved raib and jben gave similar or even higher sensory scores than market samples. The characteristics considered in this evaluation are mainly acidity, texture, and aroma. o Chemical composition; Because standard procedures were used for making raib and jben, the samples obtained had uniform composi-
MOROCCAN DAIRY PRODUCTS 79 lions. This information will be useful in establishing standards for these products. · Microbiological quality: The use of heat-treated milk in the manufacture of raib and jben had a profound effect on the microbiologi- cal quality of the products. The improved products were free from pathogens such as S. aureus, Salmonella, L. monocytogenis, and Y. enterocolitica. They were either free of or contained very few coliforms (<10 cfu/g). Their microbiological quality was substantially improved compared with currently marketed traditional products. CONCLUSIONS Although data on all traditional dairy products are not yet available, information on the quality of laboratory-made raib and jben indicates that the use of modern technology in their manufacture has enhanced their bacteriological quality and reduced the risks of dairy-borne infections. This new technology has also begun to establish standards for these products. In addition, the manufacture of traditional dairy products at an industrial scale will increase the production of these products and assure better distribution and marketing. On the other hand, the use of dry milk, which is more economical than raw milk for preparing products such as raib and jben, has the advantage of being available any time of the year. This is very important in Morocco, where seasonal variabilities in milk production are a major problem for the dairy industry. Furthermore, the availability of dairy products that are rich in nutrients (e.g., proteins, fat) at a modest price and throughout the year will contribute to reduced malnutrition especially among children in rural areas. REFERENCES 1. El Marrakchi, A.M., M. Berrada, M. Chahboun, and M Benbou- hou. 1986. Etude chimique du smen marocain. Le Lait 66:117-133. 2. H-amama, A. 1989. Studies on the hygienic quality of certain Moroccan dairy products. Ph.D. thesis, University of Minnesota. 3. Hamama, A., and M. Bayi. 1991. Composition and microbiological profile of two Moroccan traditional dairy products: raib and jben. Journal of the Society of Dairy Technology. 4. Tantaoui Elaraki, A., M. Berrada, A. El Marrakchi, and A. Berramou. 1983. Etude du Iben marocain. Le Lait 63:230-245.
11 Fermented Milk Products in Zimbabwe Sara Feresu Fermentation is the oldest means of preserving milk (11. Originally, unpasteurized milk was left to ferment naturally, and fermentation involved microorganisms present in the raw milk and surrounding air. With the development of modern technologies, specific lactic-acid- producing microorganisms are now introduced to carry out fermenta- tions under controlled conditions. In this way fermented products of superior nutritional, physical, chemical, and sanitary qualities are produced. In Zimbabwe one finds the modern fermented products such as yogurt and different types of cheese. The rural population, however, still ferment their milk traditionally. Fresh unpasteurized cow's milk is allowed to stand, at ambient temperature, in an earthenware pot loosely covered by a plate. This allows microorganisms inherent in the milk, from the pot, and from the surrounding air to ferment the milk. Fermentation takes 1 to 2 days depending on the ambient temperature (20 to 25°C). The fermented milk is not refrigerated and has an estimated shelf life of 3 days at ambient temperature. In response to the urban population's desire for fermented milk, the Zimbabwe Dairy Marketing Board produces a fermented milk called Lacto on an industrial scale. Milk is standardized, pasteurized at 92°C for 20 minutes, cooled to 22°C, and inoculated with 1.2 percent of an imported mesophilic starter culture, similar to that used to produce "filmjolk," a Scandinavian fermented milk. The milk is immediately packaged into sachets, left to ferment at ambient temperature for 18 hours, and stored at 5°C ready for the market. The shelf life of refrigerated Lacto is 7 days. Our studies have compared traditionally fermented milk with Lacto. We included traditionally fermented pasteurized milk, since substitu- tion of unpasteurized with pasteurized milk might be an alternative for 80
FERMENTED MILK PRODUCTS 81 upgrading hygienic standards. The initial study was concerned with the effects of pasteurization and of the container used during fermenta- tion on the total microbial cell counts, the counts of lactic acid bacteria, the amount of lactic acid produced, and the acceptability of the fermented milk by a panel (2~. We have characterized 10 predominant lactic acid bacterial isolates from traditionally fermented milk and four isolates from Lacto (3~. We have also carried out studies to determine the fate of pathogenic and nonpathogenic Escherichia cold during fermentation of Lacto and traditionally fermented pasteurized and unpasteurized milk. The sur- vival of E. cold was also tracked during storage of the fermented products at ambient (20°C) and refrigeration temperatures (5°C) for 4 days (4), since it is possible that pathogenic bacteria may gain access to these products before, during, and after fermentation. In the case of traditionally fermented milk, coliform contamination from cattle dung or from the milker's hands is possible. Contamination with coliforms during Lacto production can occur through bulk starter cultures and from inadequately sanitized equipment. TRADITIONALLY FERMENTED MILK AND LACTO In an earlier study (2) unpasteurized milk and pasteurized milk were fermented in clean nonsterile earthenware pots and sterile glass containers. At the same time, Lacto was fermented in plastic sachets and sterile glass containers. Bacterial counts and lactic acid levels were determined. The acceptability of the fermented milks was ranked by 11 panelists. Comparisons of all parameters were made after 24 and 48 hours of fermentation, when Lacto and traditionally fermented milk are likely to be consumed. The numbers of lactic acid bacteria, lactic acid production, and acceptability were always higher for unpasteurized than pasteurized traditionally fermented milk irrespective of the container used. Earthenware pots are better containers for traditional fermentation of milk. This is because earthenware pots have micropores in their walls, which, if not sterilized, may harbor lactic acid bacteria from the previous fermentation, which then act as inocula for the next fermentation. Our results suggest that earthenware pots are good containers to ferment milk in and may still have a place in milk fermentation in the home. Unpasteurized milk fermented traditionally in either container was significantly more acceptable to the panel than Lacto, although the products were similar in all the other parameters assessed. It was therefore impossible to explain the differences in the acceptability of
82 FERMENTED FOODS traditionally fermented milk and Lacto on the basis of this work. We suggested that the differences were probably due to the types of microorganisms involved in the fermentation of the two milk products rather than pasteurization or the container used for fermentation. Thus, we set out to isolate and characterize the lactic acid bacteria in traditionally fermented milk and Lacto. ISOLATION AND IDENTIFICATION OF LACTIC ACID BACTERIA From the previous study (3), 10 predominant morphologically different lactic acid bacteria colony types from plates inoculated with traditionally fermented milk and four morphologically different types of colonies from Lacto plates were selected and isolated into pure culture. The isolates were identified using numerical taxonomic tech- niques and reference strains. The isolates and reference strains were examined for 32 characteristics. Data were analyzed using the simple matching coefficient, and clustering was by unweighted pair group average linkage (51. All the isolates from traditionally fermented milk belonged to the genus Lactobacillus. Seven of the isolates could be identified as belonging to L. helveticus, L. plantarum, L. delbrueckii subspecies lactis (two isolates), L. cased subsp. cased (two isolates) and L. cased subsp. pseudoplantarum. Three of the isolates could only be identified as either betabacteria or streptobacteria. The four isolates from Lacto were identified as Lactococcus lactis. They could not, however, be identified to subspecies level. From this study we concluded that the differences in acceptability of traditionally fermented milk and Lacto are probably due to differ- ences in the biochemical pathways and resulting types and levels of end products produced by the different bacteria responsible for fermentation of the two products. We suggested that more work should be done to determine the particular flavors and aroma present in traditionally fermented milk that are absent in Lacto and to determine whether any of our isolates are responsible for producing these desired properties. E. COLI STRAINS IN LACTO AND TRADITIONALLY FERMENTED MILK In another study (4) the growth and survival of pathogenic and nonpathogenic strains of E. cold were determined in traditionally
FERMENTED MILK PRODUCTS 83 fermented pasteurized and unpasteurized milk and Lacto. Unpasteur- ized and pasteurized milk and freshly inoculated Lacto, together with sterile control milk, were each inoculated with two strains of pathogenic and one strain of nonpathogenic E. cold to give approximately 103 cells/ milliliter. All the milk treatments were left to ferment at ambient temperature (20°C) for 24 hours. One set of the fermented products was stored at ambient temperature, and the other set was refrigerated (5°C) for another 96 hours. Samples were taken at 24-hour intervals and tested for numbers of E. coli, pH, and percentage of lactic acid. Lacto inhibited all three E. cold strains. Two strains (one pathogenic and one nonpathogenic) could not be recovered, and the third (patho- genic) survived only in very low numbers after 24 hours of storage of Lacto at both 20° and 5°C. All three E. cold strains survived and multiplied to maximum cell numbers in the range 107 to 109/milliliter during traditional fermentation of unpasteurized milk. Cell numbers decreased to 103 to 106 and 102 to 105 during storage of the fermented product at 20° and 5°C, respectively. These results indicated that traditional methods of fermenting milk in Zimbabwe pose a potential health hazard because, if milk is contami- nated during milking or fermentation, E. coli, and possibly other enteric pathogens, are able to multiply to infective doses and retain relatively high numbers during storage of the product at both refrigeration and ambient temperatures. The results also indicated that more than acid production alone is involved in the fate of E. cold during fermentation and storage of Lacto and traditionally fermented unpasteurized milk since more E. cold survived in unpasteurized fermented milk despite similar final lactic acid and pH levels of both milk products. We suggested that, since in our earlier studies we found that different lactic acid bacteria were responsible for fermentation of the two milk products, it is likely that these organisms produce different types and quantities of other inhibitory products (antibiotics, volatile acids, hydrogen peroxide) during fermentation. Higher maximum numbers, 109 to 10'° of the three strains of E. coli, were attained during traditional fermentation of pasteurized milk. The numbers decreased to 105 to 108 and 104 to 107 during storage of the fermented product at 20° and 5°C, respectively. Under our experimental conditions there appeared to be more danger in traditionally fermenting pasteurized milk than unpasteurized milk; since less acid was produced, more E. cold multiplied and survived during fermentation and during storage of the pasteurized fermented milk. The practical relevance of this result should be interpreted with caution, since pasteurization also removes milk-borne organisms such as E. cold and Salmonella spp. and since it is unlikely that airborne recontamination of the milk by E. cold would result in initial numbers as high as 103 cells/milliliter. Thus,
84 FERMENTED FOODS use of pasteurized milk in practice may not be as inappropriate as it might appear in theory. Generally, fewer E. cold survived when the fermented milk products were stored at refrigeration than at ambient temperature. However, most people in rural areas of Zimbabwe do not have access to refrigerators. CONCLUSIONS We are currently determining the amounts of some B vitamins and of aroma and flavor compounds in traditionally fermented unpasteur- ized milk and Lacto. Preliminary results indicate that traditionally fermented milk contains more thiamine, riboflavin, pyridoxine, and folic acid than Lacto. Again, traditionally fermented unpasteurized milk is performing better than Lacto. From the work we have done so far there are two options to follow in our future studies. We know that traditionally fermented milk has similar amounts of lactic acid and a pH level similar to that of Lacto and that it might also have higher amounts of some B vitamins; however, it is not hygienically acceptable. We know some of the lactic acid strains involved in the fermentation, but we also know that in a situation where raw milk is used and fermentation is carried out under conditions where asepsis is not observed, other microorganisms, in addition to lactic acid bacteria, contribute to the production of aroma and flavor compounds. Supposing we were to develop a starter culture based mainly on members of the genus Lactobacillus, it is debatable whether we would have the same organoleptic properties in a tradition- ally fermented pasteurized milk as found in traditionally fermented unpasteurized milk. If we developed and sold this starter culture for home use in fermentation of boiled milk, it is also unlikely that poor rural people would adopt such a fermentation since it has an added cost when compared with traditional fermentation. Alternatively, we could incorporate some isolates from traditionally fermented milk into the Lacto starter culture and see whether the organoleptic properties of Lacto can be improved. Such a product would have to taste much better than traditionally fermented unpasteur- ized milk so as to entice rural populations to abandon traditional fermentation and adopt Lacto. Educational programs would have to be instituted for the public to appreciate the wisdom of spending money on buying Lacto, a hygienically safer product. At present, it is unlikely that Lacto will replace traditionally fermented milk in the foreseeable future.
FERMENTED MILK PRODUCTS REFERENCES 85 1. Robinson, R. K., and A. Y. Tamime. 1981. Microbiology of fermented milks. Pp. 245-278 in: Dairy Microbiology, Vol. 2. R. K. Robinson, (Ed.~. London: Applied Science Publishers. 2. Feresu, S., and M. I. Muzondo. 1989. Factors affecting the development of two fermented milk products in Zimbabwe. MIRCEN Journal of Applied Microbiology and Biotechnology 5:349-355. 3. Feresu, S., and M. I. Muzondo. 1990. Identification of some lactic acid bacteria from two Zimbabwean fermented milk products. World Journal of Microbiology and Biotechnology 6: 178-186. 4. Feresu, S., and PI. Nyati. 1990. Fate of pathogenic and non- pathogenic Escherichia cold strains in two fermented milk products. Journal of Applied Bacteriology 69:814-821. 5. Sneath, P. H. A., and R. R. Sokal. 1973. Numerical Taxonomy: The Principles and Practice of Numerical Classifications. San Francisco: W. H. Freeman, pp. 228-234.