“Bulgarian sour milk – a unique probiotic”,
Ch. Tchomakov, professor in Chemical Engineering Institute in Sofia
Bulgarian sour milk (BSM) is the oldest probiotic known to man. Since ancient times it has been used as food which favors human health and longevity.
Its nutritive value and health beneficial properties ware veiled by legends and myths.
Exact historical evidence for the origin of BSM is unavailable. It is accepted that it was introduced by Thracian people, the earliest inhabitants of our lands. The traces of BSM are lost about 4000 years B.C. Thracia had fertile soil, abundant vegetation and wide pastures. For Thracian people the most popular domesticated animal was sheep.
The people of ancient Thracia noticed that fermented milk preserved its quality for a longer time than fresh milk. By the addition of fermented milk to boiled fresh milk they obtained a product known as “prokish” or “kvaseno mliako”. The regular and continuous preparation of “kvaseno mliako” resulted in the natural selection of the two lactic acid bacteria of BSM - Lactobacillus delbrueckii ssp. bulgaricus / L. bulgaricus / and Streptococcus thermophilus /S. thermophilus/ . The first original BSM was made from sheep’s milk. Later on it became the basic food of Slavs and ancient Bulgarians. Herodotus mentions sheep’s milk yogurt as a gift from Thracian people /5/.
First data on BSM under the name “yogurut” appear in literature in the eighth century A.D. In the 11 th century the name of BSM is changed to “yogurt”.
The word “yogurt” is from Turkish origin. Someone consideres it to belong to the ancient Bulgarians. It means “yog” – thick and “urt” – milk, i.e. thick milk
/6/.
In the 12 th century Tchengis-Khan used BSM as basic food for his troops and as biological means for preserving meat. In Western Europe BSM is mentioned for the first time in France when a Turkish doctor cured with sheep’s yogurt the French king Francois first from incurable diarrhea, keeping him on
BSM diet. Since then the name “yogurt” was established for BSM in Western
Europe. Today the word yogurt is used to describe the fermented milk product containing L. bulgaricus and S. thermophilus , a product called in its native home
Bulgarian sour milk /BSM/
The famous Russian biologist Ilya Metchnikoff was the first who put the qualities of BSM on a scientific basis. Being a scientist of world publicity and awarded a Nobel prize, Metchnikoff put forward the hypothesis for the generation of toxic components during decomposition of proteins in the gastro-intestinal tract of humans caused by putrefactive microorganisms.
These components are absorbed from the intestinal lumen and cause deep changes in the organism such as aging alterations and early death of humans.
Metchnikoff proposed that the harmful effect of putrefactive microorganisms in human intestines could be diminished or abolished by the introduction of

lactic acid bacteria to this location. They produce lactic acid and stop the growth of putrefactive microorganisms. This hypothesis of Metchnikoff is brilliantly proved by modern research.
The theory of Metchnikoff for the influence of BSM on the organism resulting in the longevity of Bulgarians was accepted with great interest. The vision of Metchnikoff is presented as “Theory for rejuvenation of man”. In that way Metchnikoff drew the attention of the world community to the qualities of
BSM. There is no other fermented milk product so well documented as BSM.
One century after the discovery of L. bulgaricus by Stamen Grigorov we become the witnesses of the scientific revealing of the valuable biological qualities of BSM. Today the definition of BSM refers to a fermented milk product obtained with Bulgarian symbiotic starter from L. bulgaricus and S. thermophilus by an original technology without any additives. The product is prepared from cow’s, sheep’s, buffalo’s or goat’s milk. The final product contains viable bacterial cells not less than 10 8 cfu/g at the end of its shelf life period. The fermented milk product obtained with symbiotic starter from L.
bulgaricus and S. thermophilus but with additives such as stabilizers is simply called yogurt. Today we cannot imagine the dietary regimen of Bulgarians without yogurt. In Bulgaria in the form of yogurt the daily intake of viable bacterial cells amounts to 1.1 tons or about 400 tons per year. There is no other food product providing our consumer with such quantities of viable lactic acid bacteria favorable for the organism.
In 2003 according to the Codex Alimentarius Standard yogurt is prepared from cow’s milk fermented by symbiosis of L. bulgaricus and S. thermophilus only. At the end of its shelf life the product must still contain an abundance of viable bacterial cells. Fermented milks, named yogurt, obtained in some countries may contain lactic acid bacteria other that L. bulgaricus and S. thermophilus such as L. casei , L. rhamnosus or species from the genus
Bifidobacterium .
During the fermentation the microflora of BSM causes deep changes in the composition of milk. The generated metabolites have beneficial properties and health effects on the organism of the individual.
It is the intake of food that represents the most intimate contact between humans and environment. There is no other system of the organism to be entered with food by such large numbers of bacteria as the digestive system. The presence of great number of bacteria in the intestines, which in the colon may reach up to 10 12 cfu/g, shows that it is the intestinal tract of the digestive system that is in the closest contact with microorganisms. This contact deeply influences human physiology /33/.
From microbiological point of view the human intestinal tract represents an extremely complex ecosystem. The possibility of changing the composition of intestinal microflora is of great interest. In that way the growth and/or the metabolism of certain microorganisms, beneficial for the organism, such as

lactobacilli and bifidobacteria can be stimulated. At the same time the suppression of the growth of harmful microorganisms such as the representatives of the Clostridium genus is essential. The main factor influencing the composition and/or the metabolism of intestinal microflora is the nutrition regimen of the individual. Hence the majority of efforts in nutrition are directed to improvement of intestinal microflora composition by the intake of probiotics, foods containing health beneficial microorganisms. In that respect
BSM has the leading role. Studies prove that the consumption of yogurt has positive effect on the balance of the microbial population in the intestinal tract.
Following intake of yogurt and under the influence of the metabolites generated by L. bulgaricus and S. thermophilus during fermentation, the cell numbers of beneficial microflora, specific for the intestinal tract, increase /21, 22/. Poor digestion of lactose is relieved, the immune system and the antitumour activity of the organism are stimulated /52,71/. The numbers of bacteria from the species belonging to the Clostridium genus substantially decrease /42/.
The representatives of the Clostridium genus decompose aminoacids to some toxic substances such as ammonia, indole and phenol. These are related to intestinal pathology. Yogurt administered to children of 10-18 months of age decreased the content of branched-chained fatty acids originating from protein decomposition in the intestinal tract. The representatives of the Clostridium genus are the major proteolytic species in the colon.
There are studies proving that consumption of yogurt results in the increase of bifidobacteria numbers in the intestinal tract of children and middleaged people /19, 20/. With children the number of enterococci also increases
/48/. The microbial population in the intestinal tract is stabilized following the consumption of yogurt. Experiments with mice /44/ demonstrate that feeding with yogurt stimulates the growth of the Lactobacillus and Bifidobacterium genera.
It is well known that L. bulgaricus and S. thermophilus are not part of the specific microflora of the human intestinal tract. They are isolated from green plants and milk. In Bulgaria they have been isolated from the plant Cropis biennis Linei from the Asteraceae family. The isolated L. bulgaricus and S. thermophilus strains possess high β-galactosidase activity /69/. The same strains decrease the content of indole in the intestinal tract, when they are applied in the form of yogurt /70/. L. bulgaricus and S. thermophilus strains are isolated from home-made yogurt with a high genetic diversity observed among the isolates
/95/.
As L. bulgaricus and S. thermophilus are not inhabitants of the intestinal tract, there is a continuous debate on their survivability in the gastro-intestinal tract. As a rule L. bulgaricus and S. thermophilus cannot tolerate the high acidity in the stomach and bile salts in the duodenum. They have a limited survivability after oral intake /31, 65/. During transition about 15% of them survive in the stomach and about 1% reaches the colon /82/. The main factor to determine the

survival of bacteria during passage through the stomach is the pH of gastric juice. The buffer capacity of food, determining the pH in the stomach and the time for empting the stomach are also essential. Another important factor for bacterial survivability is the physiological condition of bacteria /40/. Our studies show that various numbers of L. bulgaricus and S. thermophilus survive in gastric juice at the same pH and the same buffer capacity. The tolerance of probiotic species of lactic acid bacteria can be substantially increased due to the protective effect of milk components /29, 45/. In that respect an important biological property of buffalo’s milk has been established which remains to be studied in detail. It makes possible for large numbers of viable and active cells from BSM to reach the small intestines and the colon with all the beneficial effects on the organism. The survivability of L. bulgaricus at low pH is substantially increased by encapsulation in an emulsion from sesame oil /50/.
Nikolov /7/ reports that Dr. Fikov and Dr. Maer performed trials with
BSM in a pediatric clinic at the University of Hamburg. They found that feeding breast-fed infants with BSM on the second day results in the establishment of L. bulgaricus in the feces. In the Pediatric Institute in Sofia Dr. Kochkova and Dr.
Spassov fed children with BSM, prepared with starter cultures from different parts of the country. Their results show that 10 days after the end of yogurt consumption L. bulgaricus is still detected in children’s feces. For some children taking yogurt, prepared with starter cultures from the northern regions of
Bulgaria, L. bulgaricus can be detected in feces even 18-20 days after the end of
BSM consumption. Children received daily 200 to 800 ml of BSM. Nikolov /7/ reports that BSM prepared in different regions of the country always contains L. bulgaricus strains capable of surviving in the human gastro-intestinal tract.
Children receiving BSM weighed more than children from the control group fed with fresh milk.
In Bulgaria studies are performed to establish the survivability of L. bulgaricus and S. thermophilus in gastric juice and bile for three starters for production of BSM in industrial conditions. Starters are treated for 3 hours at
37°С. The results show that there is no substantial change in the number of
L. bulgaricus and S. thermophilus /13/. Data from recent research show that yogurt bacteria survive the passage through the human gastro-intestinal tract /81/.
The metabolites present in the fermented products can directly influence the pathogenic bacteria in the gastro-intestinal tract, Such metabolites with antimicrobial properties are produced by L. bulgaricus and S. thermophilus .
Bogdanov Iv., /1/ detects antibiotic substances in BSM.
Emanuilov I. and L. Nachev /2/ find that the microflora of BSM produces antimicrobial substances that inhibit selected microorganisms from the intestinal microflora. These substances are thermally stable and are not destroyed by heating at 80°С for 15 min. They are inactivated by neutralization of the medium to pH 7,0. The authors report that these antimicrobial substances are accumulated to a greater extent during microflora cultivation at 37°С.

L. bulgaricus and S. thermophilus have antagonistic effect on
Mycobacterium tuberculosis . Mycobacterium tuberculosis looses its virulence 6 hours after inoculation of milk at 45˚С /3/. L. bulgaricus and S. thermophilus possess bacteriostatic properties with respect to Brucella bovis and
Erysipolothryx rhusiopathiae /3/.
BSM has bacteriostatic effect on Salmonella typhimurium , S.enteritidis
, S. choleraesuis, S. abortus equil, S. newport, Staphylococcus aureus and other microorganisms /4/. Beside the low pH of yogurt, the effect on Esch. coli , S. typhimurium, Ersinia pseudotuberculosis and Shigella sonnei is caused by specific substances produced by L. bulgaricus and S. thermophilus at viable counts of 10 8 , 10 9 cfu/ml. Thermally treated yogurt has only weak bacteriostatic effect /59/.
Domestic studies show that the inhibitory activity of L. bulgaricus and S. thermophilus towards species of the Salmonella genus is best expressed by cocultures of the two yogurt species, in the form they exist in BSM /16/. S. thermophilus has stronger inhibitory activity against S.enteritidis
and S. gallinarum compared to L. bulgaricus . The inhibitory activity of lactate against
S. typhimurium, S. enteritidis and S. gallinarum is negligible compared to the effect of the antimicrobial components produced by L. bulgaricus and S. thermophilus /17/.
Rachev et al. /8/ find that some L. bulgaricus strains beside lactate produce four substances with antimicrobial effect. With its metabolites BSM alters the physiological properties of Esch.coli
and it looses its ability to form gas /10/.
Pulusani et al /85/ isolate three fractions ~ 700 Da from milk fermented with S. thermophilus . These fractions suppress the growth of species from the
Pseudomonas, Bacillus and Flavobacterium genera and Esch. coli . Chemical analysis indicates that the isolated fractions are aromatic amines liberated in the growth medium. The synthesis of these compounds requires the presence of glucose and lactose. The cells themselves do not possess antimicrobial activity.
L. bulgaricus synthesizes an antimicrobial substance named “bulgarican” /87,
88/. The substance is thermally stable and is active against several strains from the Bacillus, Streptococcus, Sarcina, Pseudomonas, Escherichia and Serratia genera in neutral and alkaline environment.
There is no other fermented milk product than BSM with better effect on individuals with poor lactose digestion. This disorder is typical for 70% of the world’s population. In these individuals the enzyme lactase /β-galactosidase/ is absent and lactose is not decomposed to glucose and galactose. Lactose reaches the colon intact. Under the activity of intestinal microflora lactose is fermented, a process that causes osmotic diarrhea. Gasses are generated (meteorism) with swelling feeling. The presence of L. bulgaricus and S. thermophilus in yogurt improves lactose digestion, provided that bacteria are viable /60, 62/. Moreover the number of bacterial cells should be not less than 10 8 cfu/ml. The enzyme

lactase, present in the cells of L. bulgaricus and S. thermophilus causes decomposition of lactose /63/. Coagulated milk has longer transition time in the intestinal tract compared to fresh milk. The slower transition of yogurt in the intestinal tract ensures longer time for lactose digestion by L. bulgaricus and S. thermophilus /90/.Bacterial cells of S. thermophilus activate their metabolism in the intestinal tract and hydrolyze lactose /39/. Such effect is barely detected for heat treated yogurt.
Basic parameter related to the health beneficial effect of the probiotic on the organism is its ability to adhere to intestinal mucosa. This property is related to the effect of the probiotic on the immune system. The adhesion stimulates the immune system. There is a straightforward relation between adhesion ability and antibody titers in blood serum of individuals taking probiotics. Adhesion can be influenced by genetic factors such as blood group determinants /73/. Vast differences are observed in the extent of adhesion of strains of the same species.
It is interesting to note that L. bulgaricus and Lactococcus lactis ssp cremoris have strong binding ability to intestinal mucosa, although they are not considered colonizing microorganisms /73/. Of all tested species L. bulgaricus has the strongest adhesion ability /21/. The nature of L. bulgaricus receptors is not clear. There is no correlation between the hydrophobicity of the cell surface and the adhesion to intestinal mucosa. The growth phase and the culture medium in which bacteria are cultivated may prove decisive for adhesion. Data are missing on the adhesion of L. bulgaricus and S. thermophilus when grown as coculture. Co-culturing of probiotics increases adhesion. The difference in the adhesion of separate species and strains may be one of the reasons for the different health effect on the individual.
Assessment of adhesion is most often performed with the use of cell lines and the intestinal mucosa model. A new, more versatile method for determination of adhesion to intestinal mucosa is developed /74/. It is based on resection of part of colon tissue during surgery. In the trials L. bulgaricus strain
ATCC 11842 is used in cell numbers of 10 7 _ 10 8 cfu/ml. Tissue samples are taken from patients with colon cancer. By its adhering ability L. bulgaricus does not differ from LGG and Bifidobacterium lactis B6 I2. Lactobacilli may adhere to epithelial cells via proteins similar to lectins.
For many years BSM has been applied as protective and therapeutic means against diarrhea. Yogurt shortens the duration of particular types of diarrhea, especially in children /9, 26, 92/. The World Health Organization recommends the substitution of fresh milk with yogurt for treatment of diarrhea
/96/. Yogurt is better tolerated than fresh milk and makes restoration of normal feeding possible. In Bulgaria assoc. prof. Fikov is the first to apply BSM as effective means against summer diarrhea in children /9/. BSM prepared from skim cow’s milk applied in the form of clisma gives exceptionally good results.
Daily yogurt consumption by 6-36 months old children with infantile diarrhea in quantities of 2x10 8 cfu for 5 days reduced occurrence of the disease

from 42% to 14% /26/.
The oral intake of antibiotics very often causes diarrhea. Antibiotics change intestinal microflora. The quantity of antibiotic sensitive bacteria such as lactobacilli and bifidobacteria decreases while that of antibiotic resistant enterococci and coli-forms increases. The normal microflora of the intestinal tract contains low numbers of Clostridium difficile . When antibiotics are applied this species causes diarrhea. In such cases yogurt is effective means for normalization of intestinal microflora and cessation of diarrhea. For individuals who frequently take antibiotics and those with suppressed immune system yogurt consumption may have good results against traveler’s diarrhea. S. thermophilus is an effective means against traveler’s diarrhea, but not for all individuals.
The health effect of lactobacilli and other probiotic bacteria on the organism is related to their direct influence on the immune system of the individual /43/. L. bulgaricus and S. thermophilus in the form of yogurt starter stimulate the immune system in vitro and in vivo. L. bulgaricus contains oligonucleotide which activates the immune system. DNA from L. bulgaricus and S. thermophilus strongly activate the function of B-cells. The mitogenic activity of B-cells is induced by the oligonucleotide contained in L. bulgaricus
NIAI B6. L. bulgaricus and its metabolites stimulate to different extent the human immune system /56, 57, 58/. L. bulgaricus strain NIAI В6, which contains an immunomodulating nucleotide is used for the development of new type of foods named “Bio defense Food”. They are used for enhancement of the natural and adaptive immunity. The mechanism of immunomodulation by the oligonucleotide OL-LB7 is related to Peyer’s patches. They are part of the lymphoid tissue, which plays an essential role in mucosal immunity /58/. L. bulgaricus has antimutagenic activity and infection-protective effect /76, 77,
83/. It induces the synthesis of cytokines in the organism /34, 36, 47, 80, 83/. A fermented milk product from skim cow’s milk and S. thermophilus increases the phagocytic activity of blood peripheral leucocytes /91, 92/. The minimal daily effective dose for immune reactivity is 10 9 cfu /51/.
Domestic research on 30 L. bulgaricus strains and 10 S. thermophilus strains isolated from industrial starters and plants show that they induce TNF α synthesis and stimulate non-specific immunity of the organism /37/.
In the intestines a specialized lymph system is present that is connected to mucosa. It is called Gut associated lymfoid tissue /GALT/ and it is in continuous contact with the general immune system. This system is the most important tissue that produces secretory immunoglobuline sIgA and distributes it mainly on the mucosa surface. The intake of yogurt /78, 84/, as well as cell-lysates /83/ results in substantial changes in the different parameters of immune response.
These changes include increase of sIgA-producing cell numbers, increase of macrophages and IgG. The specific responses to infectious agents are enhanced.
The intake of yogurt results in increased levels of gamma-interferon IFN,

increased numbers of lymphocytes and natural killer cells /36, 49, 92/. Presently a component of the cell wall of L. bulgaricus is considered responsible for the stimulation of cytokines production by the organism /83, 92/.
Dr. I. Bogdanov was the first to establish the antitumour properties of L. bulgaricus cell wall /24/. Later this property was confirmed by other authors /18,
41/. It is no secret that colon cancer takes second place in mortality in many industrial countries. It develops very slowly in the mucosa of the colon or rectum. During this multistep process cell division goes out of control. Cells accumulate to form polyps which later transform into malignant formations.
Several mechanisms for the protective effect of BSM against colon cancer are proposed: direct suppression of carcinogens; activation of the immune system and changes in colon microflora. Decisive role in the colon cancer risk has the dietary regimen. The risk of colon cancer is low when fermented milk products with low energy values are consumed /32/. In India the low incidence of colon cancer is attributed to high yogurt and other fermented milk consumption, while other studies do not establish such correlation /54, 61/.
In France an epidemiologic study found that people consuming yogurt have low risk for the development of enlarged adenomas of the colon /27/. S. thermophilus counteracts against DNA changes induced by chemical mutagens in the colon /25/. The intake of increased quantities of calcium results in decrease of free fatty acids in the intestinal lumen. Calcium forms molecular complexes with the free fatty and bile acids facilitating their disposal. Calcium reduces the toxicity of these acids and decreases the risk of colon cancer. Hence
BSM reduces the risk of colon cancer not only due to its microflora but also for its abundance of calcium. Tavan et al /94/ find decrease in the numbers of abnormal crypt formation in rats, induced by heterocyclic amines, when animals receive milk fermented with Bif. animalis or S. thermophilus . Antitumour effect of yogurt against colon cancer in mice is also established by Perdigen et al /79/.
The authors report that 10 days after yogurt intake and digestion the production of IgA in the colon mucosa is stimulated, which is effective against cancer cells.
Yogurt possesses antithrombotic activity /28/. During fermentation by L. bulgaricus and S. thermophilus functional peptides are liberated in yogurt, which prohibit the formation of thrombs in blood vessels. After daily intake of
500 ml of yogurt by adults functional peptides can be detected in the stomach, duodenum and blood. Domestic studies reveal that hydrolysis of milk proteins by selected strains of L. bulgaricus results in the formation of functional peptides, which lower blood pressure /93/. The consumption of yogurt does not increase cholesterol content and does not change lipid levels in blood plasma
/42, 67/.
Consumption of BSM normalizes the physiological processes during aging. Twenty to forty percent of the individuals over 60 years old suffer progressive atrophy of stomach mucosa. This is caused by decrease of hydrochloric acid production and increase of stomach pH. As a result calcium

absorption of elderly people is reduced to 30-50%. People who consume BSM have higher calcium absorption compared to individuals who do not take yogurt.
The former are in better health condition and are better protected against osteoporosis.
We ought to mention the role of BSM as starter in the production of our national varieties of cheese – white brined cheese (WBC) and kashkaval.
WBC belongs to the semi-hard cheese varieties, while kashkaval is hard type cheese with curd steaming.
In the practice of WBC or kashkaval production BSM is used as starter individually or in combination with Lactococcus lactis ssp. lactis , L. lactis ssp. cremoris and L. casei . Cheese ripening is caused by the enzymes liberated after autolysis of lactic acid bacteria. They contain the complete range of proteinases and peptidases that decompose caseins to peptides. Intracellular peptidases are responsible for further hydrolysis of peptides to aminoacids. Aminoacids are the basic precursors of taste and aroma components of cheese /35/. Peptidases are liberated during autolysis of bacterial cells. The extent of autolysis is influenced by salt content in cheese, cheese pH and the temperature of environment. The rate and extent of autolysis is strain specific /53/.
The autolysis of L. bulgaricus is best expressed at 2% of salt. At this concentration autolysis reaches 78% /53/. The autolysis of lactic acid bacteria is maximal at pH=5.5. The decomposition of aminoacids methionin, cystein and cystin by L. bulgaricus results in the formation of sulphur compounds, participating in taste formation of WBC /12/. In WBC L. bulgaricus produces acetate and ethanol forming the taste buckeye of cheese. WBC produced with
BSM as starter contains the highest amounts of acetate and free fatty acids /12/, compared to cheese produced with L. lactis ssp. lactis и L. casei as starter. To avoid fast acidification when BSM is used as starter S. thermophilus must prevail in its microflora. This requires the starter to be cultivated at 36-38° С. S. thermophilus has an important biological peculiarity. It forms more acetaldehyde and diacetyl than other homofermentative streptococci. S. thermophilus is applied in the production of soft cheese which carries probiotic bacteria /68/. When simply prepared BSM is used as starter for WBC poor consistency and strong acidic taste of cheese are obtained /15/. The formation of large quantities of D-lactate by L. bulgaricus has negative effect on WBC quality /12/. Maintaining high pH of WBC in the first days has favorable effect on cheese taste, aroma and consistency. High pH however is favorable for the growth of staphylococci and salmonellas which may cause food poisonings. An effective biological means of protection is the application of BSM as starter. The microflora of BSM also suppresses gas formation by colibacteria /10/.
Although L. bulgaricus and S. thermophilus are described as species that do not grow at temperatures bellow 22°С and at 2% of salt, these bacteria in
WBC grow at 4% of salt in the curd and 7,5% in the water phase of cheese /11/.
The number of L. bulgaricus cells in cow’s WBC prepared with BSM starter

reaches 10 7 cfu/g cheese and remains at 10 5 cfu/g for 60 days. BSM starter influences the formation of the microbial population of WBC. In WBC prepared with BSM starter, after 3 days the presence of L. casei is detected, the cell count of which for 15 days reaches 10 7 cfu/ g and for 30 days - 10 8 cfu/g. The major
Lactobacillus species in WBC prepared with BSM starter is L. casei /12/. The prevailing species from the Streptococcus genus is S. thermophilus and from that of enterococci – Ent. faecium . Whether BSM microflora with its metabolites stimulates the growth of Ent. faecium in WBC remains to be established. Ent. faecium has an antienterotoxic activity against enterotoxic Esch. coli . The established microbial population of the ripe WBC prepared with BSM starter has favorable effect on the ecosystem of the human intestinal tract and especially of children. This is confirmed by practical observations.
When BSM starter is used for WBC production together with slow acidifiers such as Lactococcus lactis ssp. lactis the quality of WBC is improved.
For Bulgaria the second important cheese is kashkaval. The two basic processes in the technology of this type of cheese are the cheddarization of curd and steaming of cheddarized curd. When BSM is used as kashkaval starter S. thermophilus must prevail in the microflora. This species has active role in curd cheddarization. The prevalence of L. bulgaricus in the kashkaval starter has negative effect on cheddarized curd. The BSM starter suppresses the growth of coli-forms that cause mixed fermentation of kashkaval. The metabolites resulting from that fermentation worsen the quality of kashkaval. The predominant species in the steamed curd is S. thermophilus . During curd steaming autolysis of bacterial cells occurs, causing kashkaval to ripen. In ripe kashkaval the major representative of lactobacilli is L. casei .
BSM is especially effective means for preservation of thermally treated milk at low temperatures (6-8°С). The addition of 0,1 – 0,2 % BSM to thermally treated milk suppresses the growth of psychrophilic bacteria /14/. This has favorable effect on the quality and shelf life of dairy products prepared from such milk.
Revealing of the qualities of this ancient probiotic as functional food for man is still ahead. This product completely complies with the brilliant thought of Hippocrates: “Food to be our medicine and our medicine – food”.
References
1.
Bogdanov I. The antibiotic of yogurt. [Nauchni trudove na ISUL god1 kn II],
1952 (in Bulgarian)
2.
Emanuilov I. and Nachev L., [Izvestiya na otdelenieto za biologichni I medicinski nauki I, 57], 1957 (in Bulgarian)
3.
Katrandzhiev K., Bulgarian yogurt. Edited by the Bulgarian Academy of
Sciences, Sofia, 1962 (in Bulgarian)
4.
Kondratenko M., Gruev P., Andreev N., Mutafova N., Kozareva M., Enikova

R., Todorov K., Pendelashka S., Bulgarian yogurt. [Zemizdat], Sofia, 1985
(in Bulgarian)
5.
Kondratenko M. and Simov Zh. Bulgarian yogurt. Sofia 2003 (in Bulgarian)
6.
Minkov T., Bulgarian yogurt across the world. Sofia 2002 (in Bulgarian)
7.
Nikolov M., Bulgarian yogurt and other dairy products. [Zemizdat], Sofia
1962 (in Bulgarian)
8.
Rachev R. et al., Scientific session on dairy industry, Vidin, 16-17, XI. 1982
(in Bulgarian)
9.
Fikov A., Bulgarian yogurt and its application for dietetics and therapy of breast-fed infants. [Lekoopizdat], Sofia, 1945 (in Bulgarian)
10.
Tchomakov C., Study of the effect of Bulgarian yogurt on gas formation by certain colibacteria. Zootechnical faculty, [Nauchni trudove, XXII],745-756,
1973 (in Bulgarian)
11.
Tchomakov C., Microbiology of milk and dairy products. [Zemizdat], Sofia,
1978 (in Bulgarian)
12.
Tchomakov C., Study of the microflora of cow’s yogurt and white brined cheese. Doctoral thesis. Stara Zagora, 1978 (in Bulgarian)
13.
Tchomakov C. and Boicheva S., Effect of stomach juice and bile salts on the microflora of Bulgarian yogurt. [Zhivotnovadni nauki, XXI], 5, 91-96, 1984
(in Bulgarian)
14.
Tchomakov C. and Boicheva S., Microbiological studies on thermal treatment of cow’s milk. Institute of Dairy Research, Vidin, [Nauchni trudove, XIII], 137-148, [Zemizdat], 1984 (in Bulgarian)
15.
Tchomakov C., Bulgarian yogurt. Health and longevity. [Zemizdat], Sofia,
1987 (in Bulgarian)
16.
Tchomakov C. and Boicheva S., Study of the inhibitory activity of L. delbrueckii ssp. bulgaricus and Str. salivarius ssp. thermophilus cultured separately or in co-culture against species from the Salmonella genus.
[Zhivotnovadni nauki, god XXX, №3], 55-58, Sofia, 1992 (in Bulgarian)
17.
Tchomakov C. and Boicheva S., Study of the participation of lactate in the inhibitory activity of L. delbrueckii ssp. bulgaricus and Str. salivarius ssp. thermophilus against species from the Salmonella genus. [Zhivotnovadni nauki, god XXX, №3], 59-62, Sofia, 1992 (in Bulgarian)
18.
Balanski, R., Gyosheva, B., Ganchev, G., Mircheva L., Minkova, S.,&
Georgiev G. /1999/ Inhibitory effects of freezed dried milk fermented by selected Lactobacillus bulgaricus strains on carcinogenesis induced by I, 2, dimethylhydrazine in rats and by diethylnitrosamine in hamsters. Cancer
Letters 147, 125-137
19.
Bartram H.P., Scheppach W., Gerbach S., Ruckdeched G., Kelber E., Kasper
H., Does yogurt enrich with Bifidobacterium longum affect colonic microbiology and fecal metabolits in healthy subjects? Amer. Clin. Nutr. 59/
1994/ 428-432
20.
Bianchi S.B., Gotti M., Brughera F., Polinelli U. Etude sur les variantions de

la flora lactique of bifide intestinal par rapport a l’administration de cellules lactiques du yogurt. Lait 58 /1978/
21.
Bianchi S. B., Intestinal Microflora: the role of yogurt in the eqiulibrum of the gut ecosystem. Int. J. Immunoth. 3 /Suppl II, 9-18, 1986
22.
Bianchi S., Vesely R., Lactic asid bacteria and intestinal microflora.
Microecol Ther. 25 /1995/ 247-255
23.
Bodana, A. R., Kao, D.R. /1990/. Antimutagenic activity of milk fermented by Streptococcus thermophilus and Lactobacillus bulgaricus. J. Dairy Sci. 73,
3379-3384
24.
Bogdanov I, G, Dalev P. G., Gurevich A, I. Kolosov, M.N., Malekova V. P.,
Plemyannikova L.A., Sorokina I.B. 1975. Antitumour glycopeptides from
Lactobacillus bulgaricus cell wall. FEBS Lett, 54, 259-261
25.
Bolognani F., Rumney C. J., Pool Zobel BL., Rowland I R. Effect of
Lactobacillus, Bifidobacteria and inulin on the fermentation of abberant crypt foci in rats. Eur. J. Nutr. 2001: 40: 293-300
26.
Boudraa G., Touhami M. Pochart P., Soltana R., Marry Y & Desjeux J. F.
/1990/ Effects of feeding of yogurt versus milk in children with persistant diarrhea. J. Pediatr. Gastroenterol. Nutr. II, 509-512
27.
Boutron Mc., Faivre J., Marteau P., Cauillault C., Senesse P & Quipourt V.
/1996/ Calcium, phosphorus, vitamin D dairy products and colorectal carcindgenesis: a French-case controlled study. Brit. J. Cancer, 74, 145-151
28.
Chabance B., Marteau P., Rambaud J.C., Migliere S.D., Boynard M.,
Perrotin P., Guillet R., Jollis P. & Fiat A. M. /1998/. Casein peptide release and passage to the blood in humans during digestion of milk or yogurt.
Biochemie 80, 156-165
29.
Charteris, W. P., Kelly P.M., Morelli L. & Collins J.K. /1998/ Development and application of in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper gastrointestinal tract. I. Appl. Microbiol. 84, 759-768
30.
Codex Standard for fermented Milks. Codex Stan. 243-2003
31.
Conway P.L., Gorbach S.L. & Goldin B.R. /1987/ Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells. J. Dairy Sci.
70, 1-12
32.
Danone Nutritopics N 26 March 2003
33.
Danone Nutritopics N 29 March 2004
34.
Davidkova G., Popova P., Guencheva G., Bogdanov A., Pacellie E., Antery
A., Mincheva V. /1992. Endogenous production of tumor necrosis factor in normal mice orally treated with Deodan a preparation from Lactobacillus bulgaricus “LB 51” Int. J. Immunopharmacol, 14, 1355-1362
35.
Deschamps B.M., Dominique Le Bars, Mireil Yvon, Marie Piere
Chapotchartier. Autolysis of Lactococcus lactis AM2 stimulates the formation of certain aroma compounds from amino acids in a cheese model.
Int. Dairy J. 14/2004/ 791-800

36.
De Simmone C., Veseley R., Bianchi S.B. & Jirillo E. /1993/ The role of probiotics in modulation of the immune system in man and animals. Int. J.
Immunother. IX, 23-28
37.
Dimitrov Z., Screening of lactobacilly and bifidobacteria for their potential to induce TNF alpha production from human monocytic U-937 cells. The food
GI-tract Functionality and Human Health Cluster. Proenhealth VTT-
Symposium 232 p.61, Spain 15-17, March 2004
38.
Dronaults S., Corthier G. Health effects of lactic acid bacteria ingested in fermented milk. Vet, Res. 2001: 32/2/101-117
39.
Dronault S., Anba J., Corthier G., Streptococcus thermophilus is able to produce a beta galaktosidase active during its transit in the digestive tract of germ free mice. Appl. Environ. Microbiol. 2002 68/2/938-941
40.
Eliza B.M., A. Benini M., M. Marzotto, A. Sbarbati, O. Kuz Lenente, R.
Ferrario, H. Hendrics, F. Dellaglio /2004/. Ssesement of novel probiotic
Lactobacillus casei strain for the production of functional dairy foods. Int.
Dairy J. 14, 723-736
41.
Elina T., Ogama N., Murata K. /1995/. Antitumor effects of Lactobacillus bulgaricus 878 k. Biotherapy, 9: 65-70
42.
Enrica Canzi, Maria Cristina Casiraghi, Raffaella Laudi, Raffaela Gandolfi,
Annamaria Ferrari, Furio Brighenti, Ricardo Bosia, Anna Cripta, Paolo
Maestrio, Rennata Vesely, Bruno Bianchi Salvadori. Yogurt in the diet of elderly: a preliminary investigation into its effect on the gut ecosystem and lipid metabolism. Lait /2002/ 82, 713-723
43.
Erisen KL & Hubbard N, E, 2000 Probiotic immunomodulation in health and disease. J. of Nutr. 130, 403 S-409S
44.
Garvie E.I., Cole C.B., Fuller R. & Hewitt D. /1984/. The effect of yogurt on some components of the gut microflora and on the metabolism of lactise in the rat. J. Appl. Bacteriol. 50, 237-245
45.
Gardiner G., Stanton C., Linch P.B., Collins J.K. Fitzgerald G. & Ross R. P.
/1999/ Evaluation of Cheddar cheese as food carrier for delivery of probiotic to gastrointestinal tract. J. Dairy Sci. 82, 1379-1387
46.
Gorbach S. L. The discovery of Lactobacillus GG. Nutrition Today 1996 vol
31/6/ Suppl. 2S4S
47.
Guencheva G., Popova P., Davidkova G., Mincheva V., Michailova S.,
Bogdanov A., Pacellie E., Anteri A. 1992. Determination of cytokine release after in vivo and in vitro administration of Deodan /a preparation from
Lactobacillus bulgaricus “LB 51”/ by the rabbit pyrogen test. Int. J.
Immunopharmacol. 14, 1429-1436
48.
Guerin-Dunan C., Chabanet C., Pendone C., Popot F., Vais Sade P., Bouley
C. Szylit O., Andreux C., Milk fermented with yogurt cultures and
Lactobacillus casei compared with yogurt and gelled milk: influence on intestinal microflora in healthy infants. Amer. J. Clin. Nutr. 67 /1998/, 111-
117

49.
Halpern G.M., Vruwink K.G., Vande Water, Keen CL & Gershwin M. E.
/1991/ Influence of long term yogurt consumption in young adults. Int. J.
Immunother. VII, 205-210
50.
Hou W.C.R., M.Y.Lin., M.M.C.Wang and J.T.C. Tzen. Increase of viability of Entrapped Cells of Lactobacillus delbrueckii ssp. bulgaricus in Artificial
Sesame Oil Emulsions J. Dairy Sci. 2003, 86: 424-428
51.
Huges D.A., F. Rochat., P. Serrant, J, M., Aeschilmann and E.J.Schiffrin.
Modulation of Nonspecific Mechanisms of Defense of Lactic Acid Bacteria.
Effective Dos. J. Dairy Sci. 1999, 82: 863-869
52.
Kalantzo P, G., Fermented Products with probiotic qualities. Anaerobe 3/
1997/ 185-190
53.
Kang J. O., L.P. Verinz, S. Laberge and R.E. Simarad. Some Factors
Influencing the Autolysis of Lactobacillus bulgaricus and Lactobacillus casei. J. Dairy Sci. /1997/, 81: 639-646
54.
Kampman E., Giovannucci E., Veer P et al. Calcium, vitamin D. Dairy Foods and the occurrence of colorectal adenomas amang men and women in two prospective studies. Amer. J. Epidemiol. 1994: 139: 16-29
55.
Kampman E., Van’t Veer P., Hiddink G., J., Van Aken Schneijder P., Kok
E.J., Hermus K. J. Fermented dairy products, dietary calcium and colon cancer: a case control study in the Netherlands. Int. J. Cancer 1994: 59: 174-6