RawHealth Kefir Curds II

Abstract
       Kefir is a refreshing fermented milk with a slightly acidic taste. It is made only from kefir grains or mother cultures prepared from grains, although attempts to produce kefir with pure cultures are in progress.

       Kefir grains are a complex and specific mixture of bacteria and yeasts held together by a polysaccharide matrix. The lactic acid bacteria and yeast fermentation of milk results in the production of numerous components, including lactic acid, CO2, a small amount of alcohol, and an array of aromatic molecules, all of which provide kefir with its unique organoleptic properties. Many health benefits related to the consumption of kefir have been observed, but rigorous research using modern scientific methods is in its early stages.
 

Introduction
       Kefir is a fermented drink which has been consumed for thousands of years. It originated in the Caucasus mountains in the former Soviet Union where the drink was fermented naturally in bags made of animal hides. Its use is currently being expanded because of its unique organoleptic properties and its long tradition of health benefits.

       Kefir distinguishes itself from the more known fermented milk yogurt in that it is traditionally made only from kefir grains which contain a complex mixture of both bacteria and yeasts. The resulting kefir possesses unique organoleptic characteristics. Research on its health benefits is just beginning, and there remain many questions unanswered.

        Two types of kefir exist: sugary, a fermented sweetened water; and milky, a fermented milk beverage. This article addresses the milky variety, whose norm has been established by the International Dairy Federation (2) , and it will provide an overview of its characteristics and a discussion of its potential health benefits.
 
 

Characteristics and consumption of kefir
        Kefir is a refreshing slightly carbonated and acidic fermented milk (3) . It can be consumed as is, or can be used in cooking (in soups, sauces, and cakes). The distinctive organoleptic properties differ from yogurt in that small amounts of CO2, alcohol, and aromatic molecules are produced as a result of a dual fermentation of lactic acid bacteria and yeasts.

       Although kefir is just being discovered in some areas of the world, it has been very popular in the former Soviet Union, Hungary, and Poland for many years. In the former Soviet Union, kefir accounts for 70% of the total amount of fermented milk consumed (4). It is also well known in Sweden, Norway, Finland, and Germany (5) , as well as in Greece, Austria, Brazil, and Israel (6). It is currently available in the United States, primarily as an ethnic drink, and is growing in popularity in Japan.
 
 

Kefir grains
       While yogurt can readily be made from the lactic acid bacteria present in fresh yogurt, kefir can only be made from kefir grains and mother cultures prepared from grains. The grains contain a relatively stable and specific balance of microorganisms which exist in a complex symbiotic relationship. The grains are formed in the process of making kefir and only from pre-existing grains. They resemble small cauliflower florets, and each grain is 3 to 20 mm in diameter (7). Kefir grains are clusters of microorganisms held together by a matrix of polysaccharides. The grains include primarily lactic acid bacteria (lactobacilli, lactococci, Leuconnostoc) and yeasts, and include acetic acid bacteria and possibly other microorganisms (8).

        The overall organization of microorganisms of grains is not completely elucidated. More than a thousand years of consumption have demonstrated that the microorganisms in kefir are not pathogenic. Even further, milk inoculated with grains can suppress the growth of some pathogens such as Salmonella or Shigella (1) . The grain matrix is composed of a complex of 13% protein (by dry weight), 24% polysaccharide, plus cellular debris and unknown components (6) . The principal polysaccharide is a water-soluble substance known as kefiran. Several homofermentative Lactobacillus species including L. kefiranofaciens and L. kefir (9-11) produce this polysaccharide. They are an integral part of the grain, and without their presence, kefir grains cannot be propagated. The mechanism, however, is not fully understood.
 
 

Fabrication of kefir
       There exist several methods of producing kefir (see Figure 1). Food scientists are currently studying modern techniques to produce a kefir with the same characteristics as those found in traditional kefir, but without some of its drawbacks.

1. Traditional process
       The traditional, or artisanal, method of making kefir is currently achieved by directly adding kefir grains (2-10%) to milk that has been pasteurized and cooled to 20-25¡C. After a period of fermentation lasting around 24 hours, the grains are removed by filtration. The beverage, itself containing live microflora from the grain (see Table 1), is then ready for consumption. The grains grow in the process of kefir production, and are reused for subsequent fermentations (6). ?Grains can then be dried at room temperature and kept at cold temperature (4¡C). For a longer conservation, they can be lyophilized (freeze-dried) or frozen (14) .

        A second method, known as the "Russian method", permits production of kefir on a larger scale, and uses a series of two fermentations. The first step is to prepare the cultures by incubating milk with grains (2-3%), as just described. The grains are then removed by filtration and the resulting mother culture is added to milk (1-3%) which is fermented for 12 to 18 hours (6). Several problems associated with traditional kefir have led to a more modern method of production. The traditional method produces only small volumes of kefir, and requires several steps, each additional step increasing the risk of contamination.

        In addition, the grains themselves are not well understood, and are not well controlled.  Strong pressure from the CO2 gas content can lead to the explosion of the recipient unless appropriate containers which resist the escaping of gas are used (14). Finally, the shelf-life of traditional kefir is very short, less than three days.

2. Recent process
To resolve the above difficulties, some producers in Eastern Europe have begun using concentrated lyophilized cultures made from grains (7). These mother cultures are then used as bulk starters for direct inoculation of the milk. More control over the process and fewer steps provide a more consistent quality.

3. Current areas of research
Attention is now being turned toward producing kefir from pure, defined cultures (15-17). This method will allow for a better control of the microorganisms involved, an ease of production, and a more consistent quality. The product will also have a longer shelf-life (14) of 10 to 15 days at 4¡C. It will also permit various modifications of the product to achieve certain health or nutritional benefits.

        Two basic procedures for manufacturing kefir have been developed using pure cultures isolated from kefir grains. Milk can be inoculated simultaneously with lactic acid bacteria and yeast, or it can undergo two fermentations, the first with lactic acid bacteria and the second with yeast. Results have been encouraging, but finding the right equilibrium of bacterial and yeast strains to create a product with the characteristic properties of traditional kefir including both the organoleptic qualities and the health benefits - is a difficult task. The major difficulty is understanding the microbiology of kefir.
 
 

The microbiological, chemical, and nutritional composition of kefir
       The composition and flavor of kefir vary significantly, depending on a variety of factors including the source (cow, ewe, goat, mare) (18) and the fat content (regular fat, low fat, nonfat) of the milk used, the composition of the grains or starters, and the technological conditions of production (14).
Table 2 lists some of the biochemical components of the range of kefir. The major products formed during fermentation are lactic acid, CO2, and alcohol. Many aromatic compounds, including diacetyl and acetaldehyde are present in kefir (14) .

        Diacetyl is produced by Str. lactis subsp. diacetylactis and Leuconostoc sp. (7). The pH of kefir is 4.2 to 4.6 (19). As in yogurt, the lactose content is reduced in kefir (14), and the b-galactosidase level is increased as a result of fermentation. Information on vitamin and mineral content is limited and sometimes contradictory, but overall, there do not seem to be significant variations from that of the milk used. There is also a small increase in proteolysis, leading to an increase in free amino acids (2).
 
 

Health properties of kefir
       Kefir enjoys a rich tradition of health claims. In the former Soviet Union, it is used in hospitals and sanatoria for a variety of conditions, including metabolic disorders, Atherosclerosis, and allergic disease (1). It has even been used for the treatment of tuberculosis, cancer, and gastrointestinal disorders when no modern medical treatment was available. Its consumption has also been associated with longevity in Caucasus (20) . Various scientists have observed digestive benefits of kefir (21, 22) , but controlled studies have yet to confirm their empirical findings.

        Various research teams around the world have reported encouraging results, but several methodological difficulties still need to be resolved. Most studies to date have been performed in vitro or using animal models, and human studies are not available.  Further, the effects of kefir grains or their isolates are often studied, rather than the product kefir, and there is no evidence that the observed effects would occur using the drink itself. Also, kefir products vary significantly according to the composition of the grains used and even according to the region in which it is made, and therefore specific effects may not be demonstrated in all kefirs. Given these caveat, a variety of health benefits are being investigated. Table 3 presents recent studies using kefir products.

        Several studies have investigated the antitumor activity of kefir (20, 23, 24) and of kefir grains (25, 26) . Specific cultures isolated from kefir were also shown to bind to mutagenic substances such as indole and imidazole (27, 28). Immune system stimulation with kefir (24) and with sphingomyelin isolated from the lipids of kefir (29) have been demonstrated in both in vitro and in vivo studies.

        Kefir (30) possesses antimicrobial activity in vitro against a wide variety of gram-positive and gram-negative bacteria (20, 31), and against some fungi (20) . In Zacconi et al.ís recent study (30), the antagonistic effects of kefir against Salmonella kedougou were attributed to the complexity and vitality of the kefir microflora. De Vrese et al . (32) demonstrated that fresh, but not heat treated, disintegrated kefir grains suspended in kefir directly enhanced intestinal lactose digestion in minipigs. This effect was attributed to microbial b-galactosidase activity of kefir.  The above studies provide encouraging results, but much more research is necessary in order to demonstrate similar effects using kefir in humans. Further, a standardized, well-defined product must be used in order to provide useful information.
 

Abstract
Research on fermented milks (FM) has grown dramatically in the past 20 years. FM have probiotic effects since their consumption leads to the ingestion of large numbers of live bacteria which exert health benefits beyond basic nutrition. Major results of research are as follows. Yogurt consumption reduces symptoms of lactose maldigestion compared to milk.  FM, may have antibacterial and immunological properties. Ingestion of the lactic acid bacteria bifidobacteria improves the colonic microflora by increasing bifidobacteria levels. Lactobacillus casei reduces the duration of some types of diarrhea. Future research conducted using human subjects, with rigorous methodology and modern statistical analysis, will provide further information on the health benefits of FM.
Keywords: fermented milk, probiotic, yogurt, kefir, Lactobacillus streptococcus, Streptococcus thermophilus, Lactobacillus casei, bifidobacterium
 

Glossary
       Lactic acid bacteria (LAB): a large group of bacteria with the common characteristic of producing lactic acid as the principal end product of metabolism; found in milk and other natural environments LAB can be: a. homofermentative: produce 70-90% lactic acid; e.g., L. bulgaricus, S. thermo-philus, L. acidophilus b. heterofermentative: produce at least 50% lactic acid plus other compounds such as acetic acid, CO2, and ethanol; e.g., L. casei, bifidobacteria a. mesophilic: grow best at a temperature range of 25-30¡C; e.g., L. casei b. thermophilic: prefer a range of 40-44¡C; e.g., L. bulgaricus, S. thermophilus a. Facultatively prefer anaerobic anaerobic: conditions for metabolism, but are aero-tolerant (most LAB fit in this b. Strictly anaerobic: survive only in anaerobic conditions; e.g., bifidobacteria

Functional foods:
       Foods that, by virtue of physiologically active food components, provide health benefits beyond basic nutrition (Working definition of ILSI Functional Food Task Force, Brussels, February 17,1997). Interleukin, interferon, tumor necrosis factor: examples of cytokines, which serve as signals between cells involved in immune response. sIgA: secretory immunoglobulin A; principal antibody produced by the gut immune system.

        Azoreductase,§-glucuronidase, glycocholic acid hydrolase, nitroreductase: colonic enzymes implicated in the conversion of procarcinogens to carcinogens.  LDL/HDL: ratio between blood levels of low density lipoprotein and high density lipoprotein; level above 3 indicates increased risk of cardiovascular disease. Breath hydrogen test: measurement of hydrogen expired after oral lactose load of 12-50 g compared to base level; > 10-20 ppm indicates malabsorption.

From legend to science: Historical perspective
       For centuries, fermented milks have been purported to provide a large gamut of health benefits, from improving well-being to increasing longevity. One story recounts that in the sixteenth century, King Fran*ois the First of France suffered from persistent diarrhea, and after several unsuccessful treatments, a Turkish doctor was sent in. He brought with him sheep and a secret recipe for yogurt. The king was soon cured of his intestinal infection.

Scientific interest began much later, in the early twentieth century, when Elie Metchnikoff, a Nobel-prize winning biologist at the Pasteur Institute in Paris, first suggested that lactobacilli might counteract the putrefactive effects of gastrointestinal metabolism (1). In the past twenty years, scientific research has blossomed, with an interest in topics ranging from antimicrobial effects to reduction of risk of cancer. Much valuable preliminary work has been done using animal or in vitro models, which allow for much greater control over variables than when studying humans, and which offer reproducible results. These models are also useful for studying the mechanisms involved.

        Studying the effects of FM on humans presents several challenges. Fermented milksare unctional foods, and as such, their impact on human physiology is of a small amplitude and not easily detected. Also, early humans studies, though numerous, were generally case reports rather than modern experimental studies (randomized). Currently, researchers are beginning to address these methodological problems.
 

Yogurt, the ever-popular fermented milk
       According to the Codex Alimentarius (5), yogurt is milk (usually cowís milk) that has been fermented by Streptococcus thermophilus and Lactobacillus bulgaricus under defined conditions of time and temperature. Each species of bacteria stimulates the growth of the other, and the products of their combined metabolism produce the characteristic creamy texture and mild acid flavor. Fermentation is stopped by cooling, and the final product, which contains100-1000 million live bacteria per ml, is refrigerated until use. As a fresh dairy product, it has a limited shelf-life.

1. Milk digestibility
       Given all the research to date on FM, the fact that lactose is better digested from yogurt than from milk by lactase-deficient individuals is the most well-established health benefit (6). Yogurt ingestion leads both to less hydrogen production in the breath hydrogen test (lactose maldigestion) (Figure 1) and to reduced symptoms (lactose intolerance) (Marteau, 1990; Lerebours,1989; Kolars, 1984). This effect is related to the living bacteria, the enzymatic content ( e.g ,§-galactosidase), and the texture of yogurt.

2. Recovery from diarrhea
       Yogurt reduces the duration of certain types of diarrhea, especially in children (Niv, 1963; Boudraa, 1990). The World Health Organization (WHO, 1995) recommends that during treatment of diarrhea, yogurt should replace milk when available since it is better tolerated than milk and can help prevent malnutrition or reestablish nutritional adequacy.

3. Immunomodulating effects
       Yogurt has been shown to enhance various parameters of the immune system in invitro models (13) and in mice (14-16). In humans, one study found an improvement in clinical symptoms of nasal allergy, but no changes in any parameters tested (17) . A recent report with atopic subjects found no significant modification of immune system parameters, showing that there was no aggravation of the immune system caused by yogurt (18) . Very high concentrations of yogurt bacteria have led to increases in IFNy, B lymphocytes, and natural killer cells (19) , and yogurt consumption increased 2í,5í-a synthetase activity (a reflection of production of IFNy) (20).

4. Reduction of risk of cancer
       A recent epidemiological study from France showed that people consuming yogurt had less risk of developing large colorectal adenomas (21). In addition, the consumption of yogurt in elderly subjects with atrophic gastritis led to a decrease in the procarcinogenic fecal enzymes nitroreductase and azoreductase (22). Research in this field is intriguing, but preliminary.

5. Blood cholesterol levels
       Mann and Spoerry (23) reported over 20 years ago that Maasai warriors consumed several liters of FM per day and yet had low serum cholesterol levels. This observation sparked a series of conflicting studies on the possible hypocholesterolemic properties of yogurt and other FM. Results have been inconsistent (24). What is clear is that regular consumption of yogurt does not increase plasma cholesterol concentration (24, 25); yogurt can be part of the daily intake of individuals who are concerned about heart disease.
 

Kefir, another traditional fermented milk
       Kefir is a stirred beverage made from milk fermented with a complex mixture of bacteria (including various species of lactobacilli, lactococci, leuconostocs, and aceterobacteria) and yeasts (both lactose-fermenting and non-lactose-fermenting). The small amount of CO2, alcohol, and aromatic compounds produced by the cultures give it its characteristic fizzy, acid taste (26). Kefir fabrication differs from that of yogurt in that kefir grains (small clusters of microorganisms held together in a polysaccharide matrix) or mother cultures from grains (27) are added to milk and cause its fermentation. Kefir is actually a family of products, in that the grains and technology used can vary significantly and thus result in products with different compositions.

        Many health benefits have been traditionally reported. Kefir has been used for the treatment of Atherosclerosis, allergic disease, and gastrointestinal disorders, among other diseases (28). Until recently, most research has been limited to studies lacking modern statistical practices or to reports written up in Slavic languages, rendering them inaccessible to most western scientists.

        Recent studies have investigated antibacterial (29), immunological (30), antitumoral (31), and hypocholesterolemic(32) effects of kefir consumption on animals. Results suggest potential benefits. Fresh, but not heat-treated grains in kefir enhanced intestinal lactose digestion in minipigs (33). While awaiting more research, it is important to remember that kefir, like yogurt, has been and continues to be a part of the regular diet in central and eastern Europe for centuries.  Bifidobacterium: a natural inhabitant of the intestines Bifidobacteria were first described in 1900 by Tissier (34) . Since that time, their classification has evolved continually, and currently includes around thirty species (35, 36) . In general, they are strictly anaerobic, Gram-positive rods which often have special nutritional needs and grow slowly in milk. Very few strains are adapted well enough to milk that they both grow in sufficient numbers and survive well throughout the shelf-life of the FM.

        Although bifidobacteria produce both lactic acid and acetic acid as major end-products of metabolism (heterofermentative), many microbiologists consider them to be lactic acid bacteria, albeit a special case.  Tissierís hypothesis almost 100 years ago that bifidobacteria might have health benefits(37) was based on the following observations. Bifidobacteria are normal inhabitants of the human intestinal tract throughout the life cycle, beginning just days after birth. Further, they are often the predominant microorganism in the gut of breast-fed infants. It has since been shown that breast-fed babies are less at risk for diarrheal disease than formula-fed infants (38).  In addition to the above inherent characteristics of bifidobacteria, some strains of the micro-organism survive intestinal transit in sufficient numbers to exert a metabolic effect in the gut (39,40).

1. Effects on the intestinal microflora
       Ingestion of milk fermented with bifidobacteria leads to an increase in fecal bifidobacteria levels, both in infants (43) and in adults (44) . Elevated levels return to normal after cessation of consumption (39). Ingestion of FM with bifidobacteria has also led to a decrease in §-glucuronidase activity, but not in other enzymes associated with colon (44).

2. Effect on mild constipation
       Slow intestinal transit can be partially corrected in women by the regular consumption of a milk fermented with yogurt cultures and bifidobacteria (41). This effect was not observed with yogurt as a control, thus demonstrating the specificity of bifidobacteria for the increased colonic motility (42).

3. Prevention of diarrhea
       Few studies have been performed. One double-blind study of infants demonstrated that a formula with added B. bifidum and S. thermophilus reduced the incidence of hospital-acquired diarrhea compared to a standard formula. It also lowered the rate of rotavirus shedding into the environment (45).

4. Immunomodulating effects
       Ingestion of milk fermented with B. bifidum led to an increase in phagocytic activity in peripheral blood compared to milk consumption (46). A mixture of B. bifidum and L. acidophilus decreased chronic inflammation of the sigmoid colon and increased humoral immunity in a group of elderly subjects (47).
 

Lactobacillus casei: new interest in an old bacteria

        The group L. casei consists of several species of facultatively anaerobic and hetero-fermentative, mesophilic lactic acid bacteria(48). Their metabolism provides organoleptic qualities to several traditional FM and cheeses, and more recently, to new fermented milks. L. casei have been detected in the feces of both infants (49) and adults (50). Their ability to survive transit through the intestinal tract in adequate numbers to have a physiological effect (50) , coupled with their potential health benefits make L. casei an ideal candidate for a probiotic.

1. Treatment of diarrhea
       Several double blind, placebo-controlled clinical trials have demonstrated that oral consumption of L. casei reduces the duration of diarrhea (51), and in particular, rotavirus gastroenteritis (52) in children. In addition, L. casei may help reduce the duration of diarrhea associated with children in day care centers (53), antibiotic treatment (54) and travelerís diarrhea (55).

2. Effects on the intestinal microflora
       In addition to increasing lactobacilli count in feces (50), milk fermented with L. casei has been shown to lower the activity of the colonic enzymes §-glucuronidase (50, 56), glycocholic acid reductase, and nitroreductase (56) in healthy adults. A recent study demonstrated a decrease in §-glucuronidase and §-glucosidase activities in infants after ingestion of a milk fermented with yogurt cultures and L. casei. This effect was not found with yogurt alone or with gelled milk (control) (57) , thus suggesting that the modification was due to L. casei or to the association between L. casei and yogurt.
 

3. Immunomodulating effects
       Challenge tests ( e.g ., using Salmonella typhimurium ) with oral ingestion of L. casei in mice has led to increased protection in animals infected with pathogenic bacteria (58, 59). A few reports using human subjects have shown an enhancement of non-specific immune system activators, such as y interferon and interleukins (ex vivo) (60) and of specific immune responses to various challenges, including rotavirus vaccine (61).  In a recent study infants with atopic dermatitis were given formula with added L. casei. Not only did the concentration of fecal tumor necrosis factor-a decrease significantly (a measure of the immune response), but clinical symptoms improved as well (62) . Viability of the bacteria is an important factor of its effectiveness (61).
 

Other probiotics
       In addition to the probiotics discussed above, other bacteria, some well known and some more recent, offer additional health benefits. In particular, much research has been conducted on L. acidophilus. Several studies suggest a hypocholesterolemic effect of L. acidophilus (63) , while others have investigated its ability to prevent various types of diarrhea (64) and to reduce the incidence of candidal vaginitis (65).

In addition, consumption of L. acidophilus has led to modifications of various parameters of the immune system (46), and to a decrease in several fecal enzymes associated with colon cancer (66). Less well-known bacteria include Lb. helveticus (67), L. plantarum(68) , and L. reuteri (69). These lactic acid bacteria have different microbiological and metabolic characteristics than the ones listed above, but may also exhibit health effects, such as stabilizing the intestinal microflora or reducing the duration of diarrhea.

        Probiotic effects of lactic acid bacteria and FM can be categorized in the following way: effects on the small intestine and digestion, direct modification of the colonic microflora and its metabolism, and general effects initiated in the colon. Thus, yogurtís main health benefit is related to improved lactose digestion; while bifidobacteria primarily affects the balance of the colonic microflora; and kefir and L. casei provide more global benefits, the first in relation to its antimicrobial effects and the second to diarrhea. In all cases, the lactic acid bacteria must be present in the FM in very large numbers, and must be live and active.  Not all effects have received as yet adequate scientific attention. Few studies have compared various types of FM. As more research is performed using human subjects and with rigorous methodology and statistically valid conditions, the variety of health benefits of FM will become more well-defined.

        Amidst the plethora of sometimes contradictory evidence, it is important to remember the nutritional and organoleptic qualities of yogurt and kefir that make them both healthful and pleasant choices in a balanced, varied diet, regardless of probiotic effects. The probiotics discussed in this report are incorporated in FM because of health benefits beyond inherent nutrition, and are appropriate for individuals with specific health goals. Taken together, they represent the best of both tradition and modern science; FM and probiotics have journeyed from nutrition practice to nutrition science and back again.

These two wonderful articles were written by  Cathy J. Saloff-Coste from Dannone at their website www.dannone.com

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