Prebiotics are non-digestible molecules produced by probiotic microorganisms . Probiotic microrganisms are generally bacteria or fungi recognized as safe, with their properties based on the production of organic acids, reduction of biogenic amines, digestion/breakdown of carbohydrates and proteins, immunomodulatory and anti-inflammatory responses, reduction of carcinogenic amines, and production of antimicrobial peptides, among others . These days probiotics are mostly consumed as probiotic yogurts and other probiotic dairy products, dietary supplements, spoonable forms, and probiotic cultured drinks for daily dosage packaging, among others. Prebiotics are also claimed to enhance wellbeing through immunomodulatory and metabolic activities, and act as a natural barrier against pathological processes . These molecules are considered to be a targeted for human and animal production and health, and represents a multimillionaire market of the functional foods. Furthermore, the increasing market of prebiotics counts today with a thousands of patented invention, related to isolation, production, preparation, methods of use, or application of newly health enhancing molecules. The global production and consumption of functional foods is a multi-billion industry, with an estimated market size around US$ 60 billion in 2008-9, several times greater than the health treatment costs only in USA in that years, in the order of US$ 832 million (Figure 1). As a comparison, the global market of probiotic products was US$ 15.9 billion in 2008 and US$ 19 billion in 2009, with a compound annual growth rate (CAGR) of 11.7 % (2009-2014). Furthermore, the probiotic market predicted by 2014 for Europe and Asia comprises, respectively, US$ 12.9 billion (11.1 % CAGR), and US$ 8.7 billion. Japan, a global leader of functional foods, devoted US$ 4.5 billion to the study and commercialization of prebiotics, with US$ 1.5 billion verted exclusively for the oligosaccharide commerce in 2009 . The USA have occupied the second position in the last decade, with a commercialization of US$ 110 million for functional oligosaccharides (35 % inulin, 20 % mannan oligosaccharides, and 10 % fructan), and with a CAGR rate of 20 % The European and the U.S. market for prebiotics is projected to reach nearly US$ 1.2 billion and US$225 million, respectively, by the year 2015 . This has reached nearly US$ 21.6 billion in 2010 and is expected to reach US$ 31.1 billion in 2015, and at a CAGR of 7.6 % for the 5-year period.
2. Studies on water kefir
In general, prebiotics are considered nondigestible but fermentable oligosaccharides, involved on health promotion for the host . Such compounds are known to provide improvements in nutritional status, besides additional health benefits such as protection against carcinogenesis, mutagenesis, prevention of injuries caused by free radicals, control of intestinal flora, gastrointestinal resistance, decrease of blood pressure induced by hypertension, production of
Historically, kefir grains (Figure 2) were considered a gift from Allah among the Muslim people of the northern Caucasian mountains . The word kefir is derived from the Turkish word
Different from the milky bacteria-encapsulated polysaccharide kefiran, AK seems to be an oligosaccharide isolated from an aqueous fraction of kefir grains .
2.1. Kefir characteristics
2.1.1. Microbial strains
Different sets of yeasts and bacteria in water kefir have been identified from several regions and sources, and with both culture-dependent or molecular methods. Notwithstanding, kefir is able to change their bacterial/yeast ratio, even their microbial strains as a function of time, experimental conditions, temperature, and neighboring microorganism, in the inner grain . A typical consortium appears to consist of mostly lactic acid bacteria plus yeasts promoting alcoholic fermentation, together with some acetic acid bacteria (Table 1), possibly oxidizing the ethanol formed . Despite the great microbial diversity found in kefir samples from different regions, there are common strains prevailing in kefir sources from different countries. The most likely strains found in kefir are
Changes in physical, chemical and microbiological parameters during continuous cultures of water kefir has been studied by several authors since 50’s . In our lab grains samples grown in molasses solutions at 50 to 200 g L-1 in distilled water have been tested for some parameters, as optima temperature and pH of development, ionic strength, some metabolites (glucose and glicerol), growth changes after freezing even at -70 °C, and bacteria/yeast proportions. The results have shown a maximum temperature of growth about 25 °C, and a continuous pH decrease for the suspensions up to 20 h (from pH 6.1 to pH 4.5). While kefir suspensions presented decreasing levels of glucose (7 times), glicerol increased 3 times during cultivation in molasses at physiological conditions for 7 days. The bacteria/yeast quotient of water kefir showed a prevalence of lactic acid bacteria in the grains (31±8 % greater), whereas yeasts have been mainly found in the suspensions (63±6 % greater). Surprisingly, water kefir grains have been demonstrated a higher resistance against extreme environment conditions. As an example, the grains were able to growth in KCl up to 5 %, or even at temperatures lower than 4 °C. At household conditions of growth, biomass curves of freezed-stored grains have shown an continuous linear trend up to the 5
As a well-structured gelatinous grains with diverse microbial strains in their composition, it was hypothesize that the bacteria and yeasts present in kefir could be protected inside the polysaccharide matrix, exhibiting a different resistance under physical and chemical stresses than freely strains in solution. Keeping this in mind it has been tested the colony resistance of kefir against three disordering factors: ultraviolet radiation exposure (UV), antibiotic administration, and gas treatment (oxygen and ozone) . After an exponential growth phase the samples were submitted to UV and chemical treatments. Far UV (15 W D2) was taken daily in tubes containing the grains during 5, 10, 30 and 60 min, up to 9 days. The growth of grains were followed gravimetrically after cutting dried grains into six layers, from the inner core to the outer shell of the grains. Antibiotic treatment was carried out with 1 mL penicillin G (20
2.1.4. Artificial symbiogenesis
The microbial flora present in kefir grains has been studied from a symbiotic community point of view by Linn Margulis since 1995 . Accordingly, it has been stated  that separated cultures of microbial kefir grains, either do not grow in milk or have a decreased biochemical activity, which further complicates the study of the microbial population of kefir grains. The mechanism of symbiogenesis of kefir grains from distinct strains of unicellular organisms is unknown, although there are some data about the recover of their structure and probiotic properties from lyophilization, and even so, about the formation of an artificial consortium produced by bits of kefir grains transferred to a yeast extract-sucrose solution . Using a simple approach, we had developed artificial cultures of kefir by trapping their strains in alginate beads . To do so, kefir grains were cultured in 200 g L-1 of molasses solution for 7 days. Then the supernatant was collected, centrifugated at 7000 rpm during 15 min, resuspended into 5 mL of molasses as above, and filtered to avoid minor grain fragments. For cell immobilization 100 mL of a 4 % sodium alginate solution was mixed with the treated kefir suspension and dropped into 1.5 % of a cold calcium chloride solution. The alginate-kefir beads resulted were then continuously cultivated with molasses replacement at 48 h intervals. Strikingly, novel kefir grains had been arisen from solution after three months of cultivation (Figure 4), resembling the ordinary household grains, as monitored by optical microscopy at low resolution, and with the commom budding property exhibited by normal grains (Figure 5).
Antimicrobial activity was chosen as a comparison index for native and artificial grains. The assays were carried out introducing 0.1 mL (3 x 108 cells) of
The anti-inflammatory activity of this modified grains, as revealed by paw edema assays in rats, showed even higher than native grains (Figure 6). This artificial process of strain internalization for kefir grains suggests a plausible strategy for incorporate some bacteria with specified purposes, e.g.,
This property of inherent modulation of kefir strains has been also reported with native grains, whenever they were stored for long periods, or even during their cultivation . In this aim, we have evaluated the bacteriocinin activity of kefir from an adaptative potential of growth against some pathogenic strains . To accomplish this, kefir samples were challenged with
2.2. Kefir properties
2.2.1. Suspension, grains and kefiran
220.127.116.11. Aqueous kefiran (AK)
There are several studies pertaining to the claimed health properties of the kefir consortium, but mainly with milky preparations. Accordingly, milky kefir is known to present a large antibacterium spectrum, gastrointestinal improvement and proliferation of normal lactic intestinal flora and bacterial colonization, anti-carcinogenic, wound healing and
2.3. On biological surfaces
2.3.1. Biomimetic membranes
Albeit kefiran has presented diverse prebiotic activities, no direct mechanism of its action on cell membranes have been understood yet. Aiming to help this, the influence of AK on biomimetic membranes composed of l-
Cellular mechanisms of action were investigated to verify the potential activity of water kefiran on the respiratory activity of isolated mitocondria . Samples from rat liver (1200 mg mL-1 protein) were preincubated with kefiran in 20 mM phosphate buffer pH 7.3 containing 70 mM sucrose, 1 mM EDTA, and 5 mM MgCl2. The oxygen consumption of mitochondria was determined by chronoamperometry at 50 rpm stirring suspensions in 2 mL using a Clark-type electrode Pt-Ag/AgCl connected to a potentiostat, and with -600 mV of applied potential. The system was previously calibrated with a N2-saturated solution and baker yeast suspensions. The current signals after successive additions of buffer, mitochondrial samples, 100 mM succinate, 100
2.4. On microorganisms
Kefir is well known to resist to a large spectrum of pathological strains, and it seems to be recognized as safe, although its culture contamination has been reported as a source of health impairments. . Antibiotic activity of both kefir and purified AK (50 mg mL-1) has been evaluated  using both the disk diffusion method and susceptibility tests against some well known pathogenic bacteria (
2.5. On animals
Despite the known probiotic and prebiotic effects of kefir and AK, little is reported about their responses in healthy individuals, e.g. a physiological status of animals naturally receiving fermented kefir suspensions . Targeting this, it was evaluated the consumption of kefir suspension byWistar rats (n=5/group) kept in metabolic cages at room temperature, and with water and commercial diet
2.5.1. Anti-inflammatory and antimicrobial activity
Anti-inflammatory responses of sugary kefir and its derivatives are poorly related in the literature. Notwithstanding, kefir may exert a beneficial effect on acute inflammatory responses, additonally improving the immune status of treated animals. In this sense an ED50 value of 12.5 mg kg -1 was found by rat paw edema, together with inhibitions values about 30±4 % and 54±8 %, for carrageenan (Figure 10) and dextran-induced inflammatory process, respectively (n=8/group). However, no changes in vascular permeability was evidenced in that experiments . When compairing with cyproheptadine, a H1-receptor blocker, these results pointed to the antiinflammatory response probably derived from serotonin receptor and arachidonic acid pathways. In another assay, the anti-edematogenic activity of both kefir suspensions and grinded grains were also evaluated with a similar approach through carrageenan, dextran or histamine. Kefir suspensions orally administered 30 min before stimulli were found to be more effective (62 % inhibition) than kefir grains mechanically disintegrated (40 %). The overall data suggest a participation of prostaglandins mediators more than just histamine and serotonine in the anti-inflammatory response as a whole.
With the use of an analgesia model of acetic acid-induced writhing reflex in mice , both kefir grains and their soured suspensions also exhibited an anti-inflammatory response through abdominal contorsions (28±2 % inhibition, n=5/group), whenever the animals were treated
Following this findings, cicatrizing activities of both kefir and purified kefiran (50 mg mL
The skin samples excised from the animals treated with kefir gel also presented a well developed granulation of the epithelium together with neovascularization areas, suggesting a partial healing in the treated group (Figure 13) .
A kefir gel prepared as above was also tested with a prior heat treatment of kefir, aiming do distinguish between probiotic and prebiotic effects of the consortium. In that job, an oitment developed from grinded grains at 70 % was topically used in cicatrizing assays, for testing their microbial resistance against different heat treatments . Cream samples were elaborated with prior treatment of kefir grains by autoclaving (15, 30, and 45 min), or by heating in a water bath at 55 °C, for 15 h. The kefir creams were then applied topically to a 8-mm wonded-induced dorsal area of rats (n=25/group), previously inoculated with
Intriguing, the group treated with autoclaved kefir grains also revealed a meaning decrease of lesion areas, greater than that presented for the negative control group (NaCl 0.9 %).
These findings happened to be so due to a nonproteic molecule taking part in the healing action to the animals, in agreement with the activities of the isolated AK molecule. Furthermore, all tested groups were able to enhance the epithelial tissue proliferation, as compared with the negative control group. In another inflammation model, anti-granuloma assays were also conducted with sugary and milk kefir, together with grinded grains (kefiran extract) and isolated AK. To do this, rats (n=5/group) were challenged with induction of granulomatous tissue by subcutaneously introduction of cotton pellets through abdominal skin incisions, following oral treatment with the agents after 2 h during 7 days  (Figure 15).
Both aqueous and milky kefir suspensions (50 g L-1) showed similar inhibition values (41±3 and 44±6 %, respectively), whereas the isolated kefiran from molasses suspension lead to a smaller inhibition (34±2 %). As kefir grains is known to stimulate innate immune responses against pathogens , we had evaluated the immune activity of neutrophils from rats treated with water kefir suspension . Then cytokine TNF-
18.104.22.168. Intestinal motility
Animal digestibility in rats has been also attempted with kefir samples . In that work it was evaluated changes in intestinal motility induced by a sugary kefir suspension daily administered (n=6/group, Wistar rats) during 15 days. After this period, the animals were kept without food during 24 h and treated with water kefir suspension, water, atropine (negative group), or acetylcholine (positive group). Following, the animals received orally 10 % active charcoal after 30 min. The animals were then submitted to euthanasia after 45 min and the intestinal tracts were exposed from the pylorus to cecum. As a result, kefir suspension was able to enhance intestinal transit up to 65±2 % (Figure 18), closer to the acetylcholine group, and greater than the negative groups. These results indicated an improvement of the peristaltic activity of the intestinal tract of the rats treated with kefir, and evoke its plausible use on treating bowel diseases and gut problems.
Based on the promising findings obtained with rodents, we had inspect some
22.214.171.124. Lipidemic activity
The intake of soured kefir was tested in the healthy rabbits to identify its plausible effects in serum cholesterol levels. Rabbits (n=10/group) were fed with kefir grains in natura mixed with reconstituted pelletized industrial rations during 30 days, following their growth and serum lipid assessments (total cholesterol, triglycerides, HDL, LDL, and VLDL) . The rabbits who received kefir grains in natura had significantly lesser growth than the control group. Besides, the fraction of total cholesterol and HDL had significant increases, with a mean reduction of the Castelli II index (LDL/HDL ratio) for the kefir group. This datum suggest the increase of total cholesterol as due to the increase of serum HDL, as measured from the rabbit auricular veins. As reported before  the total cholesterol levels has been reduced in broiler chicks fed with milk-fermented kefir, in agreement with above findings. In conclusion, these results would suggest that the probiotic can be thought for weight control therapies and prophylactic actions against dyslipidemies.
2.6. On plant
The addition of diverse compounds to plant culture medium has been successfully used for different species in tissue culture techniques. Banana and malt extract, as well as coconut water, e.g., is related to promote the growth of different species of orchids in micropropagation studies . Although the action of kefir in plant physiology is unknown, recent studies demonstrated that kefir was able to induce the synthesis of phytoalexins in soy cotyledons, and also inhibits germination in uredioniospores of
Furthermore, the biochemical data assessed from the micropropagated orchids (Figure 21) evidenced a meaningful increase of the carotene level (up to 24 times greater than control), total phenolic (33 %) and polyphenol oxidase activity (about 3 times greater than control). In this sense, the use of kefir in
The resulted treatment of micropropagated orchids (Figure 20) has been displayed a better organization and larger thickness of the mesophile as observed in culture media at 75 % kefir, when compared with the anatomical development of plants cultivated exclusively in Knudson medium .
Kefir can be considered an amazing example of coevolution of a microbial consortium. Their grains seems to simulate a multicellular living organism, as they are able to growth, divide, and age. From a survival point of view, kefir is very well adapted to resist to different and even extreme environments, also competing to a large spectrum of microbial strains. As kefir have acquired a strong resistance against several microorganisms, as well to improve the natural immunity of mammals since ancient ages, it is reasonable to think the consortium as a potential naturally-occurring drug able to decrease a large sort of illness afflictions.
The author gratefully acknowledge to all the students that have participated on the kefir studies summarized in this work, as well as the following Brazilian research support institutions, Minas Gerais State Research Foundation - FAPEMIG and National Council for Scientific and Technological Development - CNPq.
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