Exploring the Antimicrobial and Hepatoprotective Effects of Kefir; A Probiotic Fermented Milk

1Faculty of Post Graduate Studies for Advanced Sciences. Beni-Suef University, Beni-Suef 62511, Egypt. 2 Faculty of Pharmacy, Microbiology and Immunology Department, Beni-Suef University, Beni-Suef 62511, Egypt. 3Faculty of Veterinary Medicine, Virology Division, Dep. of Microbiology, South Valley University, Qena 83523, Egypt. 4Faculty of Veterinary Medicine, department of pathology, Beni-Suef University, Beni-Suef 62511, Egypt.

"Kefir is gotten from the Turkish word "keif" which signifies "nice feeling" 1 and the drink started in the Caucasian heaps of Russia 2, 3,4 . Kefir is obtained from the fermenting activity of Kefir grains 5 . Traditionally, it is fermented in goatskin bags for 24 hours 6 . Kefir contains many ingredients that demonstrate biological activity, such as some probiotic bacteria and bioactive peptides 7 and onsets of activity varies according to the type of kefir and the time of fermentation 8 . It is self-carbonated fermented milk with a slightly acidic taste 2 . The kefir drink is produced from cow, goat, sheep 3 , camel, buffalo or soy milk 4, 9 that could be whole fat, low-fat, skimmed or fat-free milk 1 . This difference in the milk type and methods of fermentation affects the amount of grain produced, food composition and flavor of kefir 2 . Kefir grains are considered to be the most important component in the production of fermented kefir and can be reused again 10 . It contains many types of bacteria in addition to proteins and polysaccharides 11,12,13 . Although the kefir drink can be found in many countries, in Egypt the grains are not commercially available and are culturally donated from person to person. Partial sequencing of the gene encoding 16S rRNA was used for species identification 14,15 . Fermented milk produced by kefir grains contains yeast and lactobacilli 16,17,18 . Kefir has many applications in a variety of medical conditions such as; high blood pressure, allergy problems and coronary heart disease. Also, it strengthens the immune system and improves the digestive health. Kefir antimicrobial activity is associated with the production of organic acids, peptides (bacteriocins), carbon dioxide, hydrogen peroxide, ethanol and diacetyl 15,19 .
The main objective of this study was to investigate the antimicrobial activity of the fermented kefir in vitro against different pathogens and to evaluate its hepatoprotective effect in mice.

Preparation and characterization of kefir
Kefir grains were collected from Qena City, Egypt. They were varying in size (from 0.3 to 3.0 cm in diameter), irregular in shape 20. 21 as shown in (Fig. 1), and white to yellowish-white in color 17,18,19 . Also, the grains were flexible, softer in texture, viscous 12,17,18 and insoluble in water and common solvents. When milk was added, the grains swelled and produced a jellied called Kefiran 22 . Kefir has a pH of 4.2 -4.6, an ethanol content of 0.5 -2.0% (v/v), a lactic acid content of 0.8-1.0% (m/v), a carbon dioxide content of 0.08 -0.2% (v/v) 23 The grains were recovered by sieving, mass of grain increased by 6% after the fermentation process and microorganisms found in the milk are different from those produced after the fermentation process 15, 22 . Kefiran was prepared by adding 100 g of kefir grains to 500 ml of purified milk at 25ºC in a dark place for 24 h -48 h. Kefir grains were separated from the fermented milk by plastic sieve 24,25 .

The antimicrobial activity of the fermented kefir
Screening for antibacterial activity of the Kefir fermented milk was done using agar diffusion method (26,27) against Gram-positive bacteria (Staphylococcus aureus ATCC 44330 and Bacillus subtilis), Gram-negative bacteria (Escherichia coli ATCC 5087, Salmonella enteritidis and Pseudomonas aeruginosa), and yeast (Candida albicans).
The Kefir fermented milk was filter sterilized using 0.45 um membrane filter and the indicator microorganisms were incubated overnight in brain heart infusion broth (Oxide) at 37°C. The antimicrobial activity was done based on seeding inoculation of each indicator microorganism in 20 ml Muller Hinton agar (Oxide), and then cups were prepared using Wassermann tubes with an external diameter of 5 mm. A fixed amount of 50 µl, 100 µl and 150 µl of tested kefir solution was distributed to each well. The plates were incubated for 24 h at 37°C. A positive control of antibiotic ampicillin (10 mg/ml) was also tested. Estimation of antimicrobial activities was done by measuring diameters of zone of inhibitions.

Identification of microorganisms isolated from fermented kefir by partially sequencing of 16S rRNA gene
The Kefir fermented milk was platted on MRS agar (Oxide) and Brain heart agar (Oxide) for 2 days at 25°C to isolate major lactic acid bacteria and other microorganisms in kefir and then a DNA extraction was done followed by PCR and DNA sequencing.

Genomic DNA extraction and purification
Genomic DNA extraction was done according to 27 with some modifications. Briefly, a 1.5 ml of culture was centrifuged for 10 min at 3,000 g, the supernatant was discarded and the pellets were resuspended in 200 µl spheroblast buffer (10% sucrose, 25 mM Tris pH 8.4, 25 mM EDTA pH 8.0, 2 mg/mL lysozyme and 0.4 mg/ ml RNase A), vortexes and incubated at 37 ºC for 10 minutes until cell lysis occurred. Then, 50 µl of 5% SDS (lysis buffer 1) and 5 M NaCl (lysis buffer 2) were added, mixed and incubated at 65ºC for 5 minutes. A 100 µl neutralizing buffer (60 ml 5M Potassium acetate, 11.5 ml glacial acetic acid, and 28.5 ml dH 2 O) was then added and put on ice for 5 min before centrifugation at 18,000 g at 4ºC for 15 minutes. The supernatant (approximately 400 µl) was transferred to a new tube, mixed with equal volume of isopropanol, left 5 minutes at room temperature and centrifuged at 18,000 g at room temperature for 15 minutes to precipitate the DNA. The resulting pellet was washed with 70% ethanol by centrifugation at 18,000 g at room temperature for 5 minutes. The final pellet was air-dried and resuspended in 50 µl 1 x TE buffer pH 8 and stored in the refrigerator at 4ºC.

PCR amplification and sequencing of bacterial 16S rRNA gene
PCR was carried out in 50 µl reaction volume in sterile 200 µl PCR tube. The PCR reaction mixture consisted of 500 ng genomic DNA, 10 mM dNTPs mixture, 1 µl (20 uM of each primer); forward primer 5'-TAACACATGCAAGTCGAACG-3' and reverse primer 5'-AAACTYAAAKGAATTGACGG-3', 2.5 units of Taq DNA polymerase enzyme and 10 µl 5X reaction buffer. The PCR program included template denaturation at 94 ºC (3 min), followed by 34 cycles of denaturing at 94 ºC (30 sec.), annealing at 56 ºC (30 sec.), and extension at 72 ºC (60 sec.), and followed by completion of DNA synthesis at 72 ºC (5 min). Primers were removed from the final PCR product prior to sequencing using QIAquick PCR purification kit (QIAGEN, Germany). The PCR product of interest was detected and purified by agarose gel electrophoresis using 1% (w/v) agarose gels with reference to 1 kbp DNA ladder. DNA was sequenced using the ABI Prism BigDye terminator sequencing ready reaction kit version 3.1 and analyzed with the ABI Prism 3100 generic analyzer.

Sequence manipulation and phylogenetic analysis
Searching for DNA sequence homology was done using BLAST tool at NCBI database (www.ncbi.nlm.nih.gov/blst) in order to assess the degree of DNA similarity. Multiple sequence alignment and molecular phylogeny were constructed using MEGA7 software 28 . Evaluation of the protective ability of Kefir against carbon tetrachloride CCl 4 -induced liver toxicity in mice Animal grouping and treatment Three weeks old, clinically healthy, female Swiss albino mice (n=40) weighting 26-30 g were randomly divided into 4 groups (10 mice/ group) after 7 days adaptation. They were housed in stainless-steel wire-mesh cages (four in a cage), at 24±2ºC temperature, 55% relative humidity and a 12 h light-dark cycle. The animals were provided a normal diet and tap water. The groups were separately treated for as following : Group I, animals were sham-treated with 2 ml/ kg distilled water through oral gavage, daily for 4 weeks; this group of animals served as the control. Group II, animals were treated with 1.5 ml/kg body weight (b.w.) CCl 4 dissolved in 1.5 ml corn oil through oral gavage, daily for 4 weeks. Group III, animals were treated with 1.5 ml/kg b.w. CCl 4 + 30 ml/kg b.w kefir through oral gavage, daily for 4 weeks. Group IV, animals were treated with 30 ml/kg b.w. Kefir through oral gavage daily for 4 weeks.

Preparation of Fermented Kefir to feed animals
The compound was prepared by washing the kefir grains with distilled water and raw milk, after that heated to 90 °C for 10 min in a water bath, then cooled to inoculation temperature (25 °C) and 10% active kefir grains added. The mixture was placed in a plastic container with screen cloth as a cover and incubated at room temperature for 24-48 hrs. A plastic container is used because the acidity of fermented kefir may degrade metals such as aluminum and iron which could mix with the drink thereby causing harmful effects to the body 29 . After fermentation, kefir grains were sieved by filtration through a plastic sieve and washed for another process 29 . Kefir drink was maintained at 4 ± 1ºC for 24 h and then used for microbiological and chemical analyses before feeding the animals in group III, kefir samples which were stored for more than 3 days were not used. Animal treatment was continued for 4 weeks then the experiment was concluded and animals were killed under anesthesia, blood samples were collected and livers, kidneys and spleen were rapidly removed then weighted to calculate relative liver weight to body weight.

Biochemical analysis
Each blood sample was placed in dry clean centrifuge tube, and then centrifuged for 10 min at 3000 revolutions per minute (rpm) to separate the serum. Serum was carefully separated into clean dry Wasser man tubes by using a Pasteur pipette and used for determination of serum liver function tests; aspartate aminotransferase (AST) (biolab), alanine aminotransferase (ALT) (biolab) and alkaline phosphatase (ALP) (biolab)] using standard techniques by manufactures.

Histopathological examination
Tissue samples were collected from liver, kidney and spleen of all animals (Group I-IV). These samples were fixed in neutral formalin solution 10% for 72 hrs., after that fixed samples were processed and stained by Hematoxylin and Eosin according to 30 .

The antimicrobial activity of kefir against pathogenic microorganisms
Antibacterial activity of kefir, freshly fermented for 24 hrs and 48 hrs or stored for 72 hours at 4 -8°C, was estimated by agar diffusion method. It was noted that Kefir has antibacterial activity against Staphylococcus aureus, E. coli and Salmonella enteritidis. For E.coli and S. enteritidis, the antimicrobial activity was superior to control antibiotics. The tested products exhibited no activity against P. aeruginosa and C. albican. The results demonstrated that kefir possesses high antibacterial potentials against Gram-negative and Gram-positive strains (Fig. 2).

Microorganisms identification by partially sequencing the 16S rRNA and phylogenetic analysis
Identification of isolates to the species level was based on sequencing of 16S rRNA gene. The PCR of 16S rRNA gene using specific primers was done and revealed positive reactions and correct amplicon sizes. DNA sequencing was done and the obtained sequences were analyzed using both Bioedit v. 7.0.9.0 and CLC sequence analyzer programs. The homology search of the obtained sequences using BLAST tool at NCBI database was done to categorize the microorganisms to the closest species as in (Table 1). The isolated microorganisms were identified as Micrococcus cohnii (isolate ID SK14), Lactobacillus kefiranofaciens ZW3 (isolate ID SK22) and Lactobacillus casei strain KF11 (isolate ID SK23).
The resulted sequences were aligned to the closely related microorganisms by retrieving their sequences from the NCBI GenBank database and assembled in MEGA7 software for phylogenetic analysis using the Neighbor-Joining method and the evolutionary distances were computed using the Kimura 2-parameter method as seen in (Fig. 3).

Protective effect of Kefir against risk of carbon tetrachloride-induced liver toxicity and other damages in mice Effect of treatment on body weight and relative liver weight
Effect of treatment on body weight and relative liver weight to body weight were estimated as illustrated in (Table 2); the ratio of liver weight  By comparing the total body weight at the end of experiments to its corresponding initial value, only CCl 4 group exhibited a significant decrease compared to its corresponding initial weight ( Table 2). Note northerly, the body weights exhibited by combination group had higher values compared to both initial body weight and body weights exhibited by CCl 4 -treated group, however, it is still less than the control values as illustrated in (Table 2).
Data were calculated as relative weight of liver to 100 g animal body weight at the end of the experiment. Data are presented as mean ± standard error of 10 animals/group. a, b or *, c indicates significant difference from control, kefir or corresponding initial body weight respectively at p<0.05 using Tukey's test as post ANOVA test and as showing in (Fig. 4).

Effect of treatment on liver function
The serum levels of liver functions (AST, ALT, and ALP) are presented in (  (Fig. 5).
-Data were presented as mean ± standard error of 10 animals/group. -a, b or c indicates significant difference from control, kefir or CCl 4 respectively at p <0.05 using Tukey's test as post ANOVA test. Fig. 3. Phylogenetic tree of the fermented kefir derived isolates based on partial 16S rRNA gene sequences. The phylogenetic tree was inferred using the Neighbor-Joining method (43). The distances were computed using the Kimura 2-parameter method (44) and are in the units of the number of base substitutions per site. Numbers at nodes indicate percentages of 1000 bootstrap re-samplings. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA7 (45) -ALP: Alkaline phosphatase; AST: Aspartyl aminotransferase; ALT: Alanine aminotransferase.

Control group
In the control group, the liver was histologically normal without noticeable alterations ( Fig. 6a) with well demarcated hexagonal lobules having a central vein with normal portal traits containing artery, vein and bile ducts. The hepatocytes within the hepatic lobules were arranged in cords radiating around the central veins and separated by hepatic sinusoids.

Carbon tetrachloride (CCl 4 ) group
In this group, the liver of the animal showed variable degrees of degeneration, necrosis and inflammation. Moderate to severe vacuolation of hepatocytes (hydropic degeneration to fatty changes) were seen especially at the periphery of the lobules (Fig.6b), in contrast to those around central veins which appeared normal. Mild to moderate sinusoidal dilatation with active proliferation of van Kupffer cells were noticed in most animals (Fig. 6c). Necrotic changes accompanied with fatty changes were also found in some areas; in which the hepatocytes had pyknotic nuclei with strong eosinophilic cytoplasm or severe destruction (Fig. 6d). Mild to moderate portal tracts and hepatic parenchyma infiltration with leucocytes was evident (Fig. 6e) with congested blood vessels.

In CCl 4 -kafir treated group
Histopathological examinations proved mild to moderate improvements in the form of absence of hepatic necrosis, however, some cells showed degenerative changes (Fig. 6f &g). Sings of regeneration was noticed in some areas as some cells showed mitotic activities and binucleation

Kidneys Control group
The renal tissue of this group appeared of normal structure especially the renal cortex (Fig. 7a). Renal tubules and glomeruli were histologically normal. The tubules were linned by columnar epithelium and Bowman's capsules were normal and had normal glomeruli. Minimal changes were noticed in some areas in renal medulla. The renal pelvis also appeared normal. c d e f g CCl 4 -treated group Histopathological examination revealed mild pathological alterations. The most common changes were hydropic degeneration of renal epithelium of some tubules (Fig. 7b) with mild congestion of some blood vessels (Fig. 7c). Early necrotic changes were demonstrated in outer cortex. Glomerulonephritis was seen some areas (Fig. 7d). Marked vacuolation was evident in renal medulla. No signs of an inflammatory reaction could be seen except in one case, which appeared as focal mononuclear cell infiltration in the renal cortex (Fig. 7e).

CCl 4 -Kefir treated group
The histopathological changes in these animals were much less in comparing to the CCl 4 -treated group. Renal tubules had minimal pathological changes (hydropic degeneration) in most areas with mild congestion and without inflammatory changes (Fig. 7f&g). The glomeruli showed no histopathologic alterations.

Spleen Control
In the control group, spleen appeared more or less normal at the level of white and red pulps (Fig. 8a).

CCl 4 -treated group
With regards to the spleen histopathological examination, splenic changes involved hyperplasia of lymphoid follicles of the white pulp (Fig. 8b). In some follicles mild lymphocyte destruction (rarefication) was found. Moreover, the red pulp of splenic tissues showed significant congestion of blood vessels and sinusoids with mild edematous changes (Fig. 8c).

CCl-kefir treated group
The remedy effect of Kefir was observed in splenic tissues that revealed no prominent lymphoid hyperplasia in the white pulp or congestion in the red pulp; so it appeared to be more or less normal (Fig. 8d).

Statistical analysis
All data were expressed as means ± standard error of the mean (S.E.M). Statistical analysis was done using statistical packages for social sciences (SPSS) computer software (version 22), IBM software, USA. One-way analysis of variance (ANOVA) test was used to elucidate significance among group means, followed by Tukey's post-hoc test to compare mean values pair-wise. Differences were considered significant at p <0.05.

DISCUSSION
Recently, there is a strong focus on beneficial foods with probiotic microorganisms and functional organic substances especially the commercial use of kefir. It may act as a matrix in the effective delivery of probiotic microorganisms in different types of products as it has a biological activity due to the presence of kefir's exopolysaccharides, known as kefiran 22 . Kefir is mainly considered a probiotic resource because of its composition 31 . According to definition "Probiotics are microbial cell preparations or components of microbial cells with a beneficial effect on the health of the host". Some studies suggest that probiotic bacteria in kefir consumers' gut are increased and play an important role in health improvement 9,32 .
The probiotic species, particularly lactobacilli are equipped for creating an extensive variety of antimicrobial mixes, counting natural acids (lactic and acidic acids), carbon dioxide, hydrogen peroxide, ethanol, diacetyl and peptides (bacteriocins) that can be helpful not just in diminishing sustenance pathogens and bacterial harm amid capacity and sustenance consumption, additionally in the treatment and counteractive action of gastrointestinal and vaginal infection 25 kefiran has more advantages, comparing to other polysaccharides, such as bactericidal, fungicidal, antitumor properties 33,34 anti-inflammatory and promote healing 35,36 , immunomodulation or epithelium protection 37 and antioxidant activity 38 .
Our results demonstrated that after 24 h as well as 48h, fermented kefir possesses high antibacterial activity against Gram-negative and Gram-positive including Staphylococcus aureus, E. coli and Salmonella Enteritidis. The antimicrobial activity was superior to control antibiotic, although exhibited no activity against P. aeruginosa and C. albican. These results agree with previous study which showed that kefir as a probiotic can restrain the action of coliform microscopic organisms, and some entero pathogenic microscopic organisms like Shigella sp., Salmonella sp., and of Grampositive microorganisms, for example, S. aureus, Bacillus cereus, Clostridium sp. and Listeria monocytogenes 39 .
M i c r o c o c c i , l i k e m a n y o t h e r representatives of the Actinobacteria, can be metabolically versatile, with the ability to utilize a wide range of unusual substrates, such as pyridine, herbicides, chlorinated biphenyls, and oil. They are likely involved in detoxification or biodegradation of many other environmental pollutants Other Micrococcus isolates produce various useful products, such as long-chain (C 21 -C 34 ) aliphatic hydrocarbons for lubricating oils 40 .
An exopolysaccharide (EPS) producing strain, ZW3, was isolated from Tibet kefir grain and was identified as Lactobacillus kefiranofaciens. FT-IR spectroscopy revealed the presence of carboxyl, hydroxyl, and amide groups, which correspond to a typical hetero polymeric polysaccharide. The GC analysis of ZW3 EPS revealed that it was glucogalactan in nature 34 .
Lactobacillus casei is a species of the genus Lactobacillus found in the human intestine and mouth. This particular species of Lactobacillus is documented to have a wide pH and temperature range, and complements the growth of L. acidophilus, a producer of the enzyme amylase (a carbohydrate-digesting enzyme) 25 .
Kefir was found to have a protective effect against CCI4-induced damage in liver, spleen and kidney. Histopathologically, compared to CCl 4 treated mice, mild to moderate improvements in the form of absence of neither hepatic degeneration nor necrosis with sings of regeneration (increased mitotic activities and bi-nucleation). The hepatic sinusoids appeared also of normal appearance with mild. Renal tissues showed minimal degeneration. Spleen also showed marked improvement comparing to CC1 4 treated animals.
Kefir has a histopathological preventive attribute in animal model as it lower the necrobiotic changes in acute renal injury. The adverse findings of CCl 4 (hepatocellular damage and apoptosis) were reduced with kefir administration; this indicating that kefir has a protective role at liver damage 41 . Also it has been found that no toxic effect of L. kefiranofaciens M1 was seen at the gross and microscopic histopathology of the organs (heart, liver, kidney, adrenal glands, spleen, ovary, and testis) 42 .
Kefir was chosen in our study as a potential protective agent because of its antioxidant and hepatoprotective activity. According results of this study, CCl 4 induced liver toxicity in mice and it is harmful to other organs such as kidney and spleen which was in the form of increased liver weight to body weight, elevated liver enzymes and alkaline phosphatase, an indication of structural and functional defects in liver cells. Marked improvement was evident with treatment with kefir as indicated by estimation of body weight and relative liver weight to body weight; the ratio of liver weight to 100 g body weight was significantly increased by sole administration of CCl 4 (1.9900±.13565, p <0.05) compared to control animals (1.1070±.04842). Interestingly treatment with both kefir and CCl 4 exhibited liver weight/100 g body weight ratio (1.3900±.08741) which is significantly lower than CCl 4 group (p <0.05) and was close to normal value.
At the end of experiment, comparing the animal total body weight to its corresponding initial value, only CCI 4 group exhibited a significant decrease compared to its corresponding initial weight. Note northerly, the body weights exhibited by combination group had higher values compared to both its initial body weight and body weights exhibited by CCl 4 -treated group, however, it is still less than the control value. Data were calculated as relative weight of liver to 100 g animal body weight at the end of the experiment. Data are presented as mean ± standard error of 10 animals/group.
Kefir effectively has protection against CCl 4 -induced hepatotoxicity in mice. These protections are approved via the serum levels of liver functions (AST, ALT, and ALP). In the CCl 4 treated group, the serum levels of AST, ALT, and ALP p <0.05, were increased to 1372.6367 ± 2.06498, 1410.2500 ± 2.60688 and 251.4583 ± 16.79796 respectively compared to negative control group values of 38.1200±.60255, 45.0820±.80311 and 67.8300 ±.50400 respectively. The pretreatment of CCl 4 -treated mice with kefir significantly p <0.05, decreased the CCl 4 induced elevation of these markers levels to 561.5050±2.79362, 472.8833±1.85210, 112.5600 ±2.62721, respectively. Interestingly, kefir administration does not exhibit any significant change from control values of liver functions that is mean the Kefir protects liver against carbon tetrachloride.
In conclusion, our findings revealed that kefir has antimicrobial activity against pathogenic microorganisms and protective properties against CCl 4 -induced hepatotoxicity. These protective effects included anti-inflammatory effect and inhibition of CCl 4 activity with improving of liver functions. So, kefir may have the potential for clinical applications to the prevention and/or treatment of liver toxicity.