Kefir inhibits 3T3-L1 adipocyte differentiation through down-regulation of adipogenic transcription factor expression.

BACKGROUND: Kefir, a traditional fermented milk composed of microbial symbionts, is reported to have various health benefits such as anti-tumour, anti-inflammatory, anti-neoplastic and pro-digestive effects. In this study, to elucidate the effects of kefir on adipocyte differentiation and lipid accumulation, three fractions were prepared from kefir culture broth. The inhibitory effects of kefir liquid culture broth fraction (Fr-1), soluble fraction (Fr-2) and insoluble fraction (Fr-3), prepared by sonication of kefir solid culture broth, on adipocyte differentiation in 3T3-L1 preadipocytes were examined. RESULTS: Fr-3 (0.1 mg mL(-1)) significantly decreased lipid accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity by 60 and 68% respectively without affecting cell viability. In addition, Fr-3 treatment down-regulated the mRNA expression of adipogenic transcription factors including C/EBPα (32%), PPARγ (46%) and SREBP-1c (34%) during adipocyte differentiation compared with untreated control cells. The mRNA expression of adipocyte-specific genes (aP2, FAS and ACC) was also clearly decreased. CONCLUSION: The results suggest that the insoluble fraction of kefir (Fr-3) mediates anti-adipogenic effects through the inhibition of adipocyte differentiation, partly via suppression of the C/EBPα-, SREBP-1c- and PPARγ-dependent pathways.

Review: functional properties of kefir.

Kefir is a unique cultured dairy product due to combined lactic acid and alcoholic fermentation of lactose in milk. Kefir is produced by microbial activity of "kefir grains" which have a relatively stable and specific balance of lactic acid bacteria and yeast. Due to the claimed health benefits of kefir which include reduction of lactose intolerance symptoms, stimulation of the immune system, lowering cholesterol, and antimutagenic and anticarcinogenic properties, kefir has become an important functional dairy food and consequently, research on kefir has increased in the past decade. In the following review, recent studies on the functional properties of kefir are reviewed.

Short communication: Antiproliferative effect of wild Lactobacillus strains isolated from fermented foods on HT-29 cells.

In vitro studies, animal models, epidemiology, and human intervention studies provide evidence that some lactic acid bacteria can reduce the risk of certain cancers. In this study, heat-killed bacterial cells, genomic DNA, and cell wall of 7 wild Lactobacillus strains isolated from traditional fermented foods in western China were tested in vitro for cytotoxicity on colonic cancer cell line HT-29 by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The heat-killed bacterial cells, genomic DNA, and cell wall of the 7 strains exhibited direct antiproliferative activities against HT-29 cells. Among the strains, the cellular components of Lactobacillus coryniformis ssp. torquens T3L exerted marked antiproliferative activities against HT-29 cells. The maximum inhibition rates of HT-29 cells by the heat-killed bacterial cells (1x10(7) cfu/mL), cell wall (20 microg of protein/mL) and genomic DNA (100 microg/mL) of L. coryniformis ssp. torquens T3L were 30, 44.9, and 35.9%, respectively. The results indicate that the heat-killed bacterial cells, cell wall, and genomic DNA of the 7 wild Lactobacillus strains could inhibit the growth of HT-29 cells.

Assessment of the sequential simulated gastrointestinal tolerance of lactic acid bacteria from kefir grains by response surface methodology.

The tolerance of lactic acid bacteria (LAB) from kefir grains to gastrointestinal tract conditions was evaluated in vitro. The effects of pH values and bile salts on the viability of LAB were investigated. The results demonstrated that pH value showed a significant effect on the viability. The viable counts exhibited a reduction of 1.5 to 2 log cycles in 0.3% to 0.5% bile salts after 4 h. The viability of LAB exposure to sequential simulated gastric and intestinal juices was assessed by response surface model (RSM). RSM indicated that the gastric pH and gastric contact time significantly affected the viability (P < 0.05), while the effect of intestinal contact time was not significant. Moreover, RSM revealed the interactions of pH and gastric contact time, and of pH and intestinal contact time. The LAB cells, temporarily damaged by the low pH of gastric juice (pH < 2), could recover in the intestinal juice; and the longer the intestinal contact time, the higher the viability of LAB. RSM proved to be a useful and accurate method to predict the viability of LAB under certain laboratory conditions by the model validation. This study indicated that LAB from kefir grains exhibited excellent tolerance to sequential simulated gastrointestinal tract conditions, and that kefiran possessed a significant protective effect on LAB in hostile environments.

The effect of kefir on glutathione (GSH), malondialdehyde (MDA) and nitric oxide (NO) levels in mice with colonic abnormal crypt formation (ACF) induced by azoxymethane (AOM).

In this study we investigated the effect of kefir on the levels of glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO) in the liver, stomach, spleen and colon of mice with colonic aberrant crypts formed by azoxymethane (AOM). Thirty 12 weeks old Swiss Albino mice averaging 31.5 g weight were used as experimental animals. The mice were separated into 3 groups. The first group was the control group, second group was the AOM and third group was the AOM+kefir group. We applied AOM to the second and third groups. Mice were fed ad libitum by laboratory rodent chow during the experiment period. Water was given to the first and second groups and third group received only kefir diluted with water (50%). AOM was injected subcutaneously to the second and third groups for 7 weeks (two times a week, 5 mg/kg). Six weeks after the final AOM treatment the animals were sacrificed and liver, stomach, spleen and colon samples were collected from all the groups. MDA level demonstrated an increase only in stomach for the third group (p < 0.001), while an elevation was observed for all of the four organs for the second group (spleen p < 0.001, liver p < 0.001, colon p < 0.01). GSH level showed an increase in the second group at stomach (p < 0.01) and colon (p < 0.001), while in the third group, a small increase was determined only at the colon (p < 0.05). NO level increased at all of the organs in the second group (spleen, liver, colon p < 0.001, stomach p < 0.05), but only at liver and colon in the third group 3 (p < 0.001). In conclusion these results showed that kefir plays an antioxidant role.

Inhibitory effect of fermented milk on delayed-onset muscle damage after exercise.

Milk fermented with a starter containing Lactobacillus helveticus and Saccharomyces cerevisiae is drunk on a daily basis by many people in Japan and has several beneficial effects. We studied the influence of this fermented milk product on muscle damage after prolonged exercise in rats. Wistar rats were divided into four groups: rested controls, rested rats given fermented milk diet, exercised rats and exercised rats given fermented milk diet. After 3 weeks of acclimatization, both exercise groups were made to run on a treadmill at 26 m/min for 60 min. Exercise increased the serum creatine kinase level, as well as myeloperoxidase activity and the level of thiobarbituric-acid-reactive substances in the gastrocnemius muscle after 24 h. These changes were ameliorated by intake of fermented milk. An increase of CINC-1 was also ameliorated by fermented milk. Furthermore, milk diet increased the mRNA and protein levels of protective proteins such as antioxidants and chaperone proteins. These results indicate that fermented milk can ameliorate delayed-onset muscle damage after prolonged exercise, which is associated with an increased antioxidant capacity of muscles.

Antihypertensive effect of peptides obtained from Enterococcus faecalis-fermented milk in rats.

Previous studies have demonstrated that milk fermented with Enterococcus faecalis decreases the systolic blood pressure (SBP) and the diastolic blood pressure (DBP) of spontaneously hypertensive rats. In this study, we evaluated the antihypertensive activity of the following peptide sequences: LHLPLP, LHLPLPL, LVYPFPGPIPNSLPQNIPP, VLGPVRGPFP, and VRGPFPIIV. These peptides isolated from E. faecalis-fermented milk showed in vitro angiotensin I-converting enzyme-inhibitory activity. Because the most potent angiotensin I-converting enzyme-inhibitory sequences were LHLPLP and LVYPFPGPIPNSLPQ-NIPP, we administered different doses of these peptides to spontaneously hypertensive rats. High doses of the remaining sequences were also administered to these animals. Water served as a negative control and captopril as a positive control. All products were administered orally. The SBP and DBP were measured before administration and also at 2, 4, 6, 8, and 24 h after administration. Before administration of the different products, spontaneously hypertensive rats showed SBP and DBP values of 218 +/- 2.5 and 157 +/- 5.9 mmHg, respectively (n = 30). The sequences LHLPLP, LVYPF-PGPIPNSLPQNIPP, VLGPVRGPFP, and VRGPFPIIV caused clear and significant decreases in SBP, DBP, or both in the animals. In particular, the antihypertensive effect could be clearly established when 2 or 3 mg/kg of LHLPLP was administered. These 2 doses of LHLPLP showed similar antihypertensive properties. Four hours after administration of captopril or the highest doses of the different peptides, the decreases in the SBP and the DBP (mmHg) were as follows: captopril (SBP = 52 +/- 5.8, DBP = 38.8 +/- 3.8), 3 mg/kg of LHLPLP (SBP = 25.3 +/- 8.2, DBP = 29.5 +/- 7.6), 6 mg/kg of LVYPFPGPIP-NSLPQNIPP (SBP = 14.9 +/- 3.7, DBP = 8.7 +/- 4.4), 10 mg/kg of LHLPLPL (SBP = 7.7 +/- 4.1, DBP = 9.4 +/- 3.1), 10 mg/kg of VLGPVRGPFP (SBP = 16.2 +/- 5.8, DBP = 21.64 +/- 3.2), and 10 mg/kg of VRGPFPIIV (SBP = 16.05 +/- 2.74, DBP = 9.19 +/- 3.49). The results obtained suggest that the sequences LHLPLP, LVYPFPGPIPNSLPQ-NIPP, VLGPVRGPFP, and VRGPFPIIV could be responsible, at least in part, for the antihypertensive properties described for E. faecalis-fermented milk.

Effect of milk fermented with Lactobacillus acidophilus strain L-92 on symptoms of Japanese cedar pollen allergy: a randomized placebo-controlled trial.

A placebo-controlled, single-blind study was conducted to evaluate the effects of Lactobacillus acidophilus strain L-92 (L-92) on the symptoms of Japanese cedar-pollen allergy. This study was carried out during the 2002 and 2003 seasons of Japanese cedar pollination. Twenty-three in-house volunteers were asked to drink 100 ml of heat-treated milk fermented with L-92 containing 5 x 10(10) of the bacteria, twice a day, for 6 consecutive weeks. A similar study was carried out during the 2003 season for 10 weeks, but the daily dose of bacteria was 2 x 10(10). A significant improvement of the ocular symptom-medication score (SMS) was observed in 2002 and of the score of distress of life in 2003. These data show that a daily oral intake of not less than 2 x 10(10) heat-treated L-92 cells improved the symptoms of Japanese cedar pollinosis, thereby contributing to reduce the dose of concomitant medications. However, no blood parameter was significantly affected in these trials.

Effect of fermented milk containing probiotic bacteria in the prevention of an enteroinvasive Escherichia coli infection in mice.

This study investigated the protective capacity of the oral administration of fermented milk containing the probiotic strains; Lactobacillus casei, Lb. delbrueckii subsp. bulgaricus and Streptococcus thermophilus, against enteroinvasive Escherichia coli infection in a murine (BALB/ c mice) model. Mice were fed for 2, 5 or 7 consecutive days with fermented milk diluted to a concentration of viable Lb. casei, Lb. delbrueckii subsp. bulgaricus and Strep. thermophilus of 10(7) cfu/ml. Phagocytic activity of peritoneal macrophages and the number of IgA+ cells in small and large intestine were determined at the end of the feeding periods. For the preventive effect against Esch. coli, animals were fed for 5 days (selected dose). Mice were challenged with an infective dose of enteroinvasive Esch. coli of 10(8) cfu/mouse. The colonization of liver and spleen and the secretory IgA specific for the pathogen in the intestinal fluid were determined (ELISA test). Results showed that the unspecific immune response enhanced itself after 5 consecutive days of the administration of this fermented milk (increase in the percentage of phagocytosis and number of IgA+ cells in the small intestine). Treated animals showed less Esch. coli colonization of liver than control mice and a higher secretory anti-Esch. coli IgA in the intestinal fluids. These results suggest that the protection against enteroinvasive Esch. coli infection observed for the fermented milk containing probiotic bacteria may be associated with an enhance of the intestinal mucosa immunity.