Graciela L. Garrote

Inhibitory Power of Kefir: The Role of Organic Acids

Milk and MRS broth fermented with kefir grains from different households were examined for inhibitory activity toward gram-negative and gram-positive strains. Fermented milk obtained with 10 g per 100 ml of inoculum (final pH 3.32 to 4.25) and MRS broth fermented with 1 and 10 g per 100 ml of inocula (final pH 4.18 to 5.25) had inhibitory power demonstrated by spot test and agar well diffusion assay. This inhibitory effect could be assigned to the undissociated form of lactic and acetic acid produced during the fermentation process. Kefir supernatants inhibited the growth of Escherichia coli 3 in nutrient broth at 37 degrees C for 24 h. However, supernatants of yogurt or milk artificially acidified with lactic and acetic acids allowed the growth of E. coli 3 in the same conditions. A bacteriostatic effect of milk fermented with kefir grains over E. coli 3 was also demonstrated.

Lactate and short chain fatty acids produced by microbial fermentation downregulate proinflammatory responses in intestinal epithelial cells and myeloid cells.

The use of short chain fatty acids to modulate gastrointestinal inflammatory conditions such as ulcerative colitis has produced encouraging results either in animal models or also in clinical trials. Identifying the key cellular and molecular targets of this activity will contribute to establish the appropriate combinations/targeting strategies to maximize the efficacy of anti-inflammatory interventions. In the present work, we evaluated in vitro the interaction of lactate, acetate, propionate and butyrate on cells relevant for innate immune response of the gastrointestinal tract. All molecules tested regulate the production of proinflammatory cytokines by TLR-4 and TLR-5 activated intestinal epithelial cells in a dose response manner. Furthermore SCFAs and lactate modulate cytokine secretion of TLR-activated bone marrow derived macrophages and also TLR-dependent CD40 upregulation in bone marrow derived dendritic in a dose-dependent manner. Butyrate and propionate have been effective at concentrations of 1 to 5mM whereas acetate and lactate produced modulatory effects at concentrations higher than 20-50mM in different assays. Our results indicate that in concentrations similar to found in large bowel lumen, all SCFAs tested and lactate can modulate activity of relevant sentinel cell types activated by TLR signals. Modulatory activity was not inhibited by pertussis toxin treatment indicating that the effects are not related to Gi signaling. The use of these molecules in combined or separately as intervention strategy in conditions where epithelial or myeloid cells are main triggers of the inflammatory situation seems appropriate.

Kefir grains as a starter for whey fermentation at different temperatures: chemical and microbiological characterisation

We report here a comparative analysis of the growth, acidification capacity, and chemical and microbiologic composition between kefir grains after 20 subcultures in whey at 20, 30, and 37°C and the original kefir grains coming from milk along with a determination of the microbiological composition of the fermented whey as compared with that of traditional fermented milk. When fermentation was carried out repeatedly at 30 or 37°C, kefir grains changed their kefir-like appearance, exhibited reduced growth rates, had a lower diversity of yeasts and water content, and a higher protein-to-polysaccharide ratio compared with the original kefir grains. In contrast, at 20°C kefir grains could remain in whey for prolonged periods without altering their acidification capacity, growth rate, macroscopic appearance or chemical and microbiologic composition-with the only difference being a reduction in certain yeast populations after 20 subcultures in whey. At this incubation temperature, the presence of Lactobacillus kefiranofaciens, Lb. kefir, Lb. parakefir, Lactococcus lactis, Kluyveromyces marxianus, Saccharomyces unisporus, and Sac. cerevisiae was detected in kefir grains and in fermented whey by denaturing-gradient-gel electrophoresis (DGGE). In whey fermented at 20°C the number of lactic-acid bacteria (LAB) was significantly lower (P<0·05) and the number of yeast significantly higher (P<0·05) than in fermented milk. Since the DGGE profiles were similar for both products, at this temperature the microbiologic composition of fermented whey is similar to that of fermented milk. We therefore suggest a temperature of 20°C to preserve kefir grains as whey-fermentation starters.

Inhibitory Activity of Cheese Whey Fermented with Kefir Grains

We investigated the chemical and microbiological compositions of three types of whey to be used for kefir fermentation as well as the inhibitory capacity of their subsequent fermentation products against 100 Salmonella sp. and 100 Escherichia coli pathogenic isolates. All the wheys after fermentation with 10% (wt/vol) kefir grains showed inhibition against all 200 isolates. The content of lactic acid bacteria in fermented whey ranged from 1.04 × 10(7) to 1.17 × 10(7) CFU/ml and the level of yeasts from 2.05 × 10(6) to 4.23 × 10(6) CFU/ml. The main changes in the chemical composition during fermentation were a decrease in lactose content by 41 to 48% along with a corresponding lactic acid production to a final level of 0.84 to 1.20% of the total reaction products. The MIC was a 30% dilution of the fermentation products for most of the isolates, while the MBC varied between 40 and 70%, depending on the isolate. The pathogenic isolates Salmonella enterica serovar Enteritidis 2713 and E. coli 2710 in the fermented whey lost their viability after 2 to 7 h of incubation. When pathogens were deliberately inoculated into whey before fermentation, the CFU were reduced by 2 log cycles for E. coli and 4 log cycles for Salmonella sp. after 24 h of incubation. The inhibition was mainly related to lactic acid production. This work demonstrated the possibility of using kefir grains to ferment an industrial by-product in order to obtain a natural acidic preparation with strong bacterial inhibitory properties that also contains potentially probiotic microorganisms.

Cheese whey fermented with kefir micro‐organisms: Antagonism against Salmonella and immunomodulatory capacity

The antagonistic effect against Salmonella spp. and the immunomodulatory capacity of whey fermented with kefir grains and with three strains isolated from them – Lactobacillus plantarum CIDCA 8327, Lactobacillus kefiri CIDCA 8348 and Kluyveromyces marxianus var. marxianus CIDCA 8154 – were evaluated. Both fermented products interfered with the capacity of Salmonella spp. to associate and invade Caco-2/TC7 cells and reduced up to the basal level the expression of the CCL20 in response to a pro-inflammatory stimulus on Caco-2 CCL20:luc cells. The products with potential application as probiotics obtained by fermentation of whey with kefir micro-organisms represent an alternative to increase whey economic value.

Local Treatment with Lactate Prevents Intestinal Inflammation in the TNBS-Induced Colitis Model

Lactate has long been considered as a metabolic by-product of cells. Recently, this view has been changed by the observation that lactate can act as a signaling molecule and regulates critical functions of the immune system. We previously identified lactate as the component responsible for the modulation of innate immune epithelial response of fermented milk supernatants in vitro. We have also shown that lactate downregulates proinflammatory responses of macrophages and dendritic cells. So far, in vivo effects of lactate on intestinal inflammation have not been reported. We evaluated the effect of intrarectal administration of lactate in a murine model of colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS). The increase in lactate concentration in colon promoted protective effects against TNBS-induced colitis preventing histopathological damage, as well as bacterial translocation and rise of IL-6 levels in serum. Using intestinal epithelial reporter cells, we found that flagellin treatment induced reporter gene expression, which was abrogated by lactate treatment as well as by glycolysis inhibitors. Furthermore, lactate treatment modulated glucose uptake, indicating that high levels of extracellular lactate can impair metabolic reprograming induced by proinflammatory activation. These results suggest that lactate could be a potential beneficial microbiota metabolite and may constitute an overlooked effector with modulatory properties.

Fermented whey as poultry feed additive to prevent fungal contamination

BACKGROUND Fungal contamination of poultry feed causes economic losses to industry and represents a potential risk to animal health. The aim of the present study was to analyze the effectiveness of whey fermented with kefir grains as additive to reduce fungal incidence, thus improving feed safety. RESULTS Whey fermented for 24 h at 20 °C with kefir grains (100 g L(-1) ) reduced conidial germination of Aspergillus flavus, Aspergillus parasiticus, Aspergillus terreus, Aspergillus fumigatus, Penicillium crustosum, Trichoderma longibrachiatum and Rhizopus sp. Poultry feed supplemented with fermented whey (1 L kg(-1) ) was two to four times more resistant to fungal contamination than control feed depending on the fungal species. Additionally, it contained kefir microorganisms at levels of 1 × 10(8) colony-forming units (CFU) kg(-1) of lactic acid bacteria and 6 × 10(7) CFU kg(-1) of yeasts even after 30 days of storage. CONCLUSION Fermented whey added to poultry feed acted as a biopreservative, improving its resistance to fungal contamination and increasing its shelf life.

Biological activity of the non-microbial fraction of kefir: antagonism against intestinal pathogens

Kefir is a fermented milk obtained by the activity of kefir grains which are composed of lactic and acetic acid bacteria, and yeasts. Many beneficial health effects have been associated with kefir consumption such as stimulation of the immune system and inhibition of pathogenic microorganisms. The biological activity of kefir may be attributed to the presence of a complex microbiota as well as the microbial metabolites that are released during fermentation. The aim of this work was to characterise the non-microbial fraction of kefir and to study its antagonism against Escherichia coli, Salmonella spp. and Bacillus cereus. During milk fermentation there was a production of organic acids, mainly lactic and acetic acid, with a consequent decrease in pH and lactose content. The non-microbial fraction of kefir added to nutrient broth at concentrations above 75% v/v induced a complete inhibition of pathogenic growth that could be ascribed to the presence of un-dissociated lactic acid. In vitro assays using an intestinal epithelial cell model indicated that pre-incubation of cells with the non-microbial fraction of kefir did not modify the association/invasion of Salmonella whereas pre-incubation of Salmonella with this fraction under conditions that did not affect their viability significantly decreased the pathogens ability to invade epithelial cells. Lactate exerted a protective effect against Salmonella in a mouse model, demonstrating the relevance of metabolites present in the non-microbial fraction of kefir produced during milk fermentation.

Kefir micro-organisms: their role in grain assembly and health properties of fermented milk

Kefir is a homemade viscous and slightly effervescent beverage obtained by milk fermentation with kefir grains, which are built up by a complex community of lactic acid and acetic acid bacteria and yeasts confined in a matrix of proteins and polysaccharides. The present review summarizes the role of kefir micro‐organisms in grain assembly and in the beneficial properties attributed to kefir. The use of both culture‐dependent and independent methods has made possible to determine the micro‐organisms that constitute this ecosystem. Kefir consumption has been associated with a wide range of functional and probiotic properties that could be attributed to the micro‐organisms present in kefir and/or to the metabolites synthetized by them during milk fermentation. In this context, the role of micro‐organisms in kefir health promoting properties is discussed with particular attention to the contribution of yeast as well as bioactive metabolites such as lactic and acetic acid, exopolysaccharides and bioactive peptides. Even though many advances on the knowledge of this ancient fermented milk have been made, further studies are necessary to elucidate the complex nature of the kefir ecosystem.