Kefir is a fermented-milk beverage originating and widely consumed in the Caucasus as well as in Eastern Europe and is a source of bacteria with potential probiotic properties. Enterohaemorrhagic Escherichia coli producing Shiga toxin is commonly associated with food-transmitted diseases; the most prevalent serotype causing epidemics is Esch. coli O157:H7. The aim of this study was to evaluate the antagonism of Lactobacillus plantarum isolated from kefir against the action on Vero cells of supernatants of the Esch. coli O157:H7 strain 69160 expressing the type-II Shiga toxin (Stx2) and to study the role of the Lactobacillus cell wall in that inhibition. Spent culture supernatants of Esch. coli O157:H7 strain 69160 led to cytotoxic effects on cultured eukaryotic cells as evidenced by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide–cleavage assay or by lactate-dehyrogenase release. Lb. plantarum CIDCA 83114 reduced the cytotoxic activity of Stx present in strain-69160 supernatants, and this protection was markedly higher than those of Lactobacillus kefir CIDCA 83113 and 8348 and Lb. delbrueckii subsp. bulgaricus CIDCA 333. This antagonism of cytotoxicity was mimicked by Lb. plantarum cell walls but was reduced after heating or protease treatments, thus indicating a protein or peptide as being involved in the protection mechanism. The cell surface of the lactobacilli bound the subunit B of Stx thereby decreasing the cytotoxicity. These interactions could constitute the first step in preventing the damage induced by Esch. coli O157:H7 supernatants, thus representing a valuable means of potentially mitigating the noxious effects of this food pathogen.

An effective strategy for enhancing the bioactive fatty acids (FA) in sheep milk could be dietary supplementation with a moderate level of a combination of soybean oil with fish oil (SFO) without negative effects on milk yield and its chemical composition. Thus, the objective of this study was to determine the effects of a moderate forage diet supplementation with SFO on milk chemical composition and FA profile, as well as on plasma FA. Twelve dairy sheep were assigned to two homogenous sub-groups. Treatments involved a control diet without added oil, and a diet supplemented with 23·6 g soybean oil and 4·7 g fish oil per kg dry matter (DM) of the total ration. The results showed that SFO diet had no effect on milk yield and chemical composition. In blood plasma the concentrations of trans-11 C18:2 (VA), C18:2n-6, C20:5n-3 (EPA) and C22:6n-3 (DHA) were significantly higher while those of C14:0, C16:0 and C18:0 were lower in sheep fed with SFO diet compared with control. The SFO supplementation of sheep diet increased the concentrations of VA, cis-9, trans-11 C18:2 CLA, trans-10, cis-12, C18:2 CLA, EPA, DHA, monounsaturated FA (MUFA), polyusaturated fatty acids (PUFA) and n-3 FA and decreased those of short chain FA (SCFA), medium chain FA (MCFA), the saturated/unsaturated ratio and the atherogenicity index value in milk compared with the control. In conclussion, the SFO supplementation at the above levels in a sheep diet, with moderate forage to concentrate ratio, improved the milk FA profile from human health standpoint without negative effects on its chemical composition.

The aim of this study was to evaluate the effect of storage time (4 °C) on milk samples and the use of azidiol as preservative on the results of microbial inhibitor tests used to detect antimicrobials in milk. For this purpose, 16 milk bulk samples divided into two aliquots, preservative-free and with azidiol, spiked with 12 concentrations of amoxicillin, ampicillin, penicillin G and oxytetracycline, were used. The milk samples were analysed using the BRT MRL, Delvotest MCS Accelerator and Eclipse 100 at 0, 24, 48 and 72 h stored at 4 °C. The logistic regression model was applied to study the effect of storage time (ST), preservative (P) and their interaction (ST×P). At a concentration equivalent to the Detection Limit (DL), the positive results of microbial inhibitor tests do not remain stable during storage time. These results are more reproducible if samples are stored with a preservative. At Maximum Residue Limits (MRLs) concentration microbial inhibitor tests can detect penicillin for up to 72 h of storage. For oxytetracycline, the BRT MRL and Delvotest MCS tests presents DL exceeding the MRL, therefore they are not sensitivity to this tetracycline (0% positive results). By contrast Eclipse 100, whose DL is lower than the MRL, gave 100% positive results during the 72 h storage period. It can be concluded that it would be convenient to store milk samples with azidiol at 4 °C and to carry out analyses within the first 48 h since milk sampling.

Goat milk protein was hydrolysed with subtilisin and trypsin. As input variables, temperature was assayed in the interval 45–70 °C for subtilisin and 30–55 °C for trypsin, while the enzyme-substrate ratio varied from 1 to 5%. The effect of the input variables on the degree of hydrolysis and ACE-inhibitory activity (output variables) was modelled by second order polynomials, which were able to fit the experimental data with deviations below 10%. The individual maximum values of the degree of hydrolysis and the ACE-inhibitory activity were found at conflicting conditions of temperature and enzyme-substrate ratio. Since such maximum values could not be reached simultaneously, a bi-objective optimisation procedure was undertaken, producing a set of non-inferior solutions that weighted both objectives.

The shortcomings of Staphylococcus aureus vaccines to control bovine mastitis have been attributed to insufficient capacity of the vaccines to induce opsonizing antibodies and to stimulate cellular immune responses. Types of antigen, administration route and adjuvant used in a vaccine formulation have been identified as critical factors for the development of opsonic antibodies. Current commercially available vaccines for Staph. aureus bovine mastitis control are formulated with Al(OH)3 and oil-based adjuvants. The aim of this study was to evaluate the immune response of heifers immunized with a Staph. aureus CP5 whole cell vaccine formulated either with Al(OH)3 or ISCOMATRIX™. Twenty primigravid Holstein dairy heifers in the last trimester of gestation were immunized either with a vaccine formulated with ISCOMATRIX™ (n = 6), Al(OH)3 (n = 7), or saline solution (placebo) (n = 7). Immunization was carried out 38 and 10 d before calving. Heifers vaccinated with Staph. aureus adjuvanted with ISCOMATRIX™ responded with significantly higher levels of anti-bacterin and anti-CP5 IgG and IgG2 in sera than animals in the Al(OH)3 or control groups. Animals in the ISCOMATRIX™ group responded with significantly higher anti-bacterin specific IgG in whey than animals in the Al(OH)3 and control groups, detected from the first week post calving until 60 d of lactation. Sera from animals inoculated with Staph. aureus in ISCOMATRIX™, obtained 7 d post partum, significantly increased both the number of neutrophils ingesting bacteria and the number of bacteria being ingested by the neutrophils, compared with sera obtained from heifers vaccinated with Al(OH)3 or non-vaccinated controls. These features coupled to safety of the ISCOMATRIX™ formulation, warrant additional studies.

This investigation analysed the applicability of principal component analysis (PCA), a latent variable method, for the early detection of mastitis and lameness. Data used were recorded on the Karkendamm dairy research farm between August 2008 and December 2010. For mastitis and lameness detection, data of 338 and 315 cows in their first 200 d in milk were analysed, respectively. Mastitis as well as lameness were specified according to veterinary treatments. Diseases were defined as disease blocks. The different definitions used (two for mastitis, three for lameness) varied solely in the sequence length of the blocks. Only the days before the treatment were included in the blocks. Milk electrical conductivity, milk yield and feeding patterns (feed intake, number of feeding visits and time at the trough) were used for recognition of mastitis. Pedometer activity and feeding patterns were utilised for lameness detection. To develop and verify the PCA model, the mastitis and the lameness datasets were divided into training and test datasets. PCA extracted uncorrelated principle components (PC) by linear transformations of the raw data so that the first few PCs captured most of the variations in the original dataset. For process monitoring and disease detection, these resulting PCs were applied to the Hotelling's T2 chart and to the residual control chart. The results show that block sensitivity of mastitis detection ranged from 77·4 to 83·3%, whilst specificity was around 76·7%. The error rates were around 98·9%. For lameness detection, the block sensitivity ranged from 73·8 to 87·8% while the obtained specificities were between 54·8 and 61·9%. The error rates varied from 87·8 to 89·2%. In conclusion, PCA seems to be not yet transferable into practical usage. Results could probably be improved if different traits and more informative sensor data are included in the analysis.

Fresh buckwheat (Fagopyrum esculentum) and chicory (Cichorium intybus) had been shown to have the potential to improve certain milk quality traits when fed as forages to dairy cows. However, the process of ensiling might alter these properties. In the present study, two silages, prepared from mixtures of buckwheat or chicory and ryegrass, were compared with pure ryegrass silage (Lolium multiflorum) by feeding to 3 × 6 late-lactating cows. The dietary dry matter proportions realised for buckwheat and chicory were 0·46 and 0·34 accounting also for 2 kg/d of concentrate. Data and samples were collected from days 10 to 15 of treatment feeding. Buckwheat silage was richest in condensed tannins. Proportions of polyunsaturated fatty acids (PUFA) and α-linoleic acid in total fatty acids (FA) were highest in the ryegrass silage. Feed intake, milk yield and milk gross composition did not differ among the groups. Feeding buckwheat resulted in the highest milk fat concentrations (g/kg) of linoleic acid (15·7) and total PUFA (40·5; both P < 0·05 compared with ryegrass). The concentration of α-linolenic acid in milk fat was similar across treatments, but its apparent recovery in milk relative to the amounts ingested was highest with buckwheat. The same was true for the occurrence of FA biohydrogenation products in milk relative to α-linolenic acid intake. Recovery of dietary linoleic acid in milk remained unaffected. Feeding buckwheat silage shortened rennet coagulation time by 26% and tended (P < 0·1) to increase curd firmness by 29%. In conclusion, particularly buckwheat silage seems to have a certain potential to modify the transfer of FA from feed to milk and to contribute to improved cheese-making properties.

The objective of the study was to determine the effect of varying degrees of overmilking on teat-end hyperkeratosis, milk production variables and indicators of udder health during late lactation. This was examined by assessing the effect of four end-of-milking criteria on 181 spring-calving, mixed-age Holstein-Friesian cows, at an average 217±24 d in milk, over a six-week period. The four treatments were: remove cluster once milk flow rate fell to 0·2 kg/min plus 5 s (Ovr0), plus 120 s (Ovr2), plus 300 s (Ovr5), and plus 540 s (Ovr9). Daily measurements included individual cow milk yield, milking duration, overmilking duration, maximum milk flow rate, milk flow rate at cluster removal and the number of cluster re-attachments. Individual cow bulk milk samples were collected weekly at AM and PM milkings to determine composition (fat, protein and lactose) and somatic cell count (SCC; AM only). Teat-end hyperkeratosis score was assessed at weeks 0, 3, 5 and 6. At week 6 mean teat-end hyperkeratosis score of the Ovr2 treatment was not greater than Ovr0, whilst Ovr5 was greater than Ovr2 and Ovr9 was greater than Ovr5 and Ovr2. Milk production, milking characteristics and SCC were not different between treatments, except milking duration and milk flow rate at cluster removal. However, higher teat-end hyperkeratosis scores may have a longer-term impact on indicators of udder health if teat-end condition reaches severe levels. Results indicate that to minimise changes in teat-end condition overmilking should be limited to 2 min, which has implications for milking management in large parlours not fitted with automatic cluster removers.

The aim of the present study was the examination of the boot swab sampling technique for the collection of environmental material in order to identify Mycobacterium avium ssp. paratuberculosis (MAP)-infected herds. Eight dairy herds were included into the study. Four of them had a well-known history of MAP-infection from a herd surveillance programme conducted since 2006. Cows in these herds were repeatedly tested positive in Pourquier® MAP-ELISA (Pourquier, Montepellier, France); in some MAP could be isolated in individual faecal culture despite that symptoms of paratuberculosis were never reported. In four presumably negative herds nearly all cows were repeatedly tested serologically negative for MAP. The pathogen was never isolated from faecal samples of cows by culture. The study was initiated with the aim of standardising environmental samples as a herd diagnostics, in which overall 130 pairs of boot swab samples from the cows’ surroundings were taken In 58 of 64 swab samples (90·6%) from confirmed MAP-infected herds the organism could be isolated by mycobacterial culture of the boot swab. Contrarily, in 66 samples from presumably MAP-negative herds only one swab was positive (1·5%). The utilisation of boot swabs as a standardised technique for environmental sampling offers an effective and inexpensive tool for identifying herds infected with MAP. This is the first report of using boot swabs for the collection of environmental samples for MAP- detection in cattle herds. This easy to perform technique enables the economical detection of MAP herd status.

Staphylococcus aureus isolates from dairy cow mastitis are not always consistent with the characteristic morphology described, and molecular investigation is often needed. The aim of the study was to develop a duplex real-time PCR assay for rapid identification of Staph. aureus isolates, targeting both nuc and Sa442. Overall, 140 isolates collected from dairy cow mastitis in 90 different herds, were tested. All strains had been identified using morphological and biochemical characteristics. DNA from each strain was amplified in real-time PCR assay, to detect nuc or Sa442. Thereafter, a duplex real-time PCR assay was performed, and specificity of the amplified products was assessed by high resolution melting curve analysis. Out of 124 Staph. aureus isolates, 33 did not show the typical morphology or enzymic activity; in 118 strains, the two melt-curve peaks consistent with nuc and Sa442 were revealed, while 2 isolates showed only the peak consistent with Sa442. Four isolates bacteriologically identified as Staph. aureus, were PCR-negative and were further identified as Staph. pseudintermedius by sequencing. Staph. pseudintermedius and coagulase-negative staphylococci did not carry nuc or Sa442. The results showed the correct identification of all isolates, comprehending also coagulase—or nuc-negative Staph. aureus, while other coagulase-positive Staphylococci were correctly identified as non-Staph. aureus. Both sensitivity and specificity were 100%. High resolution melting analysis allowed easy detection of unspecific products. Finally, the duplex real-time PCR was applied directly to 40 milk samples, to detect infected mammary quarters. The assay confirmed the results of bacteriological analysis, on Staph. aureus-positive or—negative samples. Therefore, the proposed duplex real-time PCR could be used in laboratory routine as a cost-effective and powerful tool for high-throughput identification of atypical Staph. aureus isolates causing dairy cow mastitis. Also, it could be applied directly to milk samples, to detect Staph. aureus mammary infections avoiding bacteriological analysis.

The dairy industry uses lipase extensively for hydrolysis of milk fat. Lipase is used in the modification of the fatty acid chain length, to enhance the flavours of various chesses. Therefore finding the unlimited source of lipase is a concern of dairy industry. Due to the importance of lipase, this study was an attempt to express the lipase from Burkholderia cepacia in Lactococcus lactis. To achieve this, a gene associated with lipase transport was amplified and subcloned in inducible pNZ8148 vector, and subsequently transformed into Lc. lactis NZ9000. The enzyme assay as well as SDS-PAGE and western blotting were carried out to analysis the recombinant lipase expression. Nucleotide sequencing of the DNA insert from the clone revealed that the lipase activity corresponded to an open reading frame consisting of 1092 bp coding for a 37·5-kDa size protein. Blue colour colonies on nile blue sulphate agar and sharp band on 37·5-kD size on SDS-PAGE and western blotting results confirm the successful expression of lipase by Lc. lactis. The protein assay also showed high expression, approximately 152·2 μg/ml.h, of lipase by recombinant Lc. lactis. The results indicate that Lc. lactis has high potential to overproduce the recombinant lipase which can be used commercially for industrially purposes.

Indirect comparisons from studies in vivo have suggested that caprine mammary tissue is less sensitive than bovine mammary tissue to the anti-lipogenic effect of long-chain fatty acids (LCFA), including specific rumen biohydrogenation (RBH) intermediates of polyunsaturated fatty acids (PUFA). Our objective was to investigate the effects on lipogenesis of 18-carbon LCFA differing in the degree of unsaturation and/or double bond conformation using cultured slices of bovine and caprine mammary tissues. Mammary tissues were collected from five multiparous Holstein × Normande cows and six multiparous Alpine goats in mid lactation. The expression of genes involved in milk component synthesis was measured in tissues collected at slaughter and after slice preparation: FASN, SCD1, CD36, SREBF1 and PPARG1 mRNA levels were higher in bovine than caprine samples, whereas the opposite was observed for CSN2 mRNA levels. Bovine and caprine mammary slices were incubated for 20 h in a medium with BSA (control), cis-9-18 : 1, 18 : 2n-6, 18 : 3n-3, cis-9, trans-11-CLA, or trans-10, cis-12-CLA (the latter at 3 increasing concentrations: C1 (0·11 mm), C2 (0·16 mm), C3 (0·37 mm)). Lipogenesis was estimated by measuring the incorporation of 14C-acetate into total lipid. Significant differences of individual LCFA (P < 0·05) were observed between species: bovine tissue showed a decrease in total lipogenesis with 18 : 2n-6, 18 : 3n-3, trans-10,cis-12-CLA (C2 and C3) while caprine tissue showed an increase after treatment with 18 : 3n-3, cis-9, trans-11-CLA or trans-10, cis-12-CLA (C3). These results were not related to the mRNA abundance of our set of genes in the mammary slices after incubation. In conclusion, this study demonstrates that caprine mammary slices reacted differently from bovine mammary slices to the anti-lipogenic activity of specific LCFA and suggests that regulation of lipogenesis via other genes and/or at protein level and enzyme activity may be involved.

Proper dry cow management is critical not only for subsequent milk production and fertility but also for mastitis control. A phenomenon of immunosuppression was commonly observed in transition cows, an example being the high susceptibility of the mammary gland during early the dry period to new infectious agents. Polymorphonuclear neutrophils (PMN) play important defence roles in the mammary gland of newly dried cows. One of the bactericidal mechanisms of PMN is through producing reactive oxygen species (ROS), which can be efficiently quantified by chemiluminescence (CL) assay. In the current study, the potential of intramammary application of a commercial Enterococcus faecium SF68 (SF68) product to enhance the local innate immunity of newly dried mammary glands was evaluated based on the CL assay. The preliminary experiments in vitro indicated virtual dose-responsiveness of ROS generation from three different cell preparations, bovine blood PMN, bovine blood PMN pre-conditioned with cow milk, and the post-diapedesis model somatic cells from cow milk, on their exposure to phorbol 12-myristate 13-acetate (PMA), viable SF68, and ultrasonicated SF68, but not dry-heated SF68. Because ultrasonication treatment was found to profoundly enhance the immunogenicity of SF68 in vitro, in the following animal trial, single infusion of either 5 or 10×107 original cfu of ultrasonicated SF68 was randomly applied to the front quarters and phosphate-bufferedsaline (PBS) applied to the rear quarters of each of the four experimental cows on the first day of milk stasis. The results showed that within the first post-infusion week, ultrasonicated SF68 induced a faster and greater (P<0·05) recruitment of PMN into mammary lumen with no apparent local or systemic inflammatory sign. Meanwhile, ultrasonicated SF68 also induced a greater (P<0·05) ROS production in response to PMA challenge by in situ somatic cells of mammary secretion. Taken together, ultrasonicated SF68 modulated ROS generation of bovine neutrophils, and would be a potential enhancer of udder innate immunity in drying-off dairy cows. More thorough work is warranted.

The aims of this study were to test the assumption that tissue-type plasminogen activator (t-PA) and plasminogen (PG) are closely associated with the casein micelle and form a functional complex that rules casein degradation. This assumption was essentially verified for bovine milk under conditions wherein the plasmin system was activated by treatment with casein hydrolysate. It was also shown that urokinase-type PA (u-PA), the second type of plasminogen activator present in milk, was not involved in casein degradation. In agreement with previous studies, we show that treatment with casein hydrolysate precipitously reduced mammary secretion, disrupted the tight junction integrity (increase in Na+ and decrease in K+ concentrations), induced hydrolysis of casein, and activated various elements of the innate and acquired immune system. In the present study, we have identified t-PA as the principal PA, which is responsible for the conversion of PG to plasmin. It was found that t-PA and plasminogen are present in freshly secreted milk (less than 10 min from its secretion), suggesting that they are secreted as a complex by the mammary gland epithelial cells. Further research is needed to provide the direct evidence to verify this concept.

Streptococcus uberis is the most common cause of clinical mastitis at calving in pasture-based dairy cows. Results of experimental inoculations were compared with cows' previous history of infection to help define a model for susceptibility to Str. uberis mastitis. Cows used had either no apparent history of intramammary infection (IMI) by Str. uberis or other major mastitis pathogens throughout their productive lifetime (‘apparently uninfected’; AUI), or had a confirmed history of Str. uberis IMI (‘historically infected’; HI). Cows were exposed to Str. uberis in sequential steps: dipping of the teat end (DIP; n=53 cows); a teat canal inoculation (TCI; n=33 cows); and, finally, intramammary inoculation challenge (IC; n=7 cows). Only cows that remained free of infection at each step progressed to the next phase. Infection rates were similar between AUI or HI cows following the DIP (9 and 17% respectively), or the TCI (75 and 68% respectively). Physical and biochemical traits of cows were examined. Analysis of traits prior to inoculations implied that HI cows produced more milk fat, while AUI cows tended to have longer teat canals. Analysis of traits for cows that became infected following DIP, implied that there was a positive association with milk fat production and negative association with somatic cell count (SCC), while there was a positive association with the duration of p.m. milking, and negative association with SCC in those cows that became infected following TCI. Only AUI cows became infected following the IC inoculation. Similarity in response to experimental inoculation between the two groups suggests that the current dip or teat canal inoculation (using a 3-mm depth of inoculation) models are not good predictors of natural resistance to Str. uberis. However, a population of cows was identified that remained uninfected after DIP, TCI and IC, and may comprise a resistant phenotype.

Subclinical mastitis (SM) is one of the most important diseases affecting dairy ewes worldwide, with negative impact on the animal health, farm income and public health. Animals with SM often remain untreated because the disease may not be revealed. Increase in somatic cell count (SCC) and positive bacteriology for mastitis pathogens in milk samples are indicative of SM but the evidence of only one of these alterations must suggest an uncertain SM (UM). UM is defined when positive bacteriological examination (Latent-SM) or SCC>500 000 cells/ml (non-specific-SM) are detected in milk. Nevertheless, SCC and bacteriological examination are expensive, time consuming and are not yet in use at the farm level in dairy ewes. Recently, a sensitive acute phase protein, amyloid A, displaying multiple isoforms in plasma and different body fluids including mammary secretion (milk amyloid A-MAA), has been investigated as a marker of mastitis in cows and, in a few studies, in sheep. The aim of this trial was to compare the concentration of MAA of single udder-halves in ewes with healthy udder-halves (HU-control group) and naturally occurring subclinical mastitis, both confirmed (SM group) and uncertain (UM groups: Latent-SM and non-specific-SM), for monitoring udder health. The reliability of a specific ELISA kit for the measurement of MAA was also tested. During a 3-month trial period, 153 udder halves were assigned to the experimental groups based on their health status: 25 with SM, 40 with UM (11 with latent-SM and 29 with non-specific-SM) and 88 HU. SCC and bacteriological analysis were performed to establish the control and subclinical mastitis groups. MAA concentrations in milk samples were measured using a specific commercially milk ELISA kit. The data were submitted to statistical analysis. Significant (P<0·05) differences among the groups SM, non-specific-SM and HU were detected with the SM having the highest level and HU the lowest. MAA concentration is affected by the udder health status and is a useful indicator of subclinical mastitis and increased SCC in sheep.

A concurrent determination of folate versus folic acid in milk by microbiological assay (MA) with Lactobacillus rhamnosus as the assay organism, Enzyme Linked Immuno Sorbent Assay (ELISA) by competitive binding rapid ELISA kit (RIDASCREEN®) and high-pressure-liquid chromatography (HPLC) was done for detection of the folate form and its level. MA gave total folate content as Lb. rhamnosus showed similar response to most folate isomers formed by the tri-enzyme treatment in comparison with the other two methods which specifically estimated the folic acid. In case of ELISA, specificity was apparently limited to folic acid and dihydro folic acid and thereby showed a lower response for other folate derivatives. Estimation by HPLC with UV detector was highly specific and hence only folic acid could be detected without any cross reactivity. Among the different methods HPLC was observed to be the most sensitive method for determination of folic acid and hence can efficiently determine the folic acid fortification level while MA remained highly efficient, sensitive and reproducible method for estimation of total folate indicating its potential use for dietary folate estimation.

Kefir is a dairy product obtained by fermentation of milk with a complex microbial population and several health-promoting properties have been attributed to its consumption. In this work, we tested the ability of different kefir-isolated bacterial and yeast strains (Lactobacillus kefir, Lb. plantarum, Lactococcus lactis subps. lactis, Saccharomyces cerevisiae and Kluyveromyces marxianus) or a mixture of them (MM) to antagonise the cytopathic effect of toxins from Clostridium difficile (TcdA and TcdB). Cell detachment assays and F-actin network staining using Vero cell line were performed. Although incubation with microbial cells did not reduce the damage induced by C. difficile spent culture supernatant (SCS), Lc. lactis CIDCA 8221 and MM supernatants were able to inhibit the cytotoxicity of SCS to Vero cells. Fraction of Lc. lactis CIDCA 8221 supernatant containing components higher than 10 kDa were responsible for the inhibitory activity and heating of this fraction for 15 min at 100 °C completely abrogated this ability. By dot-blot assay with anti-TcdA or anti-TcdB antibodies, concentration of both toxins seems to be reduced in SCS treated with Lc. lactis CIDCA 8221 supernatant. However, protective effect was not affected by treatment with proteases or proteases-inhibitors tested. In conclusion, we demonstrated that kefir-isolated Lc. lactis CIDCA 8221 secreted heat-sensitive products able to protect eukaryotic cells from cytopathic effect of C. difficile toxins in vitro. Our findings provide new insights into the probiotic action of microorganisms isolated from kefir against virulence factors from intestinal pathogens.