Lactobacillus salivarius CECT 5713, a potential probiotic strain isolated from infant feces and breast milk of a mother-child pair

In this study, Lactobacillus salivarius CECT 5713 was originally isolated from feces of a one-month-old breast-fed infant. Since it has been suggested that the gut microbiota of breast-fed infants reflects that of the maternal breast milk, we investigated if this specific strain was present in breast milk of the respective mother. RAPD and PFGE analysis revealed the presence of the strain L. salivarius CECT 5713 in this biological fluid. To our knowledge, this is the first report of a L. salivarius strain isolated from breast milk. L. salivarius CECT 5713 produced l-lactate, acetate and hydrogen peroxide, which may be responsible for its antimicrobial activity against most of the indicator organisms used in this study; in addition, this strain showed a high survival rate after exposition to conditions simulating those found in the gastrointestinal tract. Finally, it was strongly adhesive to Caco-2 and HT-29 cells did not produce biogenic amines and were unable to degrade gastric mucin in vitro.     

Short communication: Physicochemical features and microbial community of milk kefir using a potential probiotic Saccharomyces cerevisiae KU200284

The aim of this study was to analyze the β-glucan contents, physicochemical features, and microbial communities in milk kefir prepared using Saccharomyces cerevisiae KU200284 isolated from cucumber jangajji, a fermented vegetable commonly eaten in Korean. Three types of milk kefir were manufactured, with (1) activated kefir grain, (2) activated kefir grain with commercial S. cerevisiae BOF, and (3) activated kefir grain with S. cerevisiae KU200284. β-Glucan contents of milk kefir using kefir grain and kefir grain with S. cerevisiae strains BOF and KU200284 were 8.29, 8.59, and 8.57%, respectively. The pH, titratable acidity, viscosity, Brix level, and alcohol contents of milk kefir using kefir grain with S. cerevisiae strains were acceptable compared with milk kefir using only kefir grain. In milk kefir produced using kefir grains and S. cerevisiae strains, 16S rRNA reads showed representative strains of Lactobacillus kefiranofaciens (>72% relative abundance) and Acetobacter fabarum (>16% relative abundance). In particular, milk kefir using kefir grain with S. cerevisiae KU200284 had the highest relative abundance of L. kefiranofaciens. In addition, the internal transcribed sequence (ITS) rRNA reads in tested milk kefir showed representative strains of Kluyveromyces marxianus (>52% relative abundance) and Saccharomyces cerevisiae (>16% relative abundance). In contrast, milk kefir using S. cerevisiae strains had higher relative abundance of S. cerevisiae (>37%). The β-glucan production, physicochemical properties, and microbial community profiling indicate that S. cerevisiae KU200284 could be used in functional dairy products as a starter culture.     

Evaluation of probiotic characteristics of newly isolated Lactobacillus spp.: immune modulation and longevity

In the current study, the probiotic potential of approximately 350 strains of lactic acid bacteria isolated from Korean infant feces and Kimchi was investigated. Common probiotic properties of the bacterial strains, such as acid tolerance, bile tolerance and adhesion to human intestinal epithelial cells (HT-29 cells), were examined. Some strains were found to have immune modulatory and antimicrobial properties. Antagonistic activity against a panel of pathogenic bacteria was found to be strain dependent. To evaluate the immune modulatory activity of the strains, lymphocyte interferon (IFN)-γ secretion was determined in conjunction with cell proliferation. Some strains of Lactobacillus gasseri, L. fermentum and L. plantarum exhibited increased IFN-γ levels and lymphocyte proliferation. To evaluate the effects of these immune modulating lactobacilli on host life span, Caenorhabditis elegans was used as an in vivo model. Nematodes that were supplied heat-killed lactobacilli as a food source exhibited obvious differences in life span compared with those fed Escherichia coli OP50. The mean life span (determined as mean percent survival) of worms fed L. plantarum CJLP133 and L. fermentum LA12 was 13.89% and 13.69% greater, respectively, than that of control nematodes after 21 days (P = 0.036 and 0.043, respectively). In addition, some of safety profiles, including hemolytic type, gelatin hydration and degradation of urea, were found to be positive. These newly identified lactobacilli hold promise for use as probiotic agents, feed additives and/or in food applications.     

Characterisation of Lactobacillus fermentum SM-7 isolated from koumiss, a potential probiotic bacterium with cholesterol-lowering effects

BACKGROUND: Lactic acid bacteria (LAB) have been demonstrated to have cholesterol‐reducing effects in many studies. RESULTS: Lactobacillus fermentum SM‐7 screened from ten LAB strains isolated from koumiss, a fermented milk drink, reduced cholesterol by 66.8%. It also showed acid and bile tolerance as well as antimicrobial activity against pathogenic Escherchia coli and Staphylococcus aureus. Lactobacillus fermentum SM‐7 cells assimilated 61.5% and co‐precipitated and absorbed 38.5% of the cholesterol in the media. Co‐precipitation of cholesterol with cholic acid increased rapidly at pH levels below 6. In vivo experiments using L. fermentum SM‐7 on artificially induced hyperlipidaemial ICR mice significantly decreased serum total cholesterol and total triglyceride levels, low‐density lipoprotein cholesterol concentrations and atherogenic index (P < 0.01), while serum high‐density lipoprotein cholesterol concentrations did not increase significantly (P > 0.05). The body weight and liver weight/body weight ratio of SM‐7 groups were lower than those of mice on a high‐cholesterol diet that were not given lactobacilli. There was no bacterial translocation in the liver, spleen or kidney of experimental mice. CONCLUSION: The results suggested that L. fermentum SM‐7 is a potential probiotic bacterium with cholesterol‐lowering effects. Copyright © 2010 Society of Chemical Industry     

Prevalence,antimicrobial susceptibility,and molecular characterization of Staphylococcus aureus isolated from dairy herds in northern China

Staphylococcus aureus is one of the main pathogens involved in dairy cow mastitis. Monitoring of antibiotic use would prove useful to assess the risk of Staph. aureus in raw milk. The objective of this work was to investigate the prevalence of Staph. aureus strais isolated from raw milk in northern China, and to characterize antimicrobial susceptibility of these strains and their key virulence genes. In total, 195 raw milk samples were collected from 195 dairy farms located in 4 cities of northern China from May to September 2015. Out of 195 samples, 54 (27.7%) were positive for Staph. aureus. Among these 54 samples, 54 strains of Staph. aureus were isolated, and 16 strains were identified as methicillin-resistant Staph. aureus. The strains exhibited high percentages of resistance to penicillin G (85.2%), ampicillin (79.6%), and erythromycin (46.3%). Moreover, 72% of the strains showed resistance to more than one antimicrobial agent. Overall, 63% of penicillin-resistant strains possessed the blaZ gene, and 60% of the erythromycin-resistant strains possessed erm(A), erm(B), erm(C), msr(A), or msr(B) genes with 8 different gene patterns. All isolates resistant to gentamicin, kanamycin, and oxacillin carried the aac6'-aph2”, ant(4')-Ia, and mecA genes, respectively. Two tet(M)-positive isolates carried specific genes of the Tn916-Tn1545 transposon. The most predominant virulence genes were sec, sea, and pvl, which encode staphylococcal enterotoxins (sec and sea) and Panton-Valentine leukocidin, respectively. Thirty-two isolates (59.2%) harbored one or more virulence genes. The majority of Staph. aureus strains were multidrug resistant and carried multiple virulence genes, which may pose a risk to public health. Our data indicated that antimicrobial resistance of Staph. aureus was prevalent in dairy herds in northern China, and that antibiotics, especially penicillin G and ampicillin, to treat mastitis caused by Staph. aureus should be used with caution in northern China.     

Prevalence of Salmonella enterica,Listeria monocytogenes,and pathogenic Escherichia coli in bulk tank milk and milk filters from US dairy operations in the National Animal Health Monitoring System Dairy 2014 study

The dairy farm environment is a well-documented reservoir for zoonotic pathogens such as Salmonella enterica, Shiga-toxigenic Escherichia coli, and Listeria monocytogenes, and humans may be exposed to these pathogens via consumption of unpasteurized milk and dairy products. As part of the National Animal Health Monitoring System Dairy 2014 study, bulk tank milk (BTM, n = 234) and milk filters (n = 254) were collected from a total of 234 dairy operations in 17 major dairy states and analyzed for the presence of these pathogens. The invA gene was detected in samples from 18.5% of operations and Salmonella enterica was isolated from 18.0% of operations. Salmonella Dublin was detected in 0.7% of operations. Sixteen Salmonella serotypes were isolated, and the most common serotypes were Cerro, Montevideo, and Newport. Representative Salmonella isolates (n = 137) were tested against a panel of 14 antimicrobials. Most (85%) were pansusceptible; the remaining were resistant to 1 to 9 antimicrobials, and within the resistant strains the most common profile was resistance to ampicillin/clavulanic acid, ampicillin, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline. Listeria spp. were isolated from 19.9% of operations, and L. monocytogenes was isolated from 3.0% of operations. Serogroups 1/2a and 1/2b were the most common, followed by 4b and 4a. One or more E. coli virulence genes were detected in the BTM from 30.5% of operations and in the filters from 75.3% of operations. A combination of stx₂, eaeA, and γ-tir genes was detected in the BTM from 0.5% of operations and in the filters from 6.6% of operations. The results of this study indicate an appreciable prevalence of bacterial pathogens in BTM and filters, including serovars known to infect humans.     

Characterization and bioactivities of the exopolysaccharide from a probiotic strain of Lactobacillus plantarum WLPL04

Exopolysaccharide (EPS) was extracted and purified from Lactobacillus plantarum WLPL04, which has been confirmed previously as a potential probiotic for its antagonistic and immune-modulating activity. It has a molecular weight of 6.61 × 10⁴ Da, consisting of xylose, glucose, and galactose in an approximate molar ratio of 3.4:1.8:1. Microstructural studies demonstrated that the EPS appeared as a smooth sheet structure with many homogeneous rod-shaped lumps. The preliminary in vitro assays indicated that the EPS could significantly inhibit the adhesion of Escherichia coli O157:H7 to HT-29 cells in competition, replacement, and inhibition assays at a dose of 1.0 mg/mL, with an inhibition rate of 20.24 ± 2.23, 29.71 ± 1.21, and 30.57 ± 1.73%, respectively. Additionally, the EPS exhibited strong inhibition against biofilm formation by pathogenic bacteria, including Pseudomonas aeruginosa CMCC10104, E. coli O157:H7, Salmonella Typhimurium ATCC13311, and Staphylococcus aureus CMCC26003. Furthermore, the EPS showed good inhibitory activity against the proliferation of HT-29 cells. The characteristics and bioactivities of this EPS may make it a promising candidate in developing functional food.     

From goat colostrum to milk: Physical,chemical, and immune evolution from partum to 90 days postpartum

This study focused on the study of the changes originated in the milk from partum until d 90 of lactation. Ten multiparous Majorera goats, bred carefully under animal health standards, with a litter size of 2 kids (the average in this breed is 1.83 prolificacy) and similar gestation length (149 ± 1 d) were used. Goat kids were removed from their dams to avoid interferences with the study. Compositional content (fat, protein, and lactose) were measured, as well as some other properties, including pH, density, titratable acidity, ethanol stability, rennet clotting time, and somatic cell count. Moreover, immunity molecules (IgG, IgA, and IgM concentrations and chitotriosidase activity) received great attention. Fat and protein content were higher in the first days postpartum, whereas lactose content was lower. Density, titratable acidity, rennet clotting time, and somatic cell count decreased throughout the lactation period, whereas pH and ethanol stability increased. Relative to the immunological parameters, each measured parameter obtained its maximum level at d 0, showing the first milking as the choice to provide immunity to the newborn kids. On the other hand, this study might be used to establish what the best use is: processing or kid feeding.     

Short communication: In vitro and in vivo probiotic potential of Lactobacillus plantarum B7 and Lactobacillus rhamnosus D1 isolated from Minas artisanal cheese

Some Lactobacillus strains may contribute to the health of the host when administered in adequate concentrations, demonstrating their probiotic potential. In contrast, Listeria monocytogenes is a foodborne pathogen that can cause enteropathy, meningoencephalitis, abortion, and septicemia. The aim of this survey was to evaluate the in vitro and in vivo probiotic potential of Lactobacillus plantarum B7 and Lactobacillus rhamnosus D1, isolated from Minas artisanal cheese of the Serra da Canastra (Minas Gerais, Brazil), against Lis. monocytogenes. We submitted B7 and D1 to in vitro testing (antibiogram, tolerance to bile salts and artificial gastric fluid, and spot-on-lawn) and in vivo testing (relative weight gain in mice). Both Lactobacillus strains demonstrated in vitro inhibitory activity against Lis. monocytogenes, as well as sensitivity to antimicrobials and resistance to gastric acids and bile salts. In the in vivo assays, mice treated with D1 gained more weight than mice in the other groups. These results indicate that D1 could have higher probiotic potential than B7 because improvements in feed conversion may help animals fight infection.     

Short communication: Space allocation in intensive Mediterranean buffalo production influences the profile of functional biomolecules in milk and dairy products

The aim of the present study was to determine if space allocation influenced the concentration of biomolecules in buffalo milk and dairy products. Intensively housed buffaloes (n = 96) were randomly assigned to 2 groups according to days in milk, parity, and milk yield: group S10 had a space allocation of 10 m² per buffalo and group S15 had a space allocation of 15 m² per buffalo. Individual milk yield was recorded daily. Twice a month, a bulk milk sample was collected for each group, as well as whey, ricotta, and mozzarella cheese, to assess cheese yield and to conduct HPLC-electrospray ionization-tandem mass spectrometry, milk antioxidant activity, and cell viability analyses. We tested milk extracts from the 2 groups in vitro to evaluate their efficacy in counteracting endothelial oxidative damage induced by high glucose. We evaluated reproductive function in 28 buffaloes from each group using the Ovsynch-timed artificial insemination program. We observed no differences in milk quantity or quality in terms of fat, protein, or lactose, and reproductive function did not differ between the 2 groups. Compared with group S10, group S15 had higher concentrations of carnitine (56.7 ± 1.1 vs. 39.8 ± 0.7 mg/L in milk and 40.9 ± 0.8 vs. 31.7 ± 0.7 mg/L in whey), acetyl-l-carnitine (51.9 ± 0.3 vs. 39.7 ± 0.7 mg/L in milk and 41.1 ± 1.7 vs. 28.7 ± 2.6 mg/L in whey), propionyl-l-carnitine (34.8 ± 1.0 vs. 21.0 ± 0.9 mg/L in milk and 26.9 ± 0.8 vs. 17.6 ± 1.2 mg/L in whey), glycine betaine (23.1 ± 1.9 vs. 13.5 ± 1.6 mg/L in milk and 10.7 ± 0.4 vs. 7.9 ± 0.5 mg/L in whey), and δ-valerobetaine (24.2 ± 0.5 vs. 16.7 ± 0.5 mg/L in milk and 22.0 ± 0.9 vs. 15.5 ± 0.7 mg/L in whey). Group S15 also had higher total antioxidant activity than group S10 (56.7 ± 1.9 vs. 46.4 ± 1.13 mM Trolox equivalents). Co-incubation of high-glucose-treated endothelial cells with milk extracts from group S15 improved cell viability compared with cells treated with high glucose only; it also reduced intracellular lipid peroxidation (144.3 ± 0.4 vs. 177.5 ± 1.9%), reactive oxygen species (141.3 ± 0.9 vs. 189.3 ± 4.7 optical density units), and cytokine release (tumor necrosis factor-α, IL-1β, IL-6). Greater space allocation was associated with higher levels of biomolecules in buffalo milk. This could have been the result of improved welfare in buffaloes that were allocated more space.     

Short communication: Validation of a novel milk progesterone-based tool to monitor luteolysis in dairy cows using cost-effective,on-farm measured data

The progesterone (P4) monitoring algorithm using synergistic control (PMASC) uses luteal dynamics to identify fertility events in dairy cows. This algorithm employs a combination of mathematical functions describing the increasing and decreasing P4 concentrations during the development and regression of the corpus luteum and a statistical control chart that allows identification of luteolysis. The mathematical model combines sigmoidal functions from which the cycle characteristics can be calculated. Both the moment at which luteolysis is detected and confirmed by PMASC, as well as the model features themselves, can be used to inform the farmer on the fertility status of the cows.     

Short communication: The effect of linseed oil and DGAT1 K232A polymorphism on the methane emission prediction potential of milk fatty acids

Several in vivo CH₄ measurement techniques have been developed but are not suitable for precise and accurate large-scale measurements; hence, proxies for CH₄ emissions in dairy cattle have been proposed, including the milk fatty acid (MFA) profile. The aim of the present study was to determine whether recently developed MFA-based prediction equations for CH₄ emission are applicable to dairy cows with different diacylglycerol o-acyltransferase 1 (DGAT1) K232A polymorphism and fed diets with and without linseed oil. Data from a crossover design experiment were used, encompassing 2 dietary treatments (i.e., a control diet and a linseed oil diet, with a difference in dietary fat content of 22 g/kg of dry matter) and 24 lactating Holstein-Friesian cows (i.e., 12 cows with DGAT1 KK genotype and 12 cows with DGAT1 AA genotype). Enteric CH₄ production was measured in climate respiration chambers and the MFA profile was analyzed using gas chromatography. Observed CH₄ emissions were compared with CH₄ emissions predicted by previously developed MFA-based CH₄ prediction equations. The results indicate that different types of diets (i.e., with or without linseed oil), but not the DGAT1 K232A polymorphism, affect the ability of previously derived prediction equations to predict CH₄ emission. However, the concordance correlation coefficient was smaller than or equal to 0.30 for both dietary treatments separately, both DGAT1 genotypes separately, and the complete data set. We therefore concluded that previously derived MFA-based CH₄ prediction equations can neither accurately nor precisely predict CH₄ emissions of dairy cows managed under strategies differing from those under which the original prediction equations were developed.     

Survival of a probiotic, Lactobacillus casei strain Shirota, in the gastrointestinal tract: selective isolation from faeces and identification using monoclonal antibodies.

Lactobacillus casei strain Shirota (LCS) is a probiotic bacterium used in the production of fermented milk products and lactic acid bacteria preparations. To investigate the survival of LCS in the gastrointestinal tract, we have developed a selective medium and specific monoclonal antibodies to isolate and identify this strain. Selective LLV agar medium was prepared by modifying LBS medium, a selective medium for lactobacilli, through the replacement of glucose with lactitol as a carbon source and vancomycin as a selective antibiotic. Culture in LLV agar medium followed by ELISA using monoclonal antibodies specific for LCS was able to detect the organism in faeces. Using this method, we studied the faecal recovery of LCS in individuals who drank 125 ml of fermented milk which contained 10(10) live LCS for 3 days. The mean recovery was about 10(7) live bacteria per gram of faeces, indicating that LCS survived transit through the gastrointestinal tract after ingestion of the fermented milk.     

Assessment of probiotic characteristics of lactic acid bacteria isolated from fermented yak milk products of Sikkim,India: Chhurpi,Shyow, and Khachu

The present study documents the probiotic attributes of indigenous lactic acid bacteria (LAB) isolated from local fermented Yak milk products namely Chhurpi, Shyow and Khachu prepared in the northern and eastern region of Sikkim in the Himalayas. Samples were collected aseptically and a total of 170 LAB was isolated and screened for putative probiotic properties like hypocholesteromic effect, acid tolerance, bile tolerance, bile salt hydrolase (BSH) activity and cell surface hydrophobicity. It was observed that 70 LAB isolates showed cholesterol lowering activity, out of which 35 isolates were selected that showed 50% and less cholesterol reducing effect in vitro. Acid tolerance test revealed good tolerance of 12 isolates at pH 2.5 and pH 2.0 for up to 2 hours. The tolerance to 0.5% and 1% of three bile salts acid revealed more growth in MRS broth containing taurocholic acid with the isolates revealing good BSH activity leading to bile acid deconjugation. The cell surface hydrophobicity ranged from 20–95%. Furthermore, 16S rRNA gene sequencing revealed Lactobacillus plantarum YD5S and YD9S, L. pentosus YD8S, L. paraplantarum YD11S, Enterococcus lactis YHC20 and E. faecium YY1 as the best isolates with technological properties. The isolates may serve as potential probiotic candidates with potential for hypocholesteromic benefits in the future.     

Short communication: Use of a digital refractometer in assessing immunoglobulin G concentrations in colostrum and the first 5 transition milkings in an Irish dairy herd

Transition milk is a source of immunoglobulin G (IgG) and could potentially be used to provide calves with passive immunity, when the IgG concentration is ≥50 g/L. Assessment of IgG concentrations in transition milk would be required before feeding and could be conducted using cow-side tests such as refractometers. Currently, limited information is available on the ability of refractometers to assess transition milk quality. We hypothesized that digital refractometry could be used to provide an accurate cow-side assessment of IgG concentrations in colostrum and transition milk, and IgG concentration in colostrum and one or more transition milking in an Irish herd is >50 g/L. The objectives of this study were to determine the IgG concentrations in colostrum and first, second, third, fourth, and fifth transition milk, and determine the utility of a digital refractometer in assessing quality of colostrum and transition milk produced by cows in a pasture-based dairy production system. A convenient sample of 75 dairy cows were enrolled. Colostrum and transition milk IgG concentrations were determined by radial immunodiffusion and refractometry. Sensitivity and specificity of the refractometer were determined and cut-off points that maximized sensitivity and specificity were determined using receiver operating characteristic curves. Median (range) IgG concentrations in colostrum and first, second, third, fourth, and fifth milking were 99.6, 43.5, 12.5, 5.3, 1.9, and 1.8 g/L, respectively. The sensitivity (0.8–1) of digital refractometry in identifying samples with low IgG concentrations in colostrum, first, second, and third transition milk was acceptable. In contrast, digital refractometry was not useful for assessing IgG concentrations in the fourth and fifth milking due to low IgG concentrations.     

Interaction of probiotic Lactobacillus and Bifidobacterium strains with human intestinal epithelial cells: adhesion properties, competition against enteropathogens and modulation of IL-8 production

The human intestinal microbiota plays a pivotal role in human nutrition and health by promoting the supply of nutrients, preventing pathogen colonization and shaping and maintaining normal mucosal immunity. The depletion of the individual microbiota can result in a higher susceptibility to enteropathogenic bacteria infection. In order to reduce this risk, the use of food supplements containing probiotic bacteria has been recently addressed. In this paper, we investigate the protective role toward enteropathogen infection of probiotic strains belonging to Lactobacillus and Bifidobacterium. According to our experimental data, Lactobacillus acidophilus Bar13, L. plantarum Bar10, Bifidobacterium longum Bar33 and B. lactis Bar30 were effective in displacing the enteropathogens Salmonella typhimurium and Escherichia coli H10407 from a Caco-2 cell layer. Moreover, L. acidophilus Bar13 and B. longum Bar33 have been assessed for their immunomodulatory activity on IL-8 production by HT29 cells. Both strains showed the potential to protect enterocytes from an acute inflammatory response. These probiotic strains are potential candidates for the development of new functional foods helpful in counteracting enteropathogen infections.