Antioxidant and immunomodulatory activity of selenium exopolysaccharide produced by Lactococcus lactis subsp. lactis

Exopolysaccharide (EPS) was isolated and purified from Lactococcus lactis subsp. Lactis culture broth. Selenium chloride oxide (SeCl₂O) was added to the EPS to synthesize selenium-exopolysaccharide (Se-EPS). The in vitro and in vivo antioxidant and in vivo immunomodulatory activity of EPS and Se-EPS were compared. EPS and Se-EPS scavenged superoxide anions and hydroxyl radicals. They also increased catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, while decreasing malondialdehyde (MDA) levels in serum and in the livers of mice. Se-EPS showed stronger in vitro and in vivo antioxidant activity than were shown by EPS. The in vivo immunoenhancement activity of EPS and Se-EPS induced by cyclophosphamide (CY) treatment in immunosuppressed mice was researched. EPS and Se-EPS treatments increased macrophage phagocytosis, spleen and thymus indices and haemolytic complement activity (HC₅₀). Se-EPS showed stronger immunomodulatory activity than did EPS.     

Improved growth of bifidobacteria by cocultivation with Lactococcus lactis subspecies lactis

Cultivation of bifidobacteria in milk is a difficult and industrially valuable task. In this paper, we report the finding of a novel technique to improve the growth of bifidobacteria in dairy products and the results of mechanism studies. The growth of bifidobacteria in skim milk medium was found to be stimulated upon cocultivation with certain strains of Lactococcus lactis ssp. lactis. Bifidobacterium growth-stimulating (BGS) activity was observed on a wide range of bifidobacterial species. Bifidobacterium growth-stimulating activity was associated with the ability to grow in skim milk medium and the presence of a cell wall-anchored proteinase (PrtP) in Lc. lactis ssp. lactis. Studies on one strain, Lc. lactis ssp. lactis MCC857, showed that crude PrtP extracts and casein hydrolysates exhibited BGS activity. The casein hydrolysate BGS activity was found in the low molecular weight fraction by HPLC separation. A combination of 2 AA, Met and Leu, was found to account for a large portion of the casein hydrolysate BGS activity. In conclusion, this cocultivation system is highly efficient and industrially applicable for the production of fermented milk with high cell counts of bifidobacteria.     

Potential of bacteriocinogenic Lactococcus lactis subsp. lactis inhabiting low pH vegetables to produce nisin variants

Antimicrobial behavior of lactic acid bacteria (LAB) has been explored since many years to assess their ability to produce bacteriocin, a natural preservative, to increase the shelf life of food. This study aims to characterize bacteriocin producing strains of lactic acid bacteria isolated from acidic to slightly acidic raw vegetables including tomato, bell pepper and green chili and to investigate their potential to inhibit food related bacteria. Among twenty nine LAB screened for antimicrobial activity, three exhibited antagonism against closely related bacterial isolates which was influenced by varying temperature and pH. They were identified up to strain level as Lactococcus lactis subsp. lactis TI-4, L. lactis subsp. lactis CE-2 and L. lactis subsp. lactis PI-2 based on 16S rRNA gene sequence. Their spectrum of inhibition was observed against food associated strains of Bacillus subtilis and Staphylococcus aureus. Moreover, L. lactis subsp. lactis PI-2 selected on the basis of higher antimicrobial activity was further evaluated for bacteriocin production which was detected as nisin A and nisin Z. These findings suggest the possible use of L. lactis strains of vegetable origin as protective cultures in slightly acidic as well as slightly alkaline food by the bio-preservative action of bacteriocins.     

Quantitative PCR for the specific quantification of Lactococcus lactis and Lactobacillus paracasei and its interest for Lactococcus lactis in cheese samples

The first objective of this work was to develop real-time quantitative PCR (qPCR) assays to quantify two species of mesophilic lactic acid bacteria technologically active in food fermentation, including cheese making: Lactococcus lactis and Lactobacillus paracasei. The second objective was to compare qPCR and plate counts of these two species in cheese samples. Newly designed primers efficiently amplified a region of the tuf gene from the target species. Sixty-three DNA samples from twenty different bacterial species, phylogenetically related or commonly found in raw milk and dairy products, were selected as positive and negative controls. Target DNA was successfully amplified showing a single peak on the amplicon melting curve; non-target DNA was not amplified. Quantification was linear over 5 log units (R² > 0.990), down to 22 gene copies/μL per well for Lc. lactis and 73 gene copies/μL per well for Lb. paracasei. qPCR efficiency ranged from 82.9% to 93.7% for Lc. lactis and from 81.1% to 99.5% for Lb. paracasei. At two stages of growth, Lc. lactis was quantified in 12 soft cheeses and Lb. paracasei in 24 hard cooked cheeses. qPCR proved to be useful for quantifying Lc. lactis, but not Lb. paracasei.     

Potential production and preservation of dahi by Lactococcus lactis W8, a nisin-producing strain

Lactococcus lactis W8 produced nisin concomitantly while fermenting milk to “dahi”, a traditional Indian fermented milk. The activity of nisin was detected at 3 h of fermentation, which increased in parallel to growth of the organism and reached its maximum at 6 h. The activity remained essentially stable thereafter. At 7 h of fermentation of milk with the strain L. lactis W8 the pH of the medium dropped to 4.2, when the milk became converted to dahi. The produced dahi displayed antibacterial property against spoilage and pathogenic bacteria including Listeria monocytogenes. When L. monocytogenes was mixed with dahi at 5.2 log CFU/ml and stored at 4 °C, the number of L. monocytogenes gradually decreased and became undetectable at 10 h. L. lactis W8 appeared to be a suitable starter culture for production of dahi from milk and preservation of the dahi.     

Survival of a Lactococcus lactis strain varies with its carbohydrate preference under in vitro conditions simulated gastrointestinal tract

Lactococcus lactis G50 is a candidate probiotic bacterium with immunomodulatory activity. We evaluated the suitability of strain G50 as orally administered probiotics on the basis of its resistance to simulated gastrointestinal (GI) stress, including the presence of lysozyme, low pH, and bile, carbohydrate preference, and bacterial cell surface composition in vitro. This strain survived GI stresses but its resistance to lysozyme was affected by the type of available carbohydrate in the growth medium; it was unaffected with lactose, xylose and galactose as the carbon source but was significantly lower for fructose, sucrose and glucose. The resistance of strain G50 to low pH was unaffected by carbon source. Resistance to bile was determined by two methods; growth and survival study and varied with carbon source. The growth of strain G50 with 0.3% bile was lowest in lactose-containing broth, higher in broth containing xylose or galactose, and highest in broth containing sucrose, glucose, or fructose. In contrast, the survival of cells after 3 h incubation with 0.3% bile was highest for lactose. The hydrophobicity of bacterial cells, which can be related to epithelial adhesion in certain cases, was also highest for lactose. The fatty acid composition of cells grown on lactose differed from that of cells grown on other carbon sources. These results suggest that survival of strain G50 in the GI tract depends on the kinds of carbohydrates available. Carbohydrate preferences were observed for other strains of lactic acid bacteria under conditions of GI stress, and this preference varied with the strain and the type of GI stress. Careful consideration should be given to the selection of carbohydrates for in vitro testing of the survival of lactic acid bacteria in the GI tract.     

Effect of protein supplementation on the rheological characteristics of milk permeates fermented with exopolysaccharide-producing Lactococcus lactis subsp. cremoris

This research studied the effect of addition of whey proteins on the rheological properties of ultrafiltration permeate fermented with the exopolysaccharide (EPS)-producing strain Lactococcus lactis subsp. cremoris JFR1. Milk permeates containing 8% solids and various levels of added whey proteins (0, 2, 4, 6 and 8%) were fermented for 12 h at 30 °C. The rheological properties of the fermented samples were then evaluated and compared to controls fermented with a non-EPS producing strain. Scanning electron microscopy was also employed to confirm the existence of interactions between whey protein aggregates and EPS. The presence of EPS considerably increased the viscosity and viscoelastic properties of the media, especially in samples containing >2% whey protein added. The results obtained demonstrate the importance of EPS–protein interactions in structure formation and may help explain the viscosifying mechanism of EPS in fermented dairy products. Production of highly viscous material could potentially be employed in the future as a novel fiber-rich functional ingredient in dairy products.     

Isolation and characterization of a nisin-like bacteriocin produced by a Lactococcus lactis strain isolated from charqui,a Brazilian fermented,salted and dried meat product

A Lactococcus lactis subsp. lactis strain (L. lactis 69) capable to produce a heat-stable bacteriocin was isolated from charqui, a Brazilian fermented, salted and sun-dried meat product. The bacteriocin inhibited, in vitro, Listeria monocytogenes, Staphylococcus aureus, several lactic acid bacteria isolated from foods and spoilage halotolerant bacteria isolated from charqui. The activity of the bacteriocin was not affected by pH (2.0–10.0), heating (100 °C), and chemical agents (1% w/v). Treatment of growing cells of L. monocytogenes ScottA with the cell-free supernatant of L. lactis 69 resulted in complete cell inactivation. L. lactis 69 harbored the gene for the production of a nisin-like bacteriocin, and the amino acid sequence of the active peptide was identical to sequences previously described for nisin Z. However, differences were observed regarding the leader peptide. Besides, the isolate was able to survive and produce bacteriocins in culture medium with NaCl content up to 20%, evidencing a potential application as an additional hurdle in the preservation of charqui.     

Short communication: Recovery of viable Mycobacterium avium subspecies paratuberculosis from retail pasteurized whole milk in Brazil

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic granulomatous enteritis that affects all ruminants worldwide. Some researchers have indicated a possible role of MAP in Crohn's disease. Despite extensive research and large and important advances in the past few decades, the etiology of Crohn's disease remains indefinite. The most probable transmission route of MAP from animals to humans is milk and dairy products. Mycobacterium avium ssp. paratuberculosis has already been detected in milk samples worldwide, and some studies have reported that MAP is resistant to pasteurization. In Brazil, MAP has been reported in raw milk samples; however, Brazilian retail pasteurized milk has not yet been tested for viable MAP. The aim of this study was to investigate MAP in pasteurized milk in the region of Viçosa (Minas Gerais, Brazil). Thirty-seven samples were collected and processed for culture of MAP. One colony similar to MAP was observed and confirmed by IS900-nested PCR and sequencing. Analysis revealed 97 to 99% identity with the MAP K-10 strain. This study is the first report of the presence of MAP in retail pasteurized whole milk in Brazil.     

Short communication: Antimicrobial susceptibility profiling and genotyping of Staphylococcus aureus isolates from bovine mastitis in Poland

Staphylococcus aureus is the predominant causative agent of bovine mastitis, a disease that remains a major economic burden for the dairy industry worldwide. In this study, the antimicrobial resistance patterns and the genetic composition of 80 S. aureus mastitis isolates collected from 14 dairy farms in Eastern Poland were determined. Of the 10 antimicrobial agents evaluated, only testing for penicillin G produced drug resistance. As 41% of the S. aureus isolates were penicillin resistant, this drug along with other β-lactamase-sensitive β-lactams, should rather not be considered for the treatment of bovine mastitis caused by S. aureus. Upon genotyping, with a triplex PCR method, a total of 11 distinct PCR types were produced. The population structure of S. aureus isolates was highly clonal, with 1 predominant genotype circulating on each farm. The observed similarities in the genotype composition of S. aureus populations from geographically distant farms underscore the significance of interfarm transmission of S. aureus in Poland. This, in turn, argues for the establishment of a nationwide surveillance program for bovine mastitis due to this pathogen.     

Efficacy of vaccination on Staphylococcus aureus and coagulase-negative staphylococci intramammary infection dynamics in 2 dairy herds

The aim of this study was to evaluate vaccine efficacy of a commercial vaccine (Startvac, Hipra Spain) aimed at reducing intramammary infections (IMI) with Staphylococcus aureus and coagulase-negative staphylococci under field conditions. During the 21-mo duration of the study, 1,156 lactations from 809 cows were enrolled in 2 herds. During the first phase of the trial, all cows that were due to calve were vaccinated until approximately 50% of cows in the milking herd were vaccinated (at ~6 mo). At that point, when 50% vaccination coverage was reached, cows that were due to calve were randomly assigned to be vaccinated or left as negative controls. Cure rate, rate of new infection, prevalence, and duration of infections were analyzed. Vaccination resulted in a moderate reduction in incidence of new staphylococcal IMI and a more pronounced reduction in duration of IMI associated with reduction of the basic reproduction ratio of Staph. aureus by approximately 45% and of coagulase-negative staphylococci by approximately 35%. The utilization of vaccine in combination with other infection-control procedures, such as excellent milking procedures, treatment, segregation, and culling of known infected cattle, will result in an important reduction in incidence and duration of intramammary staphylococcal infections.     

High herd-level prevalence of Mycobacterium avium subspecies paratuberculosis in Western Canadian dairy farms,based on environmental sampling

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive enteritis in ruminants. The pathogen is present in most countries with modern dairy production, causing substantial economic losses for the industry. The objectives of this study were to estimate dairy herd prevalence of MAP in the Western Canadian provinces of Alberta and Saskatchewan, and to determine whether herd size and housing system (tie-stall versus freestall or loose housing) affected the risk of a herd testing positive for MAP. Six environmental samples were collected on 360 Alberta farms (60% of registered producers) and on 166 Saskatchewan dairy farms (99%). In total, 47% of the sampled farms in Alberta and 53% of the sampled farms in Saskatchewan had at least one environmental sample that was MAP culture positive and were, therefore, defined as infected. Sensitivity of environmental sampling was estimated using 3 subsequent annual tests performed on 82 farms. Because laboratory protocols were continuously improved throughout the project, the sensitivity increased over time. Therefore, a mean of the sensitivity estimates weighted on sampling year was constructed; this resulted in sensitivities of 68 and 69% for Alberta and Saskatchewan, respectively. Implementing those estimates in an approximate Bayesian computation model resulted in a true herd prevalence of 68% (95% probability interval: 60–80%) for Alberta and 76% (95% probability interval: 70–85%) for Saskatchewan. Herds with >200 cows had 3.54 times higher odds of being environmental sample positive and had more positive samples than herds with <50 cows (neither province nor housing system affected those results). In conclusion, the majority of Alberta and Saskatchewan dairy farms were infected with MAP and larger herds were more often MAP positive than smaller herds.     

Short communication: Epidemiology and genotyping of Candida rugosa strains responsible for persistent intramammary infections in dairy cows

The present study was undertaken during an outbreak of clinical and subclinical mastitis in 14 dairy cows caused by Candida rugosa, in which high somatic cell counts were seen and cases did not respond to antibiotic treatment. Intramammary infection cured spontaneously in 10 cows, whereas 4 cows were culled as a result of persistent infections. Repeated sampling of these cows and biomolecular analysis of the isolates showed that the infections were caused by the same genotype, even over a period of 2 lactations. Random amplification of the genome of C. rugosa milk isolates gave 3 different DNA banding patterns (genotypes G1, G2, and G3). Viable cells of C. rugosa were also isolated from various environmental sources and were present in high concentrations in total mixed ration samples, which could be considered the primary source of diffusion of viable yeast cells in the environment, as demonstrated by genotyping. The proven capacity of these microorganisms to survive in the environment of the cow, such as the total mixed ration, bedding, water, and cow skin, and to cause persistent intramammary infections highlights the importance of mycotic spread in dairy herds.     

Factors associated with the likelihood of Giardia spp. and Cryptosporidium spp. in soil from dairy farms

A study was conducted to identify factors associated with the likelihood of detecting Giardia spp. and Cryptosporidium spp. in the soil of dairy farms in a watershed area. A total of 37 farms were visited, and 782 soil samples were collected from targeted areas on these farms. The samples were analyzed for the presence of Cryptosporidium spp. oocysts, Giardia spp. cysts, percent moisture content, and pH. Logistic regression analysis was used to identify risk factors associated with the likelihood of the presence of these organisms. The use of the land at the sampling site was associated with the likelihood of environmental contamination with Cryptosporidium spp. Barn cleaner equipment area and agricultural fields were associated with increased likelihood of environmental contamination with Cryptosporidium spp. The risk of environmental contamination decreased with the pH of the soil and with the score of the potential likelihood of Cryptosporidium spp. The size of the sampling site, as determined by the sampling design, in square feet, was associated nonlinearly with the risk of detecting Cryptosporidium spp. The likelihood of the Giardia cyst in the soil increased with the prevalence of Giardia spp. in animals (i.e., 18 to 39%). As the size of the farm increased, there was decreased risk of Giardia spp. in the soil, and sampling sites which were covered with brush or bare soil showed a decrease in likelihood of detecting Giardia spp. when compared to land which had managed grass. The number of cattle on the farm less than 6 mo of age was negatively associated with the risk of detecting Giardia spp. in the soil, and the percent moisture content was positively associated with the risk of detecting Giardia spp. Our study showed that these two protozoan exist in dairy farm soil at different rates, and this risk could be modified by manipulating the pH of the soil.     

Hypocholesterolaemic effect of Bifidobacterium animalis subsp. lactis (Bb12) and trypsin casein hydrolysate

Hypercholesterolaemia is one of the most important risk factors in the development of cardiovascular disease. In this study, we report the in vitro potential of Bifidobacterium animalis subsp. lactis (Bb12) cultures and bovine casein hydrolysates formed by trypsin and Bb12 culture to reduce cholesterols levels. Cholesterol levels in vitro were reduced by up to 48% after incubation with Bb12 and up to 87% after incubation with trypsin hydrolysates, whereas unhydrolysed bovine casein did not affect cholesterol levels. Individual peptide fractions, obtained from size-exclusion chromatography, from casein hydrolysates formed by trypsin after a 48 h hydrolysis, reduced cholesterol levels by 2.7–50%. The molecular masses of these fractions, containing hypocholesterolaemic peptides, were below 1200 Da, as determined by LC-MS.     

Short communication: Survey of animal-borne pathogens in the farm environment of 13 dairy operations

A survey was conducted on 13 dairies to determine the occurrence of 5 animal-borne pathogens (Salmonella enterica, Escherichia coli O157:H7, Campylobacter jejuni, Mycobacterium avium ssp. paratuberculosis, and Cryptosporidium parvum) and their distributions across farm elements (feces, bedding, milk filters, stored manure, field soil, and stream water). Presence of C. parvum was measured only in feces and stored manure. All but one farm were positive for at least one pathogen species, and 5 farms were positive for 3 species. Escherichia coli O157:H7 was detected on 6 farms and in all farm elements, including milk filters. Mycobacterium avium ssp. paratuberculosis was detected on 10 of 13 farms and in all farm elements except for milk filters. Salmonella enterica and C. jejuni were detected at lower frequencies and were not identified in soil, stream water, or milk filters on any of the 13 farms. Cryptosporidium parvum was detected in feces but not in stored manure. Stored manure had the highest occurrence of pathogens (73%), followed by feces (50%), milk filters, bedding, soil, and water (range from 23 to 31%). Association of pathogen presence with farm management factors was examined by t-test; however, the small number of study farms and samples may limit the scope of inference of the associations. Pathogens had a higher prevalence in maternity pen bedding than in calf bedding, but total pathogen occurrence did not differ in calf compared with lactating cow feces or in soils with or without manure incorporation. Herd size and animal density did not appear to have a consistent effect on pathogen occurrence. The extent of pathogen prevalence and distribution on the farms indicates considerable public health risks associated with not only milk and meat consumption and direct animal contact, but also potential dissemination of the pathogens into the agroecosystem.     

Conjugated linoleic acid production by cells and enzyme extract of Lactobacillus delbrueckii ssp. bulgaricus with additions of different fatty acids

The objective of the study was to determine the effect of fatty acid additions to the cells and enzyme extract of Lactobacillus delbrueckii ssp. bulgaricus (CCRC14009) on CLA production. Washed cells of L. delbrueckii ssp. bulgaricus, obtained by cultivation in a MRS broth, were mixed with BSA and each of the three fatty acids: linoleic, oleic, and linolenic acids in sodium phosphate buffer at pH 6.5. After incubation at 37 °C for 108 h, CLA concentration was analyzed by HPLC. Enzyme extract from the culture was also reacted with each fatty acid at 50 °C for 10 min at pH 5 to test for CLA production. Results showed that linoleic acid addition to the culture improved CLA production, indicating the presence of linoleic acid isomerase activity in the culture. The crude enzyme extract from the culture was observed to be capable of oleic and linolenic acid conversions into CLA, demonstrating the possible presence of desaturase activity in the enzyme extract.     

Application of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and L. helveticus for sourdough bread making

The application of Kluyveromyces marxianus (IFO 288), Lactobacillus delbrueckii ssp. bulgaricus (ATCC 11842) and Lactobacillus helveticus (ATCC 15009) as starter cultures for sourdough bread making was examined. Production of lactic and acetic acids, bread rising, volatile composition, shelf-life and organoleptic quality of the sourdough breads were evaluated. The amount of starter culture added to the flour, the dough fermentation temperature and the amount of sourdough used were examined in order to optimise the bread making process. The use of mixed cultures led to higher total titratable acidities and lactic acid concentrations compared to traditionally made breads. Highest acidity (3.41 g lactic acid/kg of bread) and highest resistance to mould spoilage were observed when bread was made using 50% sourdough containing 1% K. marxianus and 4% L. delbrueckii ssp. bulgaricus. The use of these cultures also improved the aroma of sourdough breads, as shown by sensory evaluations and as revealed by GC–MS analysis.     

Short communication: Antimicrobial efficacy of intramammary treatment with a novel biphenomycin compound against Staphylococcus aureus, Streptococcus uberis, and Escherichia coli-induced mouse mastitis

Bovine mastitis undermines udder health, jeopardizes milk production, and entails prohibitive costs, estimated at $2 billion per year in the dairy industry of the United States. Despite intensive research, the dairy industry has not managed to eradicate the 3 major bovine mastitis-inducing pathogens: Staphylococcus aureus, Streptococcus uberis, and Escherichia coli. In this study, the antimicrobial efficacy of a newly formulated biphenomycin compound (AIC102827) was assessed against intramammary Staph. aureus, Strep. uberis, and E. coli infections, using an experimental mouse mastitis model. Based on its effective and protective doses, AIC102827 applied into the mammary gland was most efficient to treat Staph. aureus, but also adequately reduced growth of Strep. uberis or E. coli, indicating its potential as a broad-spectrum candidate to treat staphylococcal, streptococcal, and coliform mastitis in dairy cattle.