Human Bifidobacterium strains as adjunct cultures in Spanish sheep milk cheese.

Three bifidobacteria strains of human origin (Bifidobacterium pseudolongum INIA P2, Bifidobacterium breve INIA P734, and Bifidobacterium longum INIA P678) were used as potential probiotic adjunct cultures for the manufacture of pasteurized sheep milk cheese. Bifidobacteria were inoculated at 5 to 6 log cfu/mL in milk vats. Microbiological, physicochemical, rheological, color, and sensory characteristics were determined at 7, 28, and 60 d of ripening. Counts of B. pseudolongum INIA P2 remained above 6 log cfu/g during 60 d of ripening as well as after further simulated gastrointestinal digestion of cheeses. Bifidobacterium breve INIA P734 counts remained stable during 28 d and decreased by less than 1 log unit after simulated digestion. Bifidobacterium longum INIA P678 counts dropped sharply during cheese manufacture and ripening and were below detection level after simulated digestion. Addition of bifidobacteria strains did not affect starter viability, cheese pH, dry matter, water activity, or salt content but significantly increased overall proteolysis and the concentration of some free amino acids. Cheeses with bifidobacteria exhibited no significant differences in most sensory characteristics with respect to control cheese. According to our results, B. breve INIA P734 and B. pseudolongum INIA P2 are promising candidates as probiotic adjunct cultures in fresh and semi-hard sheep milk cheese.

Coproduction of colicin V and lactic acid bacteria bacteriocins in lactococci and enterococci strains of biotechnological interest.

AIMS: The aim of this study was the coproduction in a single strain of the Gram-negative bacteriocin colicin V with other bacteriocins from lactic acid bacteria (LAB). METHODS AND RESULTS: Colicin V was expressed in Lactococcus and Enterococcus strains by replacing the colicin V leader peptide by the leader peptide and promoter of d-alanyl-d-alanine carboxypeptidase from Lactobacillus reuteri CECT925 in pNZ8048 (pNZ:LR-colV). The antimicrobial activity of colicin V against the indicator organism Escherichia coli DH5α in transformed strains was checked by agar diffusion assay and SDS-PAGE analysis. CONCLUSIONS: Lactococcus and Enterococcus transformed with pNZ:LR-colV were able to coproduce colicin V at high levels together with other LAB bacteriocins such as nisin A, nisin Z, lacticin 481 or enterocins A and B, obtaining broad-spectrum activity strains with large potential applications. SIGNIFICANCE AND IMPACT OF THE STUDY: The construction showed in this work could be used for the heterologous expression of other bacteriocins active against Gram-negative bacteria or wide-spectrum bacteriocins from LAB.

Antimicrobial activity of reuterin produced by Lactobacillus reuteri on Listeria monocytogenes in cold-smoked salmon.

Lactobacillus reuteri INIA P579 was used for the production and purification of reuterin. The purity of reuterin was assessed by high resolution electrospray ionization mass spectrometry (HRESIMS) and nuclear magnetic resonance (NMR) spectroscopy. After purification, reuterin concentration obtained was 1.3 M. The inhibitory activity using Escherichia coli K12 as indicator strain was estimated to be 510 AU/ml. Survival curves in tryptic soy broth revealed that reuterin required to inhibit the growth of three Listeria monocytogenes strains was in the range of 2-4 AU/ml. Purified reuterin (10 AU/g) significantly reduced the growth of L. monocytogenes in cold-smoked salmon kept under moderate or strong temperature abuse conditions. After 15 d at 8 °C, cold-smoked salmon with added reuterin exhibited L. monocytogenes counts 2.0 log CFU/g lower than control smoked salmon with no reuterin added. At 30 °C, reuterin also controlled the growth of the pathogen, with counts 1.4 and 0.9 log CFU/g lower than those observed in control smoked salmon after 24 and 48 h, respectively. The addition of purified reuterin might be used as a hurdle technology to improve the safety and extend the shelf-life of lightly preserved seafood products such as cold-smoked salmon.

Short communication: Combined antimicrobial activity of reuterin and diacetyl against foodborne pathogens.

Reuterin (ß-hydroxypropionialdehyde) is a broad-spectrum antimicrobial substance produced by some strains of Lactobacillus reuteri during anaerobic fermentation of glycerol. Some of these strains are able to survive and produce reuterin in cheese and yogurt when added as adjuncts to the starter. Similarly, in fermented dairy foods, other inhibitory compounds such as lactic acid and diacetyl are produced during fermentation. In this work, we studied the combined effect of reuterin and diacetyl under different pH conditions against Escherichia coli O157:H7, Salmonella Enteritidis, and Listeria monocytogenes. Results from agar spot assays showed that the antimicrobial activity of reuterin-producing strains against the gram-negative bacteria tested was enhanced as the concentration of diacetyl increased to 50 mg/kg, and was higher under acidic conditions (pH 5.0) for the 3 pathogenic strains. The combination of reuterin and diacetyl had an additive effect against L. monocytogenes only at diacetyl concentrations of 50 mg/kg and pH 5.0. In addition, growth kinetics studies showed that the combination of 1 activity unit (AU)/mL of reuterin with 100mg/kg diacetyl increased the lag time of the 3 pathogens. In milk, synergistic antimicrobial activity was observed with the combination of 1 AU/mL reuterin and 50 or 100 mg/kg of diacetyl on the gram-negative strains tested, and with 1 AU/mL reuterin and 100 mg/kg of diacetyl on L. monocytogenes. The greatest inhibition of the 3 pathogens was achieved in acidified milk at pH 5.0 with reuterin (1 AU/mL) and diacetyl (100 mg/kg). Based on these results, the combination of reuterin and diacetyl in acidified dairy products could be a promising strategy to control food pathogens in these products.

Oral delivery of Lactobacillus casei Shirota modifies allergen-induced immune responses in allergic rhinitis.

BACKGROUND: Changes in the composition of the gut microbiota have been implicated in the pathogenesis of allergic disorders, suggesting beneficial interactions between the intestinal immune system and specific bacterial strains. Lactobacilli are naturally present within the complex gastrointestinal microbiota of humans and they are currently present in many probiotic supplements. OBJECTIVE: We sought to investigate the role that Lactobacillus casei Shirota (LcS) may play in modulating seasonal allergic rhinitis (SAR). METHODS: The study format was double-blinded, placebo-controlled with 10 SAR sufferers in each group. We have documented and compared changes in immune status arising through the daily ingestion of a milk drink with or without live LcS, over a period of 5 months. Pre-, peak- and post-grass pollen season blood samples were collected for determination of plasma total IgE and grass pollen-specific IgG and IgE levels by an enzyme immunoassay. At the same time, cytokine levels were determined by flow cytometric bead array technology following culture of peripheral blood mononuclear cells for 6 days in the presence or absence of specific grass pollen antigens. RESULTS: Volunteers treated with LcS showed a significant reduction in levels of antigen-induced IL-5, IL-6 and IFN-gamma production compared with volunteers supplemented with placebo. Meanwhile, levels of specific IgG increased and IgE decreased in the probiotic group. CONCLUSION: Changes in antigen-induced production of cytokines were observed in patients treated with probiotics. These data show that probiotic supplementation modulates immune responses in allergic rhinitis and may have the potential to alleviate the severity of symptoms.

Antimicrobial activity of nisin, reuterin, and the lactoperoxidase system on Listeria monocytogenes and Staphylococcus aureus in cuajada, a semisolid dairy product manufactured in Spain.

The inhibitory activity of nisin (N), reuterin (R), and the lactoperoxidase system (LPS), added individually or in combination, against Listeria monocytogenes and Staphylococcus aureus was investigated in "cuajada" (curdled milk), a semisolid dairy product manufactured in Spain. Cuajada was manufactured from UHT skim milk separately inoculated with L. monocytogenes and Staph. aureus, each at approximately 4 log cfu/mL, and held under conditions of temperature abuse (10 degrees C). On d 3, a synergistic bactericidal activity was observed for the combinations of biopreservatives assayed, with L. monocytogenes counts of only 0.30 log cfu/mL in cuajada made with N + R + LPS vs. 8.31 log cfu/mL in control cuajada. After 12 d, L. monocytogenes could not be detected in cuajada made with added N + LPS or N + R + LPS. Staphylococcus aureus was more resistant than L. monocytogenes to biopreservatives added individually. On d 3, the synergistic effect of the 3 biopreservatives against Staph. aureus resulted in counts of 3.03 log cfu/mL in cuajada made with N + R + LPS vs. 6.40 in control cuajada. After 12 d, Staph. aureus counts were 2.61 log cfu/mL in cuajada made with N + R + LPS, whereas they ranged from 6.11 to 7.70 log cfu/mL in control cuajada and in cuajada made with other combinations of biopreservatives. The most pronounced decrease in pathogen counts was achieved by the triple combination N + R + LPS, which acted synergistically on the inactivation of L. monocytogenes and Staph. aureus in cuajada over 12 d at 10 degrees C. The treatment combining these 3 natural biopreservatives at low concentrations, within the hurdle concept of food preservation, might be a useful tool to control the growth of pathogenic microorganisms in nonacidified dairy products.

Immunity gene pedB enhances production of pediocin PA-1 in naturally resistant Lactococcus lactis strains.

Heterologous production of the antilisterial bacteriocin pediocin PA-1 in lactococci is an attractive objective to increase the safety of dairy products. In a previous paper, we developed a system for the heterologous production of the bacteriocin pediocin PA-1 in pediocin-resistant lactococcal hosts through a leader exchange strategy. The system was based on 3 genes, 1 encoding the fusion between the lactococcin A leader and propediocin PA-1, and the other 2 encoding the lactococcin A secretion machinery. In this study, we investigated whether the addition of the pediocin PA-1 immunity gene (pedB) to this system has any effect on pediocin production. Introduction of the plasmid(s) carrying the genes described above into nisinproducing and non-nisinproducing lactococcal hosts led to a significant increase in the production of pediocin compared with the equivalent pedB-devoid systems. In addition, we obtained a nisin-producing strain with the ability to secrete pediocin PA-1 at a level equivalent to that of the parental strain Pediococcus acidilactici 347, which represents a notable improvement over our previous systems.

Bifidobacterial strains shared by mother and child as source of probiotics.

Importance of bifidobacteria as part of the infant intestinal microbiota has been highlighted. Their acquisition is influenced by the mode of birth and the feed regime afterwards, with a special role of the maternal microbiota. The presence of the same shared bifidobacterial strains between breast milk and infant faeces in 14 mother-infant pairs was assessed by means of pulsed-field gel electrophoresis (PFGE) genotyping. Four shared strains of Bifidobacterium breve (2), Bifidobacterium longum subsp. infantis and B. longum subsp. longum were found in breast milk-infant faeces pairs. Two years later, a second survey yielded four shared strains of the species Bifidobacterium adolescentis, Bifidobacterium bifidum, B. longum subsp. longum and Bifidobacterium pseudocatenulatum. Moreover, a B. bifidum strain was found to be shared by the infant faeces of the first study and the mother faeces tested two years later, pointing out a long term persistence. Some of the selected bifidobacterial strains showed probiotic potential due to their survival to gastrointestinal conditions and their ability to form biofilms.

Volatile compounds, odor, and aroma of La Serena cheese high-pressure treated at two different stages of ripening.

La Serena cheeses made from raw Merino ewe's milk were high-pressure (HP) treated at 300 or 400 MPa for 10 min on d 2 or 50 after manufacture. Ripening of HP-treated and control cheeses proceeded until d 60 at 8 degrees C. Volatile compounds were determined throughout ripening, and analysis of related sensory characteristics was carried out on ripe cheeses. High-pressure treatments on d 2 enhanced the formation of branched-chain aldehydes and of 2-alcohols except 2-butanol, but retarded that of n-aldehydes, 2-methyl ketones, dihydroxy-ketones, n-alcohols, unsaturated alcohols, ethyl esters, propyl esters, and branched-chain esters. Differences between HP-treated and control cheeses in the levels of some volatile compounds tended to disappear during ripening. The odor of ripe cheeses was scarcely affected by HP treatments on d 2, but aroma quality and intensity scores were lowered in comparison with control cheese of the same age. On the other hand, HP treatments on d 50 did not influence either the volatile compound profile or the sensory characteristics of 60-d-old cheese.

Short communication: Inactivation of microbial contaminants in raw milk La Serena cheese by high-pressure treatments.

La Serena cheese, a Spanish variety made from Merino ewes' raw milk, has a high pH value, low salt content, and high moisture, conditions that are all favorable for growth and survival of contaminating microorganisms, including pathogens. To improve its microbiological quality and safety, high-pressure treatments at 300 or 400 MPa for 10 min at 10 degrees C were applied to 2 batches of La Serena cheese on d 2 or 50 of ripening. Cheese treated on d 2 at 300 MPa showed viable aerobic counts that were 0.99 log units lower than those for control cheese on d 3 and showed counts of enterococci, coagulase-positive staphylococci, gram-negative bacteria, and coliforms that were 2.05, 0.49, 3.14, and 4.13 log units lower, respectively, than control cheese. For cheese treated on d 2 at 400 MPa, the respective reductions in counts were 2.02, 2.68, 1.45, 3.96, and 5.50 log units. On d 60, viable aerobic counts in cheese treated on d 50 at 300 MPa were 0.50 log units lower than those in control cheese, and counts of enterococci, gram-negative bacteria, and coliforms were 1.37, 2.30, and 4.85 log units lower, respectively. For cheese treated on d 50 at 400 MPa, the respective reductions in counts were 1.29, 1.98, 4.47, and > 5 log units. High-pressure treatments at 300 or 400 MPa on d 2 or 50 reduced significantly the counts of undesirable microorganisms, improving the microbiological quality and safety of La Serena cheese immediately after treatment and at the end of the ripening period.

Inactivation of Staphylococcus aureus in raw milk cheese by combinations of high-pressure treatments and bacteriocin-producing lactic acid bacteria.

AIMS: To investigate the combined effect of high-pressure treatments (HPT) and milk inoculation with bacteriocin-producing lactic acid bacteria (BP-LAB) on the survival of Staphylococcus aureus during ripening of raw milk cheese. METHODS AND RESULTS: Cheeses were manufactured from raw milk artificially contaminated with S. aureus at ca 5 log CFU ml(-1), a commercial starter culture and one of seven strains of BP-LAB, added as adjuncts at 0.1%. HPT of cheeses were performed on days 2 or 50 at 300 MPa (10 degrees C, 10 min) or 500 MPa (10 degrees C, 5 min). On day 3, S. aureus counts were 6.46 log CFU g(-1) in control cheese. Milk inoculation with different BP-LAB lowered S. aureus counts on day 3 when compared with control cheese by up to 0.46 log CFU g(-1), HPT at 300 MPa on day 2 by 0.45 log CFU g(-1) and HPT at 500 MPa on day 2 by 2.43 log CFU g(-1). Combinations of BP-LAB with HPT at 300 and 500 MPa on day 2 lowered S. aureus counts on day 3 by up to 1.02 and 4.00 log CFU g(-1) respectively. CONCLUSIONS: The combined effect of milk inoculation with some of the BP-LAB tested and HPT of cheese on S. aureus inactivation was synergistic. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of HPT at lower pressures with BP-LAB inoculation is a feasible system to improve cheese safety in case of deleterious effects on cheese quality caused by HPT at higher pressures.

Reuterin production by lactobacilli isolated from pig faeces and evaluation of probiotic traits.

AIMS: To determine the production of reuterin by lactobacilli isolates from pig faeces and to evaluate their potential as probiotic bacteria. METHODS AND RESULTS: Twenty-eight of 165 lactobacilli isolates produced reuterin in the presence of glycerol. Six isolates yielding high levels of reuterin with respect to type strain Lactobacillus reuteri CECT 925T were identified as Lact. reuteri. They were able to survive at pH 3 and subsequent exposure to cholic acid or oxgall, and presented bile salt hydrolase and bacteriocin-like activities. CONCLUSIONS: Reuterin production is a frequently found trait among lactobacilli isolated from pig faeces. Selected Lact. reuteri isolates were able to survive at conditions likely to be encountered throughout the gastrointestinal tract. SIGNIFICANCE AND IMPACT OF THE STUDY: High yields of reuterin may be obtained from selected isolates of Lact. reuteri. Probiotic characteristics of isolates studied in the present work suggest their application in food and feed.