Genetic, enzymatic and metabolite profiling of the Lactobacillus casei group reveals strain biodiversity and potential applications for flavour diversification.

AIMS: The Lactobacillus casei group represents a widely explored group of lactic acid bacteria, characterized by a high level of biodiversity. In this study, the genetic and phenotypic diversity of a collection of more than 300 isolates of the Lact. casei group and their potential to produce volatile metabolites important for flavour development in dairy products, was examined. METHODS AND RESULTS: Following confirmation of species by 16S rRNA PCR, the diversity of the isolates was determined by pulsed-field gel electrophoresis. The activities of enzymes involved in the proteolytic cascade were assessed and significant differences among the strains were observed. Ten strains were chosen based on the results of their enzymes activities and they were analysed for their ability to produce volatiles in media with increased concentrations of a representative aromatic, branched chain and sulphur amino acid. Volatiles were assessed using gas chromatography coupled with mass spectrometry. Strain-dependent differences in the range and type of volatiles produced were evident. CONCLUSIONS: Strains of the Lact. casei group are characterized by genetic and metabolic diversity which supports variability in volatile production. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a screening approach for the knowledge-based selection of strains potentially enabling flavour diversification in fermented dairy products.

Transformation of serum-susceptible Escherichia coli O111 with p16Slux plasmid to allow for real-time monitoring of complement-based inactivation of bacterial growth in bovine milk.

Complement activity has only recently been characterized in raw bovine milk. However, the activity of this component of the innate immune system was found to diminish as milk was subjected to heat or partitioning during cream separation. Detection of complement in milk relies on a bactericidal assay. This assay exploits the specific growth susceptibility of Escherichia coli O111 to the presence of complement. Practical application of the assay was demonstrated when a reduction in complement activity was recorded in the case of pasteurized and reduced-fat milks. This presented an opportunity to improve the functionality of the bactericidal assay by incorporating bioluminescence capability into the target organism. Following some adaptation, the strain was transformed by correctly integrating the p16Slux plasmid. Growth properties of the transformed strain of E. coli O111 were unaffected by the modification. The efficacy of the strain adaptation was correlated using the LINEST function analysis [r=0.966; standard error of prediction (SEy)=0.957] bioluminescence with that of bactericidal assay total plate counts within the range of 7.5 to 9.2 log cfu/mL using a combination of raw and processed milk samples. Importantly, the transformed E. coli O111 p16Slux strain could be identified in milk and broth samples using bioluminescence measurement, thus enabling the bactericidal assay-viability test to be monitored in real time throughout incubation.

Reduced-fat Cheddar and Swiss-type cheeses harboring exopolysaccharide-producing probiotic Lactobacillus mucosae DPC 6426.

Exopolysaccharide-producing Lactobacillus mucosae DPC 6426 was previously shown to have promising hypocholesterolemic activity in the atherosclerosis-prone apolipoprotein-E-deficient (apoE(-/-)) murine model. The aim of this study was to investigate the suitability of reduced-fat Cheddar and Swiss-type cheeses as functional (carrier) foods for delivery of this probiotic strain. All cheeses were manufactured at pilot-scale (500-L vats) in triplicate, with standard commercially available starters: for Cheddar, Lactococcus lactis; and for Swiss-type cheese, Streptococcus thermophilus, Lactobacillus helveticus, and Propionibacterium freudenreichii. Lactobacillus mucosae DPC 6426 was used as an adjunct culture during cheese manufacture, at a level of ~10(6) cfu·mL(-1) cheese milk (subsequently present in the cheese curd at>10(7) cfu·g(-1)). The adjunct strain remained viable at >5×10(7) cfu·g(-1) in both Swiss-type and Cheddar cheeses following ripening for 6 mo. Sensory analysis revealed that the presence of the adjunct culture imparted a more appealing appearance in Swiss-type cheese, but had no significant effect on the sensory characteristics of Cheddar cheeses. Moreover, the adjunct culture had no significant effect on cheese composition, proteolysis, pH, or instrumentally quantified textural characteristics of Cheddar cheeses. These data indicate that low-fat Swiss-type and Cheddar cheeses represent suitable food matrices for the delivery of the hypocholesterolemic Lactobacillus mucosae DPC 6426 in an industrial setting.

Characterization of a bovine isolate Lactobacillus mucosae DPC 6426 which produces an exopolysaccharide composed predominantly of mannose residues.

AIMS: To characterize Lactobacillus strains with EPS-producing ability compared with non-EPS-producing lactobacilli of the same species for technological performance including simulated gastrointestinal tract (GIT) conditions. METHODS AND RESULTS: Characterization of EPS-producing Lactobacillus mucosae DPC 6426 in detail based on 16S rRNA sequencing, and EPS production using scanning electron and atomic force microscopy. The EPS was found to consist of mannosyl residues, with mannose, glucose and galactose found to be the major sugar residues present in an approximate ratio of 3: 2: 2. The strain was compared to non-EPS-producing Lact. mucosae DPC 6420 following exposure to salt, bile, acid and heat stresses. Lact. mucosae DPC 6426 exhibited twofold increased (P < 0·05) survival during 120-min exposure to 5 mol NaCl, threefold increased survival during 90-min exposure to 0·7% (w/v) bile (P < 0·05), threefold increased survival when exposed to simulated gastric juice (P < 0·001) for 10 min and fivefold increased survival during 60-min exposure to HCl (P < 0·01) compared with Lact. mucosae DPC 6420. Furthermore, Lact. mucosae DPC 6426 was found to be more heat tolerant (P < 0·001) compared with Lact. mucosae DPC 6420 during 30-min exposure to 55°C. CONCLUSIONS: These data indicate that the EPS-producing Lact. mucosae DPC 6426 exhibits technological and biological robustness compared with a non-EPS-producing Lact. mucosae strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The data implicate the potential suitability of EPS-producing Lact. mucosae DPC 6426 in food applications and/or as a probiotic culture.

γ-Aminobutyric acid production by culturable bacteria from the human intestine.

AIMS: To assess the ability of human intestinally derived strains of Lactobacillus and Bifidobacterium to produce γ-aminobutyric acid (GABA). METHODS AND RESULTS: Strains of Lactobacillus and Bifidobacterium were grown in medium containing monosodium glutamate (MSG). Growth of the bacteria and conversion of MSG to GABA were measured. Of 91 intestinally derived bacteria assessed, one Lactobacillus strain and four strains of Bifidobacterium produced GABA. Lactobacillus brevis DPC6108 was the most efficient of the strains tested, converting up to 90% [corrected] of MSG to GABA. The ability of the cultured intestinal strains to produce GABA was investigated using a simple pH-controlled anaerobic faeces-based fermentation, supplemented with 30 mg ml⁻¹ MSG. The addition of Lact. brevis DPC6108 to a faeces-based fermentation significantly increased the GABA concentration (P < 0·001), supporting the notion that this biosynthesis could occur in vivo. CONCLUSIONS: The production of GABA by bifidobacteria exhibited considerable interspecies variation. Lactobacillus brevis and Bifidobacterium dentium were the most efficient GABA producers among the range of strains tested. The addition of Lact. brevis DPC6108 to the culturable gut microbiota increased the GABA concentration in fermented faecal slurry at physiological pH. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of optimal MSG conversion to GABA by particular cultured elements of the commensal intestinal microbiota and the demonstration that this can occur under simulated in vivo conditions offer new prospects for microbiota modulation to promote health.

A comparison of methods used to extract bacterial DNA from raw milk and raw milk cheese.

AIMS: In this study, we compare seven different methods which have been designed or modified to extract total DNA from raw milk and raw milk cheese with a view to its subsequent use for the PCR of bacterial DNA. MATERIALS AND RESULTS: Seven extraction methods were employed to extract total DNA from these foods, and their relative success with respect to the yield and purity of the DNA isolated, and its quality as a template for downstream PCR, was compared. Although all of the methods were successful with respect to the extraction of DNA naturally present in cheese, they varied in their relative ability to extract DNA from milk. However, when milk was spiked with a representative Gram-positive (Listeria monocytogenes EGDe) or Gram-negative (Salmonella enterica serovar Typhimurium LT2) bacterium, it was established that all methods successfully extracted DNA which was suitable for subsequent detection by PCR. CONCLUSIONS: Of the seven approaches, the PowerFood™ Microbial DNA Isolation kit (MoBio Laboratories Inc.) was found to most consistently extract highly concentrated and pure DNA with a view to its subsequent use for PCR-based amplification and also facilitated accurate detection by real-time quantitative PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: Accurately assessing the bacterial composition of milk and cheese is of great importance to the dairy industry. Increasingly, DNA-based technologies are being employed to provide an accurate assessment of this microbiota. However, these approaches are dependent on our ability to extract DNA of sufficient yield and purity. This study compares a number of different options and highlights the relative success of these approaches. We also highlight the success of one method to extract DNA from different microbial populations as well as DNA which is suitable for real-time PCR of microbes of interest, a challenge often encountered by the food industry.

Production of the antimicrobial peptides Caseicin A and B by Bacillus isolates growing on sodium caseinate.

AIMS: The aim of this study was to identify Bacillus isolates capable of degrading sodium caseinate and subsequently to generate bioactive peptides with antimicrobial activity. METHODS AND RESULTS: Sodium caseinate (2.5% w/v) was inoculated separately with 16 Bacillus isolates and allowed to ferment overnight. Protein breakdown in the fermentates was analysed using gel permeation-HPLC (GP-HPLC) and screened for peptides (<3-kDa) with MALDI-TOF mass spectrometry. Caseicin A (IKHQGLPQE) and caseicin B (VLNENLLR), two previously characterized antimicrobial peptides, were identified in the fermentates of both Bacillus cereus and Bacillus thuringiensis isolates. The caseicin peptides were subsequently purified by RP-HPLC and antimicrobial assays indicated that the peptides maintained the previously identified inhibitory activity against the infant formula pathogen Cronobacter sakazakii. CONCLUSIONS: We report a new method using Bacillus sp. to generate two previously characterized antimicrobial peptides from casein. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the potential to exploit Bacillus sp. or the enzymes they produce for the generation of bioactive antimicrobial peptides from bovine casein.

Novel conjugative plasmids from the natural isolate Lactococcus lactis subspecies cremoris DPC3758: a repository of genes for the potential improvement of dairy starters.

A collection of 17 natural lactococcal isolates from raw milk cheeses were studied in terms of their plasmid distribution, content, and diversity. All strains in the collection harbored an abundance of plasmids, including Lactococcus lactis ssp. cremoris DPC3758, whose 8-plasmid complement was selected for sequencing. The complete sequences of pAF22 (22,388 kb), pAF14 (14,419 kb), pAF12 (12,067 kb), pAF07 (7,435 kb), and pAF04 (3,801 kb) were obtained, whereas gene functions of technological interest were mapped to pAF65 (65 kb) and pAF45 (45 kb) by PCR. The plasmids of L. lactis DPC3758 were found to encode many genes with the potential to improve the technological properties of dairy starters. These included 3 anti-phage restriction/modification (R/M) systems (1 of type I and 2 of type II) and genes for immunity/resistance to nisin, lacticin 481, cadmium, and copper. Regions encoding conjugative/mobilization functions were present in 6 of the 8 plasmids, including those containing the R/M systems, thus enabling the food-grade transfer of these mechanisms to industrial strains. Using cadmium selection, the sequential stacking of the R/M plasmids into a plasmid-free host provided the recipient with increased protection against 936- and c2-type phages. The association of food-grade selectable markers and mobilization functions on L. lactis DPC3758 plasmids will facilitate their exploitation to obtain industrial strains with enhanced phage protection and robustness. These natural plasmids also provide another example of the major role of plasmids in contributing to host fitness and preservation within its ecological niche.

Application of whey protein micro-bead coatings for enhanced strength and probiotic protection during fruit juice storage and gastric incubation.

CONTEXT: Coated whey protein micro-beads may improve probiotic protection and provide delayed cell-release mechanisms. OBJECTIVE: Lactobacillus rhamnosus GG was encapsulated in whey protein micro-beads by droplet extrusion with coating via electrostatic deposition: primary-polysaccharide and secondary-whey protein. MATERIALS AND METHODS: Storage studies were performed in cranberry and pomegranate juice (pH 2.4; 28 days; 4 and 25°C) followed by simulated ex vivo porcine gastric (pH 1.6) and intestinal (pH 6.6) digestion. RESULTS AND DISCUSSION: After storage and simulated gastro-intestinal digestion, free cells, cells suspended in protein and cells encapsulated in alginate micro-beads, illustrated complete probiotic mortality, while coated micro-beads enhanced probiotic viability after juice storage (8.6 ± 0.1 log(10)CFUmL(-1)). Beads also showed significant binding of hydrophobic molecules. Coated micro-beads illustrated high gastric survival (9.5 ± 0.1 log(10)CFUmL(-1)) with 30 min delayed intestinal release relative to non-coated micro-beads. CONCLUSIONS: Micro-bead coatings could be applied in delayed cell-release for targeted intestinal probiotic delivery.

Modification of the technical properties of Lactobacillus johnsonii NCC 533 by supplementing the growth medium with unsaturated fatty acids.

The aim of this study was to investigate the influence of supplementing growth medium with unsaturated fatty acids on the technical properties of the probiotic strain Lactobacillus johnsonii NCC 533, such as heat and acid tolerance, and inhibition of Salmonella enterica serovar Typhimurium infection. Our results showed that the membrane composition and morphology of L. johnsonii NCC 533 were significantly changed by supplementing a minimal Lactobacillus medium with oleic, linoleic, and linolenic acids. The ratio of saturated to unsaturated plus cyclic fatty acids in the bacterial membrane decreased by almost 2-fold when minimal medium was supplemented with unsaturated fatty acids (10 µg/ml). The subsequent acid and heat tolerance of L. johnsonii decreased by 6- and 20-fold when the strain was grown in the presence of linoleic and linolenic acids, respectively, compared with growth in oleic acid (all at 10 µg/ml). Following acid exposure, significantly higher (P < 0.05) oleic acid content was detected in the membrane when growth medium was supplemented with linoleic or linolenic acid, indicating that saturation of the membrane fatty acids occurred during acid stress. Cell integrity was determined in real time during stressed conditions using a fluorescent viability kit in combination with flow cytometric analysis. Following heat shock (at 62.5°C for 5 min), L. johnsonii was unable to form colonies; however, 60% of the bacteria showed no cell integrity loss, which could indicate that the elevated heat inactivated vital processes within the cell, rendering it incapable of replication. Furthermore, L. johnsonii grown in fatty acid-enriched minimal medium had different adhesion properties and caused a 2-fold decrease in S. enterica serovar Typhimurium UK1-lux invasion of HT-29 epithelial cells compared with bacteria grown in minimal medium alone. This could be related to changes in the hydrophobicity and fluidity of the membrane. Our study shows that technical properties underlying probiotic survivability can be affected by nutrient composition of the growth medium.

A role for proline synthesis and transport in Listeria monocytogenes barotolerance.

AIMS: To assess the contribution of proline biosynthesis to listerial barotolerance. METHODS AND RESULTS: Using a Listeria monocytogenes proBA deletion mutant, incapable of synthesizing proline, together with a proline-overproducing strain, the contribution of proline synthesis to listerial barotolerance was determined. The ΔproBA strain does not survive as well as the wild type when subjected to treatment of 500 MPa in rich media and 400 MPa in minimal media (c. 1 log lower survival in both conditions). Betaine and carnitine decrease the ability of the wild type to survive at low pressures (300 MPa), but confer normal or slightly increased levels of protection at higher pressures (350 and 400 MPa). CONCLUSIONS: A functional proline synthesis system is required for optimal survival of Listeria following treatment at high-pressure (HP) levels (500 MPa in brain heart infusion and 400 MPa in defined medium), particularly where other compatible solutes are absent or limiting. SIGNIFICANCE AND IMPACT OF THE STUDY: Given the potential of HP processing as an effective food processing/safety strategy, understanding how pathogens such as Listeria have evolved to cope with such stresses is an important food safety consideration. In this context, the work presented here may help to develop safer and more effective processing regimens.

Novel listerial genetic loci conferring enhanced barotolerance in Escherichia coli.

AIMS: To identify Listeria monocytogenes genes with a role in high-pressure (HP) resistance. METHODS AND RESULTS: A L. monocytogenes genomic library constructed in an Escherichia coli background was screened for loci conferring increased HP resistance. Pressure treatments at 400 megapascals for 5 min in Luria-Bertani (LB) agar were used to screen for increased resistance to pressure. Colonies arising on the treated agar plates were isolated, the plasmid extracted and the inserts sequenced to identify the genetic loci conferring HP resistance. Seven different genetic regions were identified, which encoded proteins similar to an inorganic polyphosphate/ATP-NAD kinase, the septation ring formation regulator EzrA, flagellar motor proteins MotA and MotB, proteins similar to the quorum sensing Agr system from Staphylococcus (AgrA, AgrC and AgrD), proteins similar to a transcription regulator (RpiR family) and a fructose phosphotransferase system, proteins of unknown function, and a Fur regulator. Of the seven loci confirmed, three (EzrA, MotA/B and the Agr system) maintained significantly reproducible HP tolerance when expressed in a different E. coli background. CONCLUSIONS: Novel genetic loci from the L. monocytogenes genome confer increased HP resistance when heterologously expressed in an E. coli background. SIGNIFICANCE AND IMPACT OF THE STUDY: Molecular and functional approaches to the screening of genetic elements linked to HP resistance provide greater insights into microbial inactivation and/or survival mechanisms when using HP as a means of controlling/eliminating bacterial growth. This information will ultimately have significant implications for the use of HP processing in the food industry, in terms of both food quality and safety.

Reconstitution conditions for dried probiotic powders represent a critical step in determining cell viability.

AIMS: Resuscitation of dried cultures represents a critical control point in obtaining active and effective probiotic strains. This study examined the effects of various rehydration conditions on the viability of Bifidobacterium longum NCC3001 and Lactobacillus johnsonii La1. METHODS AND RESULTS: Reconstitution conditions for these strains were optimized using a multivariate experimental design approach. Furthermore, using flow cytometry, the cell integrity was followed during reconstitution. By adjusting the pH, availability of a metabolizable sugar, reconstitution duration, powder matrix and ratio of powder to reconstitution solution, the recovery of Bif. longum NCC3001 and Lact. johnsonii La1 following reconstitution was increased eight- and two-fold, respectively, over standard reconstitution in maximum recovery diluent. It was shown that pH had a significant effect on the recovery of Bif. longum NCC3001 and Lact. johnsonii La1. CONCLUSIONS: The recovery of dried probiotic cultures is greatly dependent on the reconstitution conditions. The maximum recovery of 11.7 (10)log CFU g(-1) Bif. longum NCC3001 was achieved at 30-min reconstitution at pH 8, in the presence of 2% L-arabinose and a ratio of 1:100 of powder to diluent. Lact. johnsonii La1 showed highest recovery (9.3 (10)log CFU g(-1)) after reconstitution, when mixed with maltodextrin at pH 4. SIGNIFICANCE AND IMPACT OF THE STUDY: To achieve accurate viable probiotic numbers from dried probiotic cultures, the reconstitution conditions should be optimized for the strain used.

Invited review: Lactobacillus helveticus--a thermophilic dairy starter related to gut bacteria.

The strain Lactobacillus helveticus DPC4571 has emerged as a promising flavor adjunct culture for Cheddar cheese given that it is consistently associated with improved flavor. The availability of the complete genome sequence of Lb. helveticus DPC4571 has enabled the search for the presence or absence of specific genes on the genome, in particular those of technological interest. Indeed, this analysis has facilitated a greater understanding into the functioning of lactic acid bacteria as a whole. The biochemical pathways of Lb. helveticus responsible for producing flavor compounds during cheese ripening are poorly understood but now with the availability of a complete genomic sequence are ripe for exploitation. Bioinformatic analysis of the genome of Lb. helveticus DPC4571 has revealed a plethora of genes with industrial potential including those responsible for key metabolic functions that contribute to cheese flavor development such as proteolysis, lipolysis, and cell lysis. In addition, it has been demonstrated that Lb. helveticus has the potential to produce bioactive peptides such as angiotensin converting enzyme inhibitory activity in fermented dairy products, demonstrating the therapeutic value of this species. A most intriguing feature of the genome of Lb. helveticus DPC4571 is the remarkable similarity in gene content with many intestinal lactobacilli, although originating from considerably different environments. Bioinformatic analysis demonstrated that 65 to 75% of genes were conserved between the commensal and dairy lactobacilli, which allowed key niche-specific gene sets to be described. This review focuses on the isolation, characterization, and exploitation of the Lb. helveticus species with particular emphesis on taking into consideration recent genome sequence data for Lb. helveticus and other Lactobacillus species.

Evaluation of an antimicrobial ingredient prepared from a Lactobacillus acidophilus casein fermentate against Enterobacter sakazakii.

Previously two antimicrobial peptides, IKHQGLPQE (caseicin A) and VLNENLLR (caseicin B), were identified following the fermentation of sodium caseinate with the proteolytic strain Lactobacillus acidophilus DPC 6026. This study evaluated the ability of these peptides to kill Enterobacter sakazakii ATCC 12868 spiked in reconstituted infant formula. The survival of E. sakazakii populations in reconstituted infant formula containing a sodium caseinate fermentate was compared with survival in formula containing positive (monocaprylin) and negative controls. The L. acidophilus DPC 6026 sodium caseinate fermentate reduced pathogen numbers by >4 log CFU/ml at 37 degrees C, comparing favorably with the activity of monocaprylin. Additionally, E. sakazakii NCTC 8155 was inoculated into pasteurized, reconstituted infant formula (6 log CFUlml) followed by the addition of increasing concentrations of the fermentate (0.21 to 6.7% [wt/vol]). At a concentration of 0.21% (wt/vol), pathogen viability was maintained over 4 h at 6.0 log CFU/ml. In contrast, pathogen numbers increased approximately 100-fold in the control formula in the same time frame. At higher final fermentate concentrations (-3.33% [wt/vol]), numbers were reduced to 0 log CFU/ml over 60 min. The spectrum of activity of the fermentate against other foodborne pathogens was also determined and shown to be effective against Escherichia coli O157:H7 and Listeria innocua. Results indicate the potential of this fermentate as a built-in protection mechanism against E. sakazakii strains in reconstituted infant formula.

The combined effects of high pressure and nisin on germination and inactivation of Bacillus spores in milk.

AIMS: The aim of this work was to investigate the germination and inactivation of spores of Bacillus species in buffer and milk subjected to high pressure (HP) and nisin. METHODS AND RESULTS: Spores of Bacillus subtilis and Bacillus cereus suspended in milk or buffer were treated at 100 or 500 MPa at 40 degrees C with or without 500 IU ml(-1) of nisin. Treatment at 500 MPa resulted in high levels of germination (4 log units) of B. subtilis spores in both milk and buffer; this increased to >6 logs by applying a second cycle of pressure. Viability of B. subtilis spores in milk and buffer was reduced by 2.5 logs by cycled HP, while the addition of nisin (500 IU ml(-1)) prior to HP treatment resulted in log reductions of 5.7 and 5.9 in phosphate buffered saline and milk, respectively. Physical damage of spores of B. subtilis following HP was apparent using scanning electron microscopy. Treating four strains of B. cereus at 500 MPa for 5 min twice at 40 degrees C in the presence of 500 IU ml(-1) nisin proved less effective at inactivating the spores of these isolates compared with B. subtilis and some strain-to-strain variability was observed. CONCLUSIONS: Although high levels of germination of Bacillus spores could be achieved by combining HP and nisin, complete inactivation was not achieved using the aforementioned treatments. SIGNIFICANCE AND IMPACT OF THE STUDY: Combinations of HP treatment and nisin may be an appealing alternative to heat pasteurization of milk.

Enumeration and identification of pediococci in powder-based products using selective media and rapid PFGE.

In this study, pediococci selective medium (PSM) was evaluated for the enumeration of Pediococcus acidilactici and Pediococcus pentosaceus from probiotic animal feed and silage inoculants. PSM is based on the complex basal medium MRS supplemented with cysteine hydrochloride, novobiocin, vancomycin, and nystatin. No significant change in electivity was observed when pediococci where recovered from culture or powder-based products following incubation at 37 degrees C under anaerobic conditions for 24 h. The medium was suitable for the enumeration of pediococci in samples also containing bacilli, bifidobacteria, enterococci, lactobacilli, lactococci, propionibacteria, streptococci, and yeast components. However, to inhibit Lactobacillus plantarum and Lactobacillus casei, ampicillin was added and the revised medium, termed PSM+A, was also considered to be suitably elective for pediococci recovered from powder. In addition, a rapid PFGE protocol is presented, which allows Pediococcus species and strain verification from colonies in less than 3 days.

Influence of GABA and GABA-producing Lactobacillus brevis DPC 6108 on the development of diabetes in a streptozotocin rat model.

The aim of this study was to investigate if dietary administration of γ-aminobutyric acid (GABA)-producing Lactobacillus brevis DPC 6108 and pure GABA exert protective effects against the development of diabetes in streptozotocin (STZ)-induced diabetic Sprague Dawley rats. In a first experiment, healthy rats were divided in 3 groups (n=10/group) receiving placebo, 2.6 mg/kg body weight (bw) pure GABA or L. brevis DPC 6108 (~10(9)microorganisms). In a second experiment, rats (n=15/group) were randomised to five groups and four of these received an injection of STZ to induce type 1 diabetes. Diabetic and non-diabetic controls received placebo [4% (w/v) yeast extract in dH2O], while the other three diabetic groups received one of the following dietary supplements: 2.6 mg/kg bw GABA (low GABA), 200 mg/kg bw GABA (high GABA) or ~10(9) L. brevis DPC 6108. L. brevis DPC 6108 supplementation was associated with increased serum insulin levels (P<0.05), but did not alter other metabolic markers in healthy rats. Diabetes induced by STZ injection decreased body weight (P<0.05), increased intestinal length (P<0.05) and stimulated water and food intake. Insulin was decreased (P<0.05), whereas glucose was increased (P<0.001) in all diabetic groups, compared with non-diabetic controls. A decrease (P<0.01) in glucose levels was observed in diabetic rats receiving L. brevis DPC 6108, compared with diabetic-controls. Both the composition and diversity of the intestinal microbiota were affected by diabetes. Microbial diversity in diabetic rats supplemented with low GABA was not reduced (P>0.05), compared with non-diabetic controls while all other diabetic groups displayed reduced diversity (P<0.05). L. brevis DPC 6108 attenuated hyperglycaemia induced by diabetes but additional studies are needed to understand the mechanisms involved in this reduction.

Biotechnological approaches to the understanding and improvement of mature cheese flavour.

There have been important milestones in biotechnological practice that have led to the determination and production of superior cheese flavours. Within the past year, the use of gas chromatographic techniques and sensory methodologies has been optimised by several groups in efforts to evaluate the organoleptic properties of a number of mature cheeses. The hydrolysis of milk caseins, small peptides, free amino acids and fatty acids, and the generation of sulfur-containing compounds are uniformly assumed to result in the formation of specific cheese aromas. Giant strides have been taken in molecular technology to aid the dissection and exploitation of the metabolic pathways that lead to the formation of these flavour constituents. Specific advances in molecular technology have included metabolic engineering of lactic acid bacteria for enhanced flavour development.

Sugar-coated: exopolysaccharide producing lactic acid bacteria for food and human health applications.

The human enteric microbiome represents a veritable organ relied upon by the host for a range of metabolic and homeostatic functions. Through the production of metabolites such as short chain fatty acids (SCFA), folate, vitamins B and K, lactic acid, bacteriocins, peroxides and exopolysaccharides, the bacteria of the gut microbiome provide nutritional components for colonocytes, liver and muscle cells, competitively exclude potential pathogenic organisms and modulate the hosts immune system. Due to the extensive variation in structure, size and composition, microbial exopolysaccharides represent a useful set of versatile natural ingredients for the food industrial sector, both in terms of their rheological properties and in many cases, their associated health benefits. The exopolysaccharide-producing bacteria that fall within the 35 Lactobacillus and five Bifidobacterium species which have achieved qualified presumption of safety (QPS) and generally recognised as safe (GRAS) status are of particular interest, as their inclusion in food products can avoid considerable scrutiny. In addition, additives commonly utilised by the food industry are becoming unattractive to the consumer, due to the demand for a more 'natural' and 'clean labelled' diet. In situ production of exopolysaccharides by food-grade cultures in many cases confers similar rheological and sensory properties in fermented dairy products, as traditional additives, such as hydrocolloids, collagen and alginate. This review will focus on microbial synthesis of exopolysaccharides, the human health benefits of dietary exopolysaccharides and the technofunctional applications of exopolysaccharide-synthesising microbes in the food industry.