Food Matrix Design for Effective Lactic Acid Bacteria Delivery.

The range of foods featuring lactic acid bacteria (LAB) with potential associated health benefits has expanded over the years from traditional dairy products to meat, cereals, vegetables and fruits, chocolate, etc. All these new carriers need to be compared for their efficacy to protect, carry, and deliver LAB, but because of their profusion and the diversity of methods this remains difficult. This review points out the advantages and disadvantages of the main food matrix types, and an additional distinction between dairy and nondairy foods is made. The food matrix impact on LAB viability during food manufacturing, storage, and digestion is also discussed. The authors propose an ideal hypothetical food matrix that includes structural and physicochemical characteristics such as pH, water activity, and buffering capacities, all of which need to be taken into account when performing LAB food matrix design. Guidelines are finally provided to optimize food matrix design in terms of effective LAB delivery.

Multilocus sequence typing of Carnobacterium maltaromaticum strains associated with fish disease and dairy products.

AIMS: Carnobacterium maltaromaticum is a lactic acid bacterium of technological interest in the field of dairy ripening and food bioprotection and is generally recognized as safe in the United States. As it is associated with fish infections, the European Food Safety Agency did not include this species in the qualified presumption safety list of micro-organisms. This implies that the risk assessment for the species has to be performed at the strain level. METHODS AND RESULTS: Multilocus sequence typing (MLST) is a tool that (i) potentially allows to discriminate strains isolated from diseased fish from apathogenic strains and (ii) to assess the genetic relatedness between both groups of strains. In this study, we characterized by MLST 21 C. maltaromaticum strains including 16 strains isolated from diseased fish and 5 apathogenic dairy strains isolated from cheese. The resulting population structure was investigated by integrating these new data to the previously published population structure (available at http://pubmlst.org), which represents an overall of 71 strains. CONCLUSIONS: This analysis revealed that none of the strains isolated from diseased fish is assigned to a clonal complex containing cheese isolates, and that 11 strains exhibit singleton genotypes suggesting that the population of diseased fish isolates is not clonal. SIGNIFICANCE AND IMPACT OF THE STUDY: This study thus provides a population structure of C. maltaromaticum that could serve in the future as a reference that could contribute to the risk assessment of C. maltaromaticum strains intended to be used in the food chain.

Lactic acid bacteria in dairy food: surface characterization and interactions with food matrix components.

This review gives an overview of the importance of interactions occurring in dairy matrices between Lactic Acid Bacteria and milk components. Dairy products are important sources of biological active compounds of particular relevance to human health. These compounds include immunoglobulins, whey proteins and peptides, polar lipids, and lactic acid bacteria including probiotics. A better understanding of interactions between bioactive components and their delivery matrix may successfully improve their transport to their target site of action. Pioneering research on probiotic lactic acid bacteria has mainly focused on their host effects. However, very little is known about their interaction with dairy ingredients. Such knowledge could contribute to designing new and more efficient dairy food, and to better understand relationships between milk constituents. The purpose of this review is first to provide an overview of the current knowledge about the biomolecules produced on bacterial surface and the composition of the dairy matter. In order to understand how bacteria interact with dairy molecules, adhesion mechanisms are subsequently reviewed with a special focus on the environmental conditions affecting bacterial adhesion. Methods dedicated to investigate the bacterial surface and to decipher interactions between bacteria and abiotic dairy components are also detailed. Finally, relevant industrial implications of these interactions are presented and discussed.

In vitro interactions between probiotic bacteria and milk proteins probed by atomic force microscopy.

Interactions between microbial cells and milk proteins are important for cell location into dairy matrices. In this study, interactions between two probiotic strains, Lactobacillus rhamnosus GG and Lactobacillus rhamnosus GR-1, and milk proteins (micellar casein, native and denatured whey proteins) were studied. The bacterial surface characterization was realized with X-ray photoelectron spectroscopy (XPS) to evaluate surface composition (in terms of proteins, polysaccharides and lipid-like compounds) and electrophoretic mobility that provide information on surface charge of both bacteria and proteins along the 3-7 pH range. In addition, atomic force microscopy (AFM) enabled the identification of specific interactions between bacteria and whey proteins, in contrast to the observed nonspecific interactions with micellar casein. These specific events appeared to be more important for the GG strain than for the GR-1 strain, showing that matrix interaction is strain-specific. Furthermore, our study highlighted that in addition to the nature of the strains, many other factors influence the bacterial interaction with dairy matrix including the nature of the proteins and the pH of the media.

Surface properties of bacteria sensitive and resistant to the class IIa carnobacteriocin Cbn BM1.

AIMS: Class IIa bacteriocins are small antimicrobial peptides synthesized by lactic acid bacteria. The proposed mechanisms of action for class IIa bacteriocins suggest that the physicochemical properties of the target bacterial surface govern the bacteriocin antimicrobial activity. The aim of this study is to decipher the relationship between both sensitivity and resistance to a class IIa bacteriocin, carnobacteriocin BM1 and physicochemical surface properties of bacteria. METHODS AND RESULTS: The study was performed on 18 strains by a microbial adhesion to solvents process and with electrophoretic mobility measurements considering bacteria as soft particles. A large variation in bacterial surface properties is observed among the bacterial populations. Electro-hydrodynamic parameters values appear to be more homogeneous for sensitive strains than for the resistant ones, which can exhibit more extreme values. CONCLUSIONS: Physicochemical surface properties of 18 strains determined show large variations between the strains. However, no direct link between these surface properties and the resistant/sensitive phenotypes of the strains can be stated. SIGNIFICANCE AND IMPACT OF THE STUDY: The surface physicochemical properties tested have a low predictive power to discriminate sensitive or resistant strains when determined at the bacterial population scale.

Short communication: impact of pH and temperature on the acidifying activity of Carnobacterium maltaromaticum.

The acidifying activity of Carnobacterium maltaromaticum LMA28, a strain isolated from French soft cheese, was studied in trypticase soy broth with yeast extract (TSB-YE) medium and in milk. In TSB-YE supplemented with lactose, glucose, or galactose, lactose and glucose were metabolized with a maximum growth rate of 0.32 h(-1) and galactose was not metabolized. During hydrolysis of lactose, the galactose moiety was not excreted. The major product was l(+) lactic acid, with no significant difference in the lactic acid yield. Glucose was not completely metabolized because cell growth stopped when pH values reached an average of 5.0. In sterilized UHT milk, the addition of 1 g/L of YE enhanced its coagulation. Compared with commercial starter lactic acid bacteria such as Lactococcus lactis DSMZ 20481 or Streptococcus thermophilus INRA 302, Carnobacterium maltaromaticum LMA 28 was shown to be a slow acidifying strain. However, in spite of this weak acidifying ability, C. maltaromaticum LMA 28 can sustain low pH values in coculture with Lc. lactis DSMZ 20481 or S. thermophilus INRA 302. The individual and interactive effects of initial pH values (5.2 to 8.0) and incubation temperatures (23 to 37 degrees C) on acidifying activity were studied by response surface methodology. The 3 strains displayed different behaviors depending on pH and temperature. The psychrotrophic lactic acid strain C. maltaromaticum LMA 28 was able to grow at alkaline pH values and during storage conditions. It could be used as a potential ripening flora in soft cheese.

Short communication: Carnobacterium maltaromaticum: the only Carnobacterium species in French ripened soft cheeses as revealed by polymerase chain reaction detection.

The PCR technique using Cb1-Cb2R and species-specific primers was applied to various French soft flowered or washed rind cheeses to identify Carnobacterium species. Thirty cheeses made from cow's, ewe's, or goat's milk (raw or pasteurized), of which 20 were Appellation d'Origine Contrôlée, were analyzed in the autumn and spring. The results revealed that, irrespective of the season, the Carnobacterium genus was initially detected in 5 cheeses and was detected in 5 others after an enrichment period. Polymerase chain reaction results using species-specific primers of Carnobacterium showed that these 10 cheeses contained only the species Carnobacterium maltaromaticum. Six different patterns of fermentation were found, and 3 of the 10 cheeses contained C. maltaromaticum isolates with anti-Listeria activity.

Optimization of the components concentrations of the lactoperoxidase system by RSM.

AIMS: The aim of this work was to use response surface methodology (RSM) approach, a statistical mathematical tool, to model effects and interactions of glucose oxidase (GOD), glucose, lactoperoxidase (LPO) and pH-values on the thiocyanate (SCN-) peroxidation, to determine the best concentrations of lactoperoxidase system (LP-s) components in order to obtain maximal SCN- peroxidation and so to enhance the LP-s antibacterial effects. METHODS AND RESULTS: Experimental design using RSM was used for modelling effects and interactions of GOD (28.5-142.5 IU l(-1)), glucose (0.55-11.11 mmol l(-1)), LPO (0-6284 IU l(-1)) concentrations, and pH-values (6.0-7.4) on thiocyanate peroxidation. A fixed SCN- concentration of 0.5 mmol l(-1) was used. Experiments were carried out at 4 or at 25 degrees C in 0.1 mol l(-1) phosphate buffer. Optimized concentrations for both temperatures (4 and 25 degrees C) were quite similar and were 85.5 IU l(-1) for GOD, 8 mmol l(-1) for glucose and 3927.5 IU l(-1) for LPO at an initial pH-value of 6.5. SCN- peroxidation was more efficient at 25 than at 4 degrees C. At 4 degrees C, no interaction between factors occurred. At 25 degrees C, thiocyanate peroxidation was affected by GOD/glucose, GOD/pH and LPO/pH. Thiocyanate peroxidation was mainly increased by glucose and LPO factors. The optimized system had a bacteriostatic effect on Listeria monocytogenes CIP 82110(T) and a strong bactericidal effect on Pseudomonas fluorescens CIP 6913(T). CONCLUSIONS: Appropriate combinations of LPO, GOD, glucose concentrations and pH-values allowed maximal thiocyanate peroxidation and enhanced the antibacterial effect of the LP-s. SIGNIFICANCE AND IMPACT OF THE STUDY: This optimization by RSM approach allowed a better understanding of the LP-s functioning, the description of the component impacts on the SCN- peroxidation, and the observation of different interactions between the factors. The antimicrobial efficiency of LP-s can be enhanced by better concentration ratios of the LP-s components.

Biological activities and structural properties of the atypical bacteriocins mesenterocin 52b and leucocin b-ta33a.

The antibacterial spectra and modes of action of synthetic peptides corresponding to mesenterocin 52B and leucocin B-TA33a greatly differ despite their high sequence homology. Circular dichroism experiments establish the capacity of each of these two peptides to partly fold into an amphiphilic helix that might be crucial for their adsorption at lipophilic-hydrophilic interfaces.

Ponericins, new antibacterial and insecticidal peptides from the venom of the ant Pachycondyla goeldii.

The antimicrobial, insecticidal, and hemolytic properties of peptides isolated from the venom of the predatory ant Pachycondyla goeldii, a member of the subfamily Ponerinae, were investigated. Fifteen novel peptides, named ponericins, exhibiting antibacterial and insecticidal properties were purified, and their amino acid sequences were characterized. According to their primary structure similarities, they can be classified into three families: ponericin G, W, and L. Ponericins share high sequence similarities with known peptides: ponericins G with cecropin-like peptides, ponericins W with gaegurins and melittin, and ponericins L with dermaseptins. Ten peptides were synthesized for further analysis. Their antimicrobial activities against Gram-positive and Gram-negative bacteria strains were analyzed together with their insecticidal activities against cricket larvae and their hemolytic activities. Interestingly, within each of the three families, several peptides present differences in their biological activities. The comparison of the structural features of ponericins with those of well-studied peptides suggests that the ponericins may adopt an amphipathic alpha-helical structure in polar environments, such as cell membranes. In the venom, the estimated peptide concentrations appear to be compatible with an antibacterial activity in vivo. This suggests that in the ant colony, the peptides exhibit a defensive role against microbial pathogens arising from prey introduction and/or ingestion.

Effects of combinations of lactoperoxidase system and nisin on the behaviour of Listeria monocytogenes ATCC 15313 in skim milk.

Individual or combined effects of nisin (100 or 200 IU/ml) and the lactoperoxidase system (LPS) were analysed against 1 x 10(4) cfu/ml Listeria monocytogenes ATCC 15313 cells in skim milk, at 25 degrees C for 15 days. Nisin induced an immediate bactericidal effect and LPS a 48 h bacteriostatic phase which in both cases was followed by re-growth of L. monocytogenes. LPS and nisin added together at t0 showed a synergistic and lasting bactericidal effect which after 8 days and until 15 days resulted in no detectable cells in 1 ml of milk. When LPS was added to cells already in contact with 100 or 200 IU/ml nisin for a period of 4 h, the inhibitory activity was enhanced with no L. monocytogenes detectable after 72 or 48 h, respectively, and until 15 days. When LPS was added after 12 h, the nisin bactericidal phase was followed by re-growth. When nisin, 100 or 200 UI/ml, was added to cells already in contact with LPS over 24 h, L. monocytogenes was not detectable after 196 and 244 h, respectively, without any re-growth. For nisin addition after 72 h, cell counts were 8 log10 cycles lower than in the control milk after 196 h, but population levels were similar to the control within 15 days. The best combination to inhibit L. monocytogenes ATCC 15313 was nisin present at t0 followed by the LPS addition 4 h later, when the maximum inhibitory effect of nisin was reached.

Response surface methodology, an approach to predict the effects of a lactoperoxidase system, Nisin, alone or in combination, on Listeria monocytogenes in skim milk.

Experimental designs using Response Surface Methodology (RSM) were used to determine effects and interactions of Nisin (0-200 i.u. ml-1), pH values (5.4-6.6), incubation time (0-36 h or 0-144 h) and the lactoperoxidase-thiocyanate-hydrogen peroxide system (LPS) on Listeria monocytogenes CIP 82110 in skim milk, at 25 degrees C. The LPS varied from level 0-2; LPS at level 1 consisted of lactoperoxidase (35 mg l-1), thiocyanate (25 mg l-1) and H2O2, which was supplied exogenously by glucose-oxidase (1 mg l-1) and glucose (0.2 g l-1); LPS activity was dependent on LPS level and incubation time. In the presence of LPS at level 1, a bacteriostatic phase was followed by growth, whereas at a higher level, a bactericidic phase was observed. Nisin response was time- and pH-dependent. Nisin was bactericidic at acidic pH values and for a short incubation time (12 h) only; then, a re-growth phase was observed. Nisin and LPS in combination gave an original response which lacked the transitory bactericidal effect of Nisin and had a continuously bactericidal affect, leading to 10 cfu ml-1 of L. monocytogenes at 144 h; the response was greatly affected by incubation time. Predicted values were in good agreement with experimental values. Response Surface Methodology is a useful experimental approach for rapid testing of the effects of inhibitors.

Influence of temperature and pH on production of two bacteriocins by Leuconostoc mesenteroides subsp. mesenteroides FR52 during batch fermentation.

The influence of temperature and pH on growth of Leuconostoc mesenteroides subsp. mesenteroides FR52 and production of its two bacteriocins, mesenterocin 52A and mesenterocin 52B, was studied during batch fermentation. Temperature and pH had a strong influence on the production of the two bacteriocins which was stimulated by slow growth rates. The optimal temperature was 20 degrees C for production of mesenterocin 52A and 25 degrees C for mesenterocin 52B. Optimal pH values were 5.5 and 5.0 for production of mesenterocin 52A and mesenterocin 52B respectively. Thus, by changing the culture conditions, production of one bacteriocin can be favoured in relation to the other. The relationship between growth and specific production rates of the two bacteriocins, as a function of the culture conditions, showed different kinetics of production and the presence of several peaks in the specific production rates during growth.

Multiple bacteriocin production by Leuconostoc mesenteroides TA33a and other Leuconostoc/Weissella strains.

Leuconostoc (Lc.) mesenteroides TA33a produced three bacteriocins with different inhibitory activity spectra. Bacteriocins were purified by adsorption/desorption from producer cells and reverse phase high-performance liquid chromatography. Leucocin C-TA33a, a novel bacteriocin with a predicted molecular mass of 4598 Da, inhibited Listeria and other lactic acid bacteria (LAB). Leucocin B-TA33a has a predicted molecular mass of 3466 Da, with activity against Leuconostoc/Weissella (W.) strains, and appears similar to mesenterocin 52B and dextranicin 24, while leucocin A-TA33a, which also inhibited Listeria and other LAB strains, is identical to leucocin A-UAL 187. A survey of other known bacteriocin-producing Leuconostoc/Weissella strains for the presence of the three different bacteriocins revealed that production of leucocin A-, B- and C-type bacteriocins was widespread. Lc. carnosum LA54a, W. paramesenteroides LA7a, and Lc. gelidum UAL 187-22 produced all three bacteriocins, whereas W. paramesenteroides OX and Lc. carnosum TA11a produced only leucocin A- and B-type bacteriocins.

Leuconostoc mesenteroides subsp. mesenteroides FR52 synthesizes two distinct bacteriocins.

Mesenterocin 52, a bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides FR52, was purified from producing cells by the adsorption-desorption method, combined with reverse-phase high-performance liquid chromatography. The elution profile revealed the presence of two inhibitory peaks of activity, each displaying different inhibitory spectra. Mesenterocin 52A possessed a broad inhibitory spectrum, including anti-Listeria activity, while Mesenterocin 52B was only active against Leuconostoc spp. The amino acid sequence and M(r) of Mesenterocin 52A appeared identical to the previously described Mesentericin Y105. In contrast, Mesenterocin 52B possessed a M(r) of 3446 Da, corresponding to 32 amino acids and a sequence that shared no homology with known bacteriocins: NH2-KGVLGWLSMASSALTGPQQPNSPWLAKIKNHK.

Purification and N-terminal amino acid sequence of dextranicin 24, a bacteriocin of Leuconostoc sp.

Leuconostoc mesenteroides subsp. dextranicum strain J24 synthesized a bacteriocin named Dextranicin 24 (Dex-24), which inhibited only other Leuconostoc sp. strains. It was purified by a two-step procedure from the fraction of the bacteriocin bound to the producer cells at the end of the growth: desorption form the cells at acidic pH, followed by reserve phase HPLC. The N-terminal sequence of Dex-24 was the following: NH2(-) K G V L G W L S M A S S A L T G P Q Q . . .