A multi-strain probiotic promoted recovery of puppies from gastroenteritis in a randomized, double-blind, placebo-controlled study.

Objective and animals: Acute diarrhea is among the most common causes of veterinary consultations for dogs. A double-blind, placebocontrolled intervention trial was done with 120 puppies with gastroenteritis. These dogs were 1 to 4 mo old, male and female, of various breeds and sizes. Procedure: Dogs were randomly allocated into 2 groups: Those in the treated group (TG) received a multi-strain probiotic with Lactobacillus johnsonii CRL1693, Ligilactobacillus murinus CRL1695, Limosilactobacillus mucosae CRL1696, and Ligilactobacillus salivarius CRL1702 (1 × 109 CFU/mL) daily for 7 d, whereas those in the control group (CG) received a placebo. All puppies received intravenous fluids, an antiparasitic, amoxicillin PO, and enrofloxacin SC. Results: At the start of the trial, the 2 groups were similar. Probiotic administration for 7 d normalized fecal consistency, with 69, 50, and 80% of small, medium, and large puppies in the TG achieving a fecal score of 1 (separate hard lumps) at 7 d, significantly better than puppies in the CG. After 7 d of treatment, most puppies (70%) in the TG had an excellent recovery, whereas in the CG, recoveries were 35.7% "bad" and 30.4% "fair." Therefore, treatment with probiotics hastened recovery (P < 0.0001). At the end of the trial, there was a significant increase of cultivable lactobacilli in the feces of TG puppies, but no significant differences between the 2 groups in numbers of total mesophylls, enterobacteria, or Gram-positive cocci. Total mortality was 5.8%, including 4 puppies from the CG and 3 from the TG. Conclusion: In a randomized, double-blind, placebo-controlled study, puppies with gastroenteritis symptoms receiving a multi-strain probiotic had rapid improvement, implying beneficial effects on the microbiota and its functionality.

Un probiotique multi-souches a favorisé la guérison des chiots de la gastro-entérite dans une étude randomisée, en double aveugle et vérifiée par placebo. Objectif et animaux: La diarrhée aiguë fait partie des causes les plus fréquentes de consultations vétérinaires pour les chiens. Un essai d'intervention en double aveugle et vérifié par placebo a été réalisé avec 120 chiots atteints de gastro-entérite. Ces chiens étaient âgés de 1 à 4 mois, mâles et femelles, de différentes races et tailles. Procédure: Les chiens ont été répartis au hasard en 2 groupes : ceux du groupe traité (TG) ont reçu un probiotique multisouches contenant Lactobacillus johnsonii CRL1693, Ligilactobacillus murinus CRL1695, Limosilactobacillus mucosae CRL1696 et Ligilactobacillus salivarius CRL1702 (1 × 109 UFC/mL) quotidiennement pendant 7 j, tandis que ceux du groupe témoin (CG) ont reçu un placebo. Tous les chiots ont reçu des liquides intraveineux, un antiparasitaire, de l'amoxicilline PO et de l'enrofloxacine SC. Résultats: Au début de l'essai, les 2 groupes étaient similaires. L'administration de probiotiques pour une durée de 7 j a normalisé la consistance fécale, avec 69, 50 et 80 % des chiots petits, moyens et grands dans le TG obtenant un score fécal de 1 (morceaux durs séparés) à 7 jours, ce qui était significativement meilleur que les chiots dans le CG. Après 7 jours de traitement, la plupart des chiots (70 %) dans le TG ont eu une excellente récupération, alors que dans le CG, les récupérations étaient de 35,7 % « mauvaises ¼ et 30,4 % « passables ¼. Par conséquent, le traitement avec des probiotiques a accéléré la récupération (P < 0,0001). À la fin de l'essai, il y avait une augmentation significative des lactobacilles cultivables dans les fèces des chiots TG, mais aucune différence significative entre les 2 groupes en nombre de mésophylles totaux, d'entérobactéries ou de coques à Gram positif. La mortalité totale était de 5,8 %, dont 4 chiots du CG et 3 du TG. Conclusion: Dans une étude randomisée, en double aveugle et vérifiée par placebo, des chiots présentant des symptômes de gastro-entérite recevant un probiotique multi-souches ont présenté une amélioration rapide, impliquant des effets bénéfiques sur le microbiote et sa fonctionnalité.(Traduit par Dr Serge Messier).

Identification and characterization of potential probiotic lactic acid bacteria isolated from pig feces at various production stages.

Lactic acid bacteria (LAB) were isolated, identified, and characterized from pig feces at various growth stages and feed rations in order to be used as probiotic feed additives. Lactic acid bacteria numbers ranged from 7.10 ± 1.50 to 9.40 log CFUs/g for growing and lactating pigs, respectively. Isolates (n = 230) were identified by (GTG)5-polymerase chain reaction and partial sequence analysis of 16S rRNA. Major LAB populations were Limosilactobacillus reuteri (49.2%), Pediococcus pentosaceus (20%), Lactobacillus amylovorus (11.4%), and L. johnsonii (8.7%). In-vitro assays were performed, including surface characterization and tolerance to acid and bile salts. Several lactobacilli exhibited hydrophobic and aggregative characteristics and were able to withstand gastrointestinal tract conditions. In addition, lactobacilli showed starch- and phytate-degrading ability, as well as antagonistic activity against Gram-negative pathogens and the production of bacteriocin-like inhibitory substances. When resistance or susceptibility to antibiotics was evaluated, high phenotypic resistance to ampicillin, gentamicin, kanamycin, streptomycin, and tetracycline and susceptibility towards clindamycin and chloramphenicol was observed in the assayed LAB. Genotypic characterization showed that 5 out of 15 resistance genes were identified in lactobacilli; their presence did not correlate with phenotypic traits. Genes erm(B), strA, strB, and aadE conferring resistance to erythromycin and streptomycin were reported among all lactobacilli, whereas tet(M) gene was harbored by L. reuteri and L. amylovorus strains. Based on these results, 6 probiotic LAB strains (L. reuteri F207R/G9R/B66R, L. amylovorus G636T/S244T, and L. johnsonii S92R) can be selected to explore their potential as direct feed additives to promote swine health and replace antibiotics.

Des bactéries lactiques (LAB) ont été isolées, identifiées et caractérisées à partir de matières fécales de porc à différents stades de croissance et de rations alimentaires afin d'être utilisées comme additifs alimentaires probiotiques. Le nombre de bactéries lactiques variait de 7,10 ± 1,50 à 9,40 log UFC/g pour les porcs en croissance et en lactation, respectivement. Les isolats (n = 230) ont été identifiés par réaction d'amplification en chaîne par la (GTG)5-polymérase et analyse partielle de la séquence de l'ARNr 16S. Les principales populations de LAB étaient Limosilactobacillus reuteri (49,2 %), Pediococcus pentosaceus (20 %), Lactobacillus amylovorus (11,4 %) et L. johnsonii (8,7 %). Des tests in vitro ont été effectués, y compris la caractérisation de surface et la tolérance aux acides et aux sels biliaires. Plusieurs lactobacilles présentaient des caractéristiques hydrophobes et agrégatives et étaient capables de résister aux conditions du tractus gastro-intestinal. De plus, les lactobacilles ont montré une capacité de dégradation de l'amidon et des phytates, ainsi qu'une activité antagoniste contre les agents pathogènes à Gram négatif et la production de substances inhibitrices de type bactériocine. Lorsque la résistance ou la sensibilité aux antibiotiques a été évaluée, une résistance phénotypique élevée à l'ampicilline, à la gentamicine, à la kanamycine, à la streptomycine et à la tétracycline et une sensibilité à la clindamycine et au chloramphénicol ont été observées dans les LAB testés. La caractérisation génotypique a montré que cinq gènes de résistance sur 15 ont été identifiés dans les lactobacilles; leur présence n'était pas corrélée aux traits phénotypiques. Les gènes erm(B), strA, strB et aadE conférant une résistance à l'érythromycine et à la streptomycine ont été signalés parmi tous les lactobacilles, tandis que le gène tet(M) était hébergé par les souches L. reuteri et L. amylovorus. Sur la base de ces résultats, six souches probiotiques LAB (L. reuteri F207R/G9R/B66R, L. amylovorus G636T/S244T et L. johnsonii S92R) peuvent être sélectionnées pour explorer leur potentiel en tant qu'additifs alimentaires directs pour promouvoir la santé des porcs et remplacer les antibiotiques.(Traduit par Docteur Serge Messier).

Effect of probiotic lactobacilli supplementation on growth parameters, blood profile, productive performance, and fecal microbiology in feedlot cattle.

Lactic acid bacteria (LAB) selected on the basis of probiotic characteristics were administered to beef feedlot catlle and the effect on body condition/growth and nutritional-metabolic status as well as on E. coli O157:H7 fecal shedding, were investigated. A feeding trials involving 126 steers were used to evaluate the effects of Lactobacillus acidophilus CRL2074, Limosilactobacillus fermentum CRL2085 and Limosilactobacillus mucosae CRL2069 and their combinations (5 different probiotic groups and control) when 107-108 CFU/animal of each probiotic group were in-feed supplemented. Cattle were fed a high energy corn-based diet (16 to 88%) and samples from each animal were taken at 0, 40, 104 and 163 days. In general, animals body condition and sensorium state showed optimal muscle-skeletal development and behavioral adaption to confinement; no nasal/eye discharges and diarrheic feces were observed. The nutritional performance of the steers revealed a steady increase of biometric parameters and weight. Animals supplied with L. mucosae CRL2069 for 104 days reached the maximum mean live weight (343.2 kg), whereas the greatest weight daily gain (1.27 ± 0.16 Kg/day) was obtained when CRL2069 and its combination with L. fermentum CRL2085 (1.26 ± 0.11 kg/day) were administered during the complete fattening cycle. With several exceptions, bovine cattle blood and serum parameters showed values within referential ranges. As a preharvest strategy to reduce Escherichia coli O157:H7 in cattle feces, CRL2085 administered during 40 days decreased pathogen shedding with a reduction of 43% during the feeding period. L. fermentum CRL2085 and L. mucosae CRL2069 show promise for feedlot cattle feeding supplementation to improve metabolic-nutritional status, overall productive performance and to reduce E. coli O157:H7 shedding, thus decreasing contamination chances of meat food products.

The Ability of Riboflavin-Overproducing Lactiplantibacillus plantarum Strains to Survive Under Gastrointestinal Conditions.

Riboflavin, vitamin B2, is essential for humans and has to be obtained from the diet. Some lactic acid bacteria (LAB) produce this vitamin, and they can be used for in-situ fortification of foods. This could be an alternative to supplementation with chemically synthesized vitamin, to palliate riboflavin deficiencies in specific groups of people. Moreover, if the producing LAB could survive in the gastrointestinal stress (GIT) they could be added as probiotics in this environment. In the present study we tested two riboflavin-overproducing Lactiplantibacillus plantarum strains (M5MA1-B2 and M9MG6-B2), spontaneous mutants of LAB isolated from chicha, a traditional Andean beverage. These two LAB, and also their isogenic strains M5MA1-B2[pRCR12] and M9MG6-B2[pRCR12], expressing the mCherry protein from the pRCR12 plasmid, were evaluated in vitro under simulated GIT conditions. Among other, specifically developed protein fluorescence assays were used. The four LAB showed similar levels of adhesion (>6.0%) to Caco-2 cells, higher than that of the probiotic Lacticaseibacillus rhamnosus GG strain (4.51%). Thus, LAB biofilm formation was assessed in the labeled cells by intracellular mCherry fluorescence and in the unlabeled parental strains by crystal violet staining. Both methods detected the formation of consistent biofilms by the L. plantarum strains. The quantification of mCherry fluorescence was also used to analyze LAB auto-aggregation properties. High levels of auto-aggregation were detected for both M5MA1-B2[pRCR12] and M9MG6-B2[pRCR12]. Survival of LAB included in a commercial cereal-based food matrix (Incaparina) under GIT conditions was also evaluated. The four LAB were resistant in vitro to the stomach and intestinal stresses, and proliferated in this environment, indicating a protective and nutritional effect of the Incaparina on the bacteria. Also, M9MG6-B2 survival in the presence or absence of Incaparina was evaluated in vivo in a BALB/c mouse model. The administration of the M9MG6-B2 strain alone or together with Incaparina had no adverse effect on the health, growth and/or well-being of the rodents. In addition, an increment in the villus length/crypt depth ratio was observed. The overall results obtained indicate that the LAB studied have probiotic characteristics of interest for the development of functional foods.

Mechanistically Inspired Kinetic Approach to Describe Interactions During Co-Culture Growth of Carnobacterium maltaromaticum and Listeria monocytogenes.

Lactic acid bacteria and Listeria monocytogenes are psychotropic organisms that can grow and compete in food such as lightly preserved fishery products. Predictive microbiology is nowadays one of the leading tools to assess the behavior of bacteria in food and to predict food spoilage. Mathematical models can be used to predict the growth, inactivation or growth probability of bacteria. Currently, the efforts in microbial modeling are oriented towards extrapolation of results beyond experiments in order to predict the growth of interacting microorganisms and develop new food preservation processes. In the present work, a model combining both heterogeneous population and quasi-chemical approaches to describe the different phases of the bacterial growth curve is presented. The model was applied to both monoculture and co-culture cases of lactic acid bacteria, Carnobacterium maltaromaticum H-17, and two Listeria monocytogenes strains in a raw fish extract. It is a highlight that our model includes novel inhibition reactions due to the accumulation of metabolites, and a general equation to take into account the effect of chemical compounds during the lag or physiological adaptation phase of the cells. Our results show that the proposed model can accurately describe the experimental data when the curve shape is a sigmoid, and when it presents a maximum. Besides, the parameters have biological interpretability since the model is mechanistically inspired.

Draft Genome Sequence of Lactobacillus plantarum CRL681, Isolated from Argentinean Artisanal Fermented Sausages.

Lactobacillus plantarum CRL681 was isolated from Argentinean artisanal fermented sausages. Here, the draft genome sequence of the CRL681 strain is described. The reads were assembled into contigs with a total estimated size of 3,370,224 bp. A total of 3,300 open reading frames (ORFs) were predicted, including 3,126 protein-coding sequences. The draft genome sequence of L. plantarum CRL681 will be useful for understanding the organism's metabolic activities and for biotechnological applications.

MLST Reveals a Separate and Novel Clonal Group for Acidovorax avenae Strains Causing Red Stripe in Sugarcane from Argentina.

Acidovorax spp. cause a wide range of economically important diseases in monocotyledonous and dicotyledonous plants, including sugarcane, corn, rice, oat, millet, foxtail watermelon, and orchid. In Argentina, the red stripe disease of sugarcane caused by Acidovorax avenae affects 30% of the milling stems with important economic losses. To explore the genetic diversity of this bacterium associated with red stripe in Argentina, multilocus sequence typing (MLST) was applied. This study included 15 local strains isolated from four different sugarcane planting regions and selected after random amplified polymorphic DNA analysis and reference strains of A. citrulli, A. avenae, and A. oryzae to investigate their phylogenetic relationships. MLST analysis resulted in five sequence types among the sugarcane A. avenae strains which constitute a clonal complex, meaning a common and close origin. Sugarcane strains were related to A. avenae from other hosts and distant to A. citrulli. Signals of frequent recombination in several lineages of A. avenae was detected and we observed that A. oryzae is closely related to A. avenae strains. This study provides valuable data in the field of epidemiological and evolutionary investigations of novel clone of A. avenae strains causing sugarcane red stripe. The knowledge of the genetic diversity and strain-host specificity are important to select the genotypes with the best response to the red stripe disease.

Safety and Growth Optimization of Lactic Acid Bacteria Isolated From Feedlot Cattle for Probiotic Formula Design.

In order to eliminate the widespread use of antibiotics in livestock production, the research for alternatives has increased lately. This study examined the safety of 40 lactic acid bacteria (LAB) isolated from bovine feedlot environment and previously selected as potential probiotics. A high sensitivity prevalence to ampicillin (AMP, 100%), gentamicin (GEN, 96.3%), kanamycin (KAN, 96.3%), clindamycin (CLI, 85.2%), chloramphenicol (CHL, 92.6%) and streptomycin (STR, 88.9%) while moderate and high resistance against erythromycin (ERY, 48%) and tetracycline (TET, 79%) respectively, were determined. Feedlot enterococci and pediococci displayed high resistance to CLI, ERY, GEN and TET (73, 100, 54.5, and 73%, respectively). Among fifteen resistance genes investigated, seven were identified in lactobacilli; their presence not always was correlated with phenotypic resistance. STR resistance genes, aadA and ant(6) were observed in 7.4 and 3.7% of isolates, respectively; genes responsible for aminoglycosides resistance, such as bla (7.4%), and aph(3")-III (3.7%) were also recognized. In addition, resistance cat and tetS genes (3.7 and 7.4%, respectively) were harbored by feedlot lactobacilli strains. The presence of ermB gene in 22.3% of isolates, including two of the six strains phenotypically resistant to ERY, exhibited the highest prevalence among the assessed antibiotics. None of the feedlot lactobacilli harbored virulence factors genes, while positive PCR amplification for ace, agg, fsrA, and atpA genes was found for enterococci. With the objective of producing large cell biomass for probiotic delivery, growth media without peptone but containing glucose and skim milk powder (Mgl and Mlac) were selected as optimal. Lactobacillus acidophilus CRL2074, L. amylovorus CRL2115, L. mucosae CRL2069, and L. rhamnosus CRL2084 were strains selected as free of antibiotic resistance and virulence determinants, able to reach high cell numbers in non-expensive culture media and being compatible among them.

Differential Proteomic Analysis of Lactic Acid Bacteria-Escherichia coli O157:H7 Interaction and Its Contribution to Bioprotection Strategies in Meat.

Human infection by Enterohemorrhagic Escherichia (E.) coli (EHEC) occurs through the ingestion of contaminated foods such as milk, vegetable products, water-based drinks, and particularly minced meats. Indeed EHEC is a pathogen that threatens public health and meat industry. The potential of different Lactic Acid Bacteria (LAB) strains to control EHEC in a meat-based medium was evaluated by using a simple and rapid method and by analyzing the growth kinetics of co-cultures (LAB-EHEC) in a meat-based medium. The activity of LAB toward EHEC in co-cultures showed variable inhibitory effect. Although, LAB were able to control EHEC, neither the produced acid nor bacteriocins were responsible of the inhibition. The bacteriocinogenic Enteroccus (Ent.) mundtii CRL35 presented one of the highest inhibition activities. A proteomic approach was used to evaluate bacterial interaction and antagonistic mechanisms between Ent. mundtii and EHEC. Physiological observations, such as growth kinetics, acidification ability and EHEC inhibitory potential were supported by the proteomic results, demonstrating significant differences in protein expression in LAB: (i) due to the presence of the pathogen and (ii) according to the growth phase analyzed. Most of the identified proteins belonged to carbohydrate/amino acid metabolism, energy production, transcription/translation, and cell division. These results contribute to the knowledge of competition strategies used by Ent. mundtii during its co-culture with EHEC setting new perspectives for the use of LAB to control this pathogen in meat.

Strategies for Pathogen Biocontrol Using Lactic Acid Bacteria and Their Metabolites: A Focus on Meat Ecosystems and Industrial Environments.

The globalization of trade and lifestyle ensure that the factors responsible for the emergence of diseases are more present than ever. Despite biotechnology advancements, meat-based foods are still under scrutiny because of the presence of pathogens, which causes a loss of consumer confidence and consequently a fall in demand. In this context, Lactic Acid Bacteria (LAB) as GRAS organisms offer an alternative for developing pathogen-free foods, particularly avoiding Listeria monocytogenes, with minimal processing and fewer additives while maintaining the foods' sensorial characteristics. The use of LAB strains, enabling us to produce antimicrobial peptides (bacteriocins) in addition to lactic acid, with an impact on quality and safety during fermentation, processing, and/or storage of meat and ready-to-eat (RTE) meat products, constitutes a promising tool. A number of bacteriocin-based strategies including the use of bioprotective cultures, purified and/or semi-purified bacteriocins as well as their inclusion in varied packaging materials under different storage conditions, have been investigated. The application of bacteriocins as part of hurdle technology using non-thermal technologies was explored for the preservation of RTE meat products. Likewise, considering that food contamination with L. monocytogenes is a consequence of the post-processing manipulation of RTE foods, the role of bacteriocinogenic LAB in the control of biofilms formed on industrial surfaces is also discussed.

Genome Sequence of Acidovorax avenae Strain T10_61 Associated with Sugarcane Red Stripe in Argentina.

Red stripe of sugarcane in Argentina is a bacterial disease caused by Acidovorax avenae. The genome sequence from the first isolate of this bacterium in Argentina is presented here. The draft genome of the A. avenae T10_61 strain contains 5,646,552 bp and has a G+C content of 68.6 mol%.

Identification of small peptides arising from hydrolysis of meat proteins in dry fermented sausages.

In this study, proteolysis and low molecular weight (LMW) peptides (<3kDa) from commercial Argentinean fermented sausages were characterized by applying a peptidomic approach. Protein profiles and peptides obtained by Tricine-SDS-PAGE and RP-HPLC-MS, respectively, allowed distinguishing two different types of fermented sausages, although no specific biomarkers relating to commercial brands or quality were recognized. From electrophoresis, α-actin, myoglobin, creatine kinase M-type and L-lactate dehydrogenase were degraded at different intensities. In addition, a partial characterization of fermented sausage peptidome through the identification of 36 peptides, in the range of 1000-2100 Da, arising from sarcoplasmic (28) and myofibrillar (8) proteins was achieved. These peptides had been originated from α-actin, myoglobin, and creatine kinase M-type, but also from the hydrolysis of other proteins not previously reported. Although muscle enzymes exerted a major role on peptidogenesis, microbial contribution cannot be excluded as it was postulated herein. This work represents a first peptidomic approach for fermented sausages, thereby providing a baseline to define key peptides acting as potential biomarkers.

Indigenous Starter Cultures to Improve Quality of Artisanal Dry Fermented Sausages from Chaco (Argentina).

Lactic acid bacteria (LAB) and coagulase negative cocci (CNC) were isolated from artisanal dry sausages sampled from the northeastern region of Chaco, Argentina. In order to evaluate their performance in situ and considering technological features of the isolated strains, two mixed selected autochthonous starter cultures (SAS) were designed: (i) SAS-1 (Lactobacillus sakei 487 + Staphylococcus vitulinus C2) and (ii) SAS-2 (L. sakei 442 + S. xylosus C8). Cultures were introduced into dry sausage manufacturing process at a local small-scale facility. Microbiological and physicochemical parameters were monitored throughout fermentation and ripening periods, while sensory attributes of the final products were evaluated by a trained panel. Lactic acid bacteria revealed their ability to colonize and adapt properly to the meat matrix, inhibiting the growth of spontaneous microflora and enhancing safety and hygienic profile of the products. Both SAS showed a beneficial effect on lipid oxidation and texture of the final products. Staphylococcus vitulinus C2, from SAS-1, promoted a better redness of the final product. Sensory profile revealed that SAS addition preserved typical sensory attributes. Introduction of these cultures could provide an additional tool to standardize manufacturing processes aiming to enhance safety and quality while keeping typical sensory attributes of regional dry fermented sausages.

Proteomic and peptidomic insights on myofibrillar protein hydrolysis in a sausage model during fermentation with autochthonous starter cultures.

The hydrolysis of myofibrillar proteins during fermentation of sausage models by an autochthonous starter culture was investigated. In order to provide a whole map of the generated products, proteomic and peptidomic were used and complemented with the amino acid profile. Beaker sausages (BS) were used as models which were inoculated or not with Lactobacillus curvatus CRL705 and Staphylococcus vitulinus GV318 as starter cultures. The hydrolysis of actin, myosin light chain 1/3 (MLC 1/3), myosin regulatory light chain-2 (MRLC-2) and myosin heavy chain (MHC) was evidenced by two-dimensional gel electrophoresis (2-DE). In addition, a total of 33 peptides arisen from troponin T, MRLC-2 and particularly from actin were identified by LC-MS/MS. These results showed that the starter culture significantly enhanced the proteolysis of the proteins named above, even when the endogenous enzymes induced a clear breakdown. L. curvatus CRL705 highly enriched both peptide pattern and amino acid concentrations. When the autochthonous starter culture was inoculated, although proteolysis was remarkably reinforced, a reduction in peptide and amino acid composition was observed. Regarding actin primary structure, three regions of this protein were highly susceptible to degradation by the starter culture. Additionally, the essential role of exopeptidases - from meat and bacteria - in diversity of actin peptides during fermentation was shown. This study improved the knowledge of the proteolysis of myofibrillar proteins and the involved enzymes, as well as, completed the previously reported degradation of sarcoplasmic proteins by the same autochthonous starter culture. The singular peptides and amino acids pattern generated might contribute to the uniqueness of produced fermented sausages while they may be used as quality markers.

Identification, technological and safety characterization of Lactobacillus sakei and Lactobacillus curvatus isolated from Argentinean anchovies (Engraulis anchoita).

In this study, the identification and characterization of Lactobacillus previously isolated from fresh anchovies (Engraulis anchoita) are investigated. 16S rDNA partial sequencing assigned all the isolates to belong to the Lactobacillus sakei/curvatus group. Fourteen out of 15 isolates were identified as L. sakei by phenotypic traits: they exhibited catalase activity and fermented melibiose, although only 10 of them hydrolyzed arginine. These results were confirmed by multiplex PCR-based restriction enzyme analysis with HindIII and by restriction fragment length polymorphic (RFLP) analysis of the 16S-23S rDNA intergenic spacer region with TaqI. Among identified isolates, four L. sakei strains and the sole L. curvatus strain showing sensitivity to chloramphenicol, erythromycin and tetracycline and exhibiting high tolerance to NaCl (10-18%) were unable to produce neither dextran nor biogenic amines. Based on technological and safety features, L. sakei SACB704 and L. curvatus SACB03a naturally present in fresh anchovies may be promising strains for the development of a starter culture to accelerate and control the fermentation of salt fermented anchovy-based products.

Characterization of a multilayer film activated with Lactobacillus curvatus CRL705 bacteriocins.

BACKGROUND: Bacteriocins produced by lactic acid bacteria offer enormous promise for food safety preservation. In this study an active multilayer film obtained by the incorporation of lactocin 705 and lactocin AL705, two bacteriocins produced by Lactobacillus curvatus CRL705 with antimicrobial activity against Lactobacillus plantarum CRL691 and Listeria innocua 7, respectively, was characterized for its potential application in active packaging technology. Film activity performance at different storage conditions, bacteriocins transfer into water and sunflower oil, and film surface properties were evaluated. RESULTS: Film activity against L. innocua 7 was maintained during 2, 4 and 6 weeks at 30, 10 and 5 °C respectively. At 30 and 10 °C, activity loss against L. plantarum CRL691 was observed on the second week of storage and after the fourth week at 5 °C. Results showed no significant difference for active multilayer film contact angle and seal properties compared to the control (without bacteriocins). A decrease in lactocin 705 inhibitory activity after sunflower oil contact was observed, while lactocin AL705 remained unaffected. After water contact, film activity was retained for both bacteriocins. CONCLUSIONS: As demonstrated by antimicrobial activity and physico-mechanical properties retention, lactocin 705 and AL705 active multilayer film present potential for application in active packaging technology.

Differential roles of the two-component peptides of lactocin 705 in antimicrobial activity.

Lactobacillus casei CRL705 produces a class IIb bacteriocin, lactocin 705, which relies on the complementary action of two components, Lac705alpha and Lac705beta. These peptides exert a bactericidal effect on the indicator strain Lactobacillus plantarum CRL691, with an optimal Lac705alpha/Lac705beta peptide ratio of 1 to 4. Electron microscopy studies showed that treated CRL691 cells have their cell wall severely damaged, with mesosome-like membranous formations protruding into their cytoplasm. Although less pronounced, a similar effect was also observed with the Lac705beta peptide alone. Furthermore, Lac705beta increased the inhibitory action of a diluted supernatant of L. casei CRL705, while Lac705alpha protected CRL691 cells from inhibition. Both peptides were required to dissipate the proton motive force (Deltapsi and DeltapH) of CRL691 cells. These data suggested that of the two components of lactocin 705, the Lac705alpha peptide is responsible for receptor recognition, and the Lac705beta peptide is the active component on the cell membrane of CRL691 cells.

Use of Bacterial Cultures in the Ripening of Fermented Sausages.

Pure bacterial cultures were used in the production of Argentina salami, the most popular cured meat product in the country. Three selected groups of two strains each of Lactobacillus plantarum and Micrococcus varians were used as a starter cultures, with two different sugar concentrations. Apart from the use of these starter cultures, the salami was manufactured under the same conditions normally used in industrial production. When 0.6% sucrose was used, the levels of L. plantarum and M. varians were 108 and 107 CFU/g, respectively, on the second day of ripening. Similar levels of lactic acid bacteria were found in non-inoculated sausages at 2 d post-preparation. Under these conditions, coliforms decreased significantly. The final pH of the inoculated and uninoculated sausage were 5.0 and 5.2, respectively, after 7 d of ripening. When 0.9% glucose plus 0.6% sucrose was added, the level of lactic acid bacteria was 109 CFU/g on the second day, a value that remained constant in the inoculated sausages to the end of the ripening period. Staphylococci showed a marked decrease in population, while coliforms disappeared on the second or third day. The final pH of 4.40 or 4.55 was reached within 4 d. The product obtained under these conditions had a firm texture, a good slicing surface and pleasant flavor and aroma. The use of starter cultures in cured dry Argentine-style sausage shortened the ripening period from 14 to 7 d.

Acid Production and Proteolytic Activity of Lactobacillus Strains Isolated from Dry Sausages.

The acid-producing capacity and proteolytic activity of 13 strains of Lactobacillus plantarum and 5 strains of Lactobacillus casei isolated from dry sausages was determined at different temperatures and at different NaCl concentrations. Most strains exhibited a maximum acid-producing rate at 30°C. According to the acidification rate at this temperature, strains were divided into three rate groups: fast (I), medium (II) and slow (III), with titratable acidity values above 1.7, between 0.7 and 1.4, and below 0.7, respectively. The decrease in pH ranged between 3.1 and 3.95 according to the group to which the strains belonged. The addition of 3% NaCl produced a marked decrease in the rate of acidification for strains in group II, a slight decrease for those in group I and no effect for those in group III. The proteolytic activity of the strains under study reached a maximum at 40°C, with values between 5.2 and 10 mg% tyrosine released. At 30°C, and in the presence of 3% NaCl, the greatest activity (5.4 mg% tyrosine) was observed in L. plantarum GV 417 and the lowest (3.4 mg% tyrosine) in L. plantarum GV 420. A decrease of approximately 80% in proteolytic activity for all strains was observed in the presence of 5% NaCl.