Functionality of the bacteriocin sakacin G produced by Lactobacillus curvatus ACU-1 on cooked sausages under industrial conditions.

Biopreservation is an alternative to prevent the growth of pathogens and reduce microbial spoilage in food based on the use of microorganisms and/or their metabolic products. The objective of this study was to determine the optimal mode of application and the effectiveness of cell-free supernatant (CFS) from Lactobacillus curvatus ACU-1, containing sakacin G, in Vienna-type sausages to control Listeria and spoilage flora. The functionality and the optimal dosage form between CFS, producing bacteria, a combination or concentrate of bacteriocin applied on Vienna-type sausages before and after stuffing the casings on an industrial scale were determined. Sakacin G was effective for the control of Listeria applied to the casing both before and after stuffing. The application of the antimicrobial on the ready sausages inhibits both lactic acid bacteria and mesophilic microorganisms from zero sampling time. The heat resistance of the bacteriocin in the food was verified under industrial manufacturing conditions. The antimicrobial activity of sakacin G was maintained throughout the period studied in all the conditions tested. In conclusion, the application of CFS containing bacteriocin is useful given both before and after casing stuffing; but the application prior to the stuffing is more practical for the process of elaboration.

Characterization, Toxicity, and Optimization for the Growth and Production of Bacteriocin-like Substances by Lactobacillus curvatus.

This study aimed to characterize, evaluate toxicity and optimize the conditions for the growth and production of bacteriocin-like substances by Lactobacillus curvatus P99. The antibacterial activity of the cell-free supernatant (CFS) containing bacteriocin-like substances was evaluated by the agar diffusion technique. The stability of the CFS was also examined after heat treatment, refrigeration, freezing, pH variations, and treatment with different chemical substances. The toxic effect of CFS on Drosophila melanogaster diet was determined by calculating the survival rate of the flies. The effects of pH, temperature, and incubation time were the parameters used to optimize cell growth and production of bacteriocin-like substances through response surface methodology. The CFS was stable for 36 weeks under freezing and refrigeration, as well as under heat treatment, in acidic and basic pH and for all chemical substances tested. Fewer than 50,000 AU/mL of CFS added to D. melanogaster diet showed no toxic effect. The optimum growth condition was pH 6.3 at 29.3 °C for 18.6 h, and the optimum production of bacteriocin-like substances was under pH 6.22 at 30.6 °C for 17.9 h. The data on the optimization of cell growth and the characterization and optimization of bacteriocin-like substances provided information to support the industrial-scale production of this microorganism and its metabolites.

Fate of Spoilage and Pathogenic Microorganisms in Acidified Cold-Filled Hot Pepper Sauces.

Consumption of spicy foods and hot sauces is currently a popular trend worldwide. Shelf-stable acidified sauces are commonly hot-filled to ensure commercial sterility, but cold-fill-hold processes might also be suitable if microbial safety and stability are ensured. For this study, model acidified hot pepper sauces were developed and characterized. The effects of sauce pH and of two different organic acids on the survival of Pichia manshurica and Lactobacillus curvatus isolated from contaminated commercial hot sauces and on pathogenic Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes were assessed. Full factorial designs with three levels for pH (3.2, 3.5, and 3.9) and two for organic acid (citric and acetic) were used to determine the effects of these factors and their interactions on the survival of the microorganisms. Commercially sterile sauces were independently inoculated and kept at ambient temperature. Microbial counts were determined at different sampling times, depending on the treatment evaluated. Sauces acidified to pH 3.2 with citric or acetic acid were inoculated with cocktails of five strains or serotypes of the three pertinent pathogens, and inactivation curves were determined. Trials were performed in triplicate. A greater than 5-log reduction of P. manshurica and L. curvatus was achieved in less than 6 h in sauces adjusted to pH 3.2 with acetic acid. Greater than 5-log reductions of pathogenic bacteria were achieved 0.5 h after inoculation in sauces acidified to pH 3.2 with acetic acid. In contrast, at least 48 h was required to guarantee the same inactivation for the most tolerant pathogen when citric acid was used. Thus, a cold-fill-hold process may be a suitable alternative for acidified hot pepper sauces. Based on survival of the microorganisms evaluated in this study, microbial safety and stability can be achieved by adjusting the pH to 3.2 or less by the addition of acetic acid.

Lactobacillus curvatus UFV-NPAC1 and other lactic acid bacteria isolated from calabresa, a fermented meat product, present high bacteriocinogenic activity against Listeria monocytogenes.

BACKGROUND: Bacteriocins produced by lactic acid bacteria (LAB) can be considered as viable alternatives for food safety and quality, once these peptides present antimicrobial activity against foodborne pathogens and spoilage bacteria. Fermented foods, such as artisanal sausages and cured meats, are relevant sources of LAB strains capable of producing novel bacteriocins, with particular interest by the food industry. RESULTS: Three LAB strains (firstly named as Lactobacillus curvatus 12, L. curvatus 36 and Weissella viridescens 23) were obtained from calabresa by presenting promising bacteriocinogenic activity, distinct genetic profiles (rep-PCR, RAPD, bacteriocin-related genes) and wide inhibitory spectrum. Among these strains, L. curvatus 12 presented higher bacteriocin production, reaching 25,000 AU/mL after incubation at 25, 30 and 37 °C and 6, 9 and 12 h. Partially purified bacteriocins from L. curvatus 12 kept their inhibitory activity after elution with isopropanol at 60% (v/v). Bacteriocins produced by this strain were purified by HPLC and sequenced, resulting in four peptides with 3102.79, 2631.40, 1967.06 and 2588.31 Da, without homology to known bacteriocins. CONCLUSIONS: LAB isolates obtained from calabresa presented high inhibitory activity. Among these isolates, bacteriocins produced by L. curvatus 12, now named as L. curvatus UFV-NPAC1, presented the highest inhibitory performance and the purification procedures revealed four peptides with sequences not described for bacteriocins to date.

Isolation of lactic acid bacteria from swine milk and characterization of potential probiotic strains with antagonistic effects against swine-associated gastrointestinal pathogens.

Probiotics are usually isolated from the gastrointestinal tract of humans and animals. The search of probiotics in human milk is a recent field of research, as the existence of the human milk microbiome was discovered only about a decade ago. To our knowledge, no reports regarding the potential probiotic effect of bacteria from swine milk have been published. In this work, we isolated several lactic acid bacteria from swine milk and evaluated them for them potential as probiotics. Among the isolated strains, Lactobacillus curvatus TUCO-5E showed antagonistic effects against swine-associated gastrointestinal pathogens. TUCO-5E was able to reduce the growth of enterotoxigenic and enterohemorrhagic Escherichia coli strains as well as pathogenic salmonella. In vitro exclusion and displacement assays in intestinal epithelial cells showed a remarkable antagonistic effect for L. curvatus TUCO-5E against Salmonella sp. strain TUCO-I7 and Salmonella enterica ATCC 13096. Moreover, by using a mouse model of Salmonella infection, we were able to demonstrate that preventative administration of L. curvatus TUCO-5E for 5 consecutive days was capable of decreasing the number of Salmonella enterica serovar Typhimurium in the liver and spleen of treated mice, compared with the controls, and prevented dissemination of the pathogen to the blood stream. Therefore, we have demonstrated here that swine milk is an interesting source of beneficial bacteria. In addition, the results of this work suggest that L. curvatus TUCO-5E is a good candidate to study in vivo the protective effect of probiotics against intestinal infection and damage induced by Salmonella infection in the porcine host.

Characterization of the spoilage lactic acid bacteria in "sliced vacuum-packed cooked ham".

The lactic acid bacteria are involved with food fermentation and in such cases with food spoilage. Considering the need to reduce the lactic acid bacteria growth in meat products, the aim of this work was to enumerated and investigated the lactic acid bacteria present on sliced vacuum-packed cooked ham stored at 4 °C and 8 °C for 45 days by phenotypic and molecular techniques. The quantification showed that the lactic acid bacteria were present from the first day with mean count of 1.98 log cfu/g for the four batches analyzed. The lactic acid bacteria grew rapidly on the samples, and plate counts around 7.59 log cfu/g and 8.25 log cfu/g were detected after 45 days of storage at 4 °C and 8 °C, respectively; storage temperatures studied showed significant influence on the microorganism in study growth. The predominant lactic acid bacteria associated with the spoilage samples at one day of storage includes Lactobacillus sp., the phenotypic overlap Leuconostoc / Weissella sp. and Enterococcus sp. At 45 days of storage at 4 and 8 °C the mainly specie was Lactobacillus curvatus , following by Lactobacillus sakei and Leuconostoc mesentereoides ; the Enterococcus sp. was not present in the samples.

Characterization of the spoilage lactic acid bacteria in “sliced vacuum-packed cooked ham”

The lactic acid bacteria are involved with food fermentation and in such cases with food spoilage. Considering the need to reduce the lactic acid bacteria growth in meat products, the aim of this work was to enumerated and investigated the lactic acid bacteria present on sliced vacuum-packed cooked ham stored at 4 °C and 8 °C for 45 days by phenotypic and molecular techniques. The quantification showed that the lactic acid bacteria were present from the first day with mean count of 1.98 log cfu/g for the four batches analyzed. The lactic acid bacteria grew rapidly on the samples, and plate counts around 7.59 log cfu/g and 8.25 log cfu/g were detected after 45 days of storage at 4 °C and 8 °C, respectively; storage temperatures studied showed significant influence on the microorganism in study growth. The predominant lactic acid bacteria associated with the spoilage samples at one day of storage includes Lactobacillus sp., the phenotypic overlap Leuconostoc/Weissella sp. and Enterococcus sp. At 45 days of storage at 4 and 8 °C the mainly specie was Lactobacillus curvatus, following by Lactobacillus sakei and Leuconostoc mesentereoides; the Enterococcus sp. was not present in the samples.

Characterization of the spoilage lactic acid bacteria in “sliced vacuum-packed cooked ham”

The lactic acid bacteria are involved with food fermentation and in such cases with food spoilage. Considering the need to reduce the lactic acid bacteria growth in meat products, the aim of this work was to enumerated and investigated the lactic acid bacteria present on sliced vacuum-packed cooked ham stored at 4 °C and 8 °C for 45 days by phenotypic and molecular techniques. The quantification showed that the lactic acid bacteria were present from the first day with mean count of 1.98 log cfu/g for the four batches analyzed. The lactic acid bacteria grew rapidly on the samples, and plate counts around 7.59 log cfu/g and 8.25 log cfu/g were detected after 45 days of storage at 4 °C and 8 °C, respectively; storage temperatures studied showed significant influence on the microorganism in study growth. The predominant lactic acid bacteria associated with the spoilage samples at one day of storage includes Lactobacillus sp., the phenotypic overlap Leuconostoc/Weissella sp. and Enterococcus sp. At 45 days of storage at 4 and 8 °C the mainly specie was Lactobacillus curvatus, following by Lactobacillus sakei and Leuconostoc mesentereoides; the Enterococcus sp. was not present in the samples.(AU)

Improving safety of salami by application of bacteriocins produced by an autochthonous Lactobacillus curvatus isolate.

The aims of this study were to isolate LAB with anti-Listeria activity from salami samples, characterize the bacteriocin/s produced by selected isolates, semi-purify them and evaluate their effectiveness for the control of Listeria monocytogenes during manufacturing of salami in a pilot scale. Two isolates (differentiated by RAPD-PCR) presented activity against 22 out of 23 L. monocytogenes strains for bacteriocin MBSa2, while the bacteriocin MBSa3 inhibited all 23 strains in addition to several other Gram-positive bacteria for both antimicrobials and were identified as Lactobacillus curvatus based on 16S rRNA sequencing. A three-step purification procedure indicated that both strains produced the same two active peptides (4457.9 Da and 4360.1 Da), homlogous to sakacins P and X, respectively. Addition of the semi-purified bacteriocins produced by Lb. curvatus MBSa2 to the batter for production of salami, experimentally contaminated with L. monocytogenes (10(4)-10(5) CFU/g), caused 2 log and 1.5 log reductions in the counts of the pathogen in the product after 10 and 20 days respectively, highlighting the interest for application of these bacteriocins to improve safety of salami during its manufacture.

Ação de bacteriocinas de bactérias láticas no controle de Listeria monocytogenes e no aumento da vida de prateleira de mortadela fatiada / Use of bacteriocins from lactic acid bacteria in the control of Listeria monocytogenes and in the shelf life extension of sliced mortadella

As bacteriocinas são peptídeos antimicrobianos produzidos por algumas bactérias, que atuam como bio-conservantes e têm grande potencial de aplicação em produtos cárneos. Estudos mostram que as bacteriocinas de bactérias lácticas (BAL) a serem utilizadas em um determinado alimento são mais eficientes quando produzidas por bactérias lácticas isoladas do mesmo tipo de alimento. Este estudo objetivou obter novas BAL produtoras de bacteriocinas a partir de mortadela fatiada, caracterizar as bacteriocinas produzidas, obter a bacteriocina semi-purificada e verificar seu potencial de aplicação in vitro e in situ no controle de Listeria monocytogenes e na vida de prateleira (VDP) de mortadela fatiada. Foram obtidos 19 isolados de BAL comprovadamente produtoras de bacteriocinas, que foram avaliadas quanto ao espectro de ação frente a uma grande variedade de bactérias Gram positivas e Gram negativas, e estabilidade em diferentes pH. As que apresentaram melhor estabilidade e inibiram maior número de bactérias foram avaliadas quanto a estabilidade térmica e resistência a diferentes agentes químicos e concentrações de NaCl. As que apresentaram maior resistência foram submetidas a testes de otimização da produção da bacteriocina em diferentes combinações de tempo/temperatura. Com base nestes resultados, selecionou-se um isolado que identificado como Lactobacillus curvatus pelo sequenciamento do gene 16S e confirmado por reações de PCR com primers específicos. A bacteriocina produzida por este isolado foi submetida a uma purificação por extração ácida e aplicada em fatias de mortadela experimentalmente contaminadas com um pool de cepas de Listeria monocytogenes, observando-se as contagens deste patógeno durante o armazenamento em refrigeração (7°C) por 40 dias em embalagem à vácuo. Paralelamente, foi realizada contagem de bactérias láticas para avaliar o efeito da bacteriocina nesta população, responsável pela VDP deste produto. A bacteriocina parcialmente purificada foi ...

Bacteriocins are peptides with antimicrobial properties produced by some bacteria, that act as bio-preservatives with great potential of application in meat products. Several studies show that bacteriocins from lactic acid bacteria (LAB) to be used in a specific food product have a better performance when produced by LAB isolated from the same kind of product. This study aimed to obtain new bacteriocinogenic LAB from sliced Bologna, to characterize the bacteriocins produced, to obtain a semi-purified bacteriocin, and to verify its potential of application in vitro and in situ in the control of Listeria monocytogenes and in the shelf-life of sliced bologna. 19 bacteriocin producer LAB, were evaluated as to spectrum of action against a great variety of Gram positives and Gram negatives bacteria and pH stability. The ones with better stability and that inhibited a larger number of bacteria were evaluated according to its thermal stability and resistance against different chemical agents and NaCl concentrations. The ones that showed better resistance were submitted to tests to optimize the bacteriocin production in different combinations of time and temperature. Based on these results, one was selected, identified as Lactobacillus curvatus by gene 16S sequencing and confirmed by PCR reactions with specific primers. The bacteriocin produced by this strain was submitted to partial purification by acid extraction and was applied in mortadella slices experimentally contaminated with a pool of L. monocytogenes strains, observing the counts of this pathogen along refrigerated (7ºC) storage fo r 40 days in vacuum package. Parallel, it was done LAB counts to evaluate the effect of the bacteriocin on this population, responsible for limiting the shelf life of this product. The semi-purified bacteriocin was able to reduce 1 log cicle of the initial population of L. monocytogenes in the sliced mortadella, which was maintained during the 40 days of analysis. Along the whole...

Utilization of supplemented sugarcane molasses for continuous production of lactic acid by Lactobacillus curvatus / Produção contínua de ácido láctico por Lactobacillus curvatus a partir de melaço de cana-de-açúcar suplementado

Several by-products and raw-materials of food and agricultural industries have been used as culture medium. It is rich nutrients sources, and it is considered low cost and high availability of molasses as a substrate can be used in lactic fermentation. Lactic acid has tradicionally been obtained by chemical process, however, lactic acid production by fermentative process is considered most viable economically. Lactic acid has application in different industries such as pharmaceutical, cosmetic, textiles, leather, chemical and food industries, where about 82% of all world production is used. In this work, lactic acid production by L. curvatus under continuous culture on sugarcane molasses supplemented with yeast extract and peptone was evaluated. Dilution rates tested were 0,05; 0,10 and 0,15h-1 at 37ºC. Samples were taken for pH, reducing sugar, lactic acid, biomass and cellular viability measurements under steady state conditions. When the D was 0,05; 0,10 and 0,15h-1, the reducing sugar consumption was 7,1; 6,4 and 4,8g/L, respectively. Highest lactic acid production (13,8g/L) was obtained when D=0,05h-1 was used, while at D=0,10 and 0,15h-1, lactic acid production reached 10,2 and 7,1g/L, respectively. The highest values of biomass (1,98g/L) and cell viability (2,03x109CFU/mL) were reached at the lowest dilution rate tested (0,05h-1). In conclusion, the results showed that u

Melaço é uma matéria prima de baixo custo e rico em nutrientes para a fermentação láctica. O ácido láctico tem aplicação na indústria farmacêutica, cosmética, têxtil, de couro, química e de alimentos, onde são utilizados cerca de 82% de sua produção mundial. Neste trabalho foi avaliada a produção de ácido láctico por Lactobacillus curvatus em fermentação contínua de melaço de cana-de-açúcar, previamente tratado com invertase e suplementado com extrato de levedura e peptona. Os valores de taxa de diluição D= 0,05; 0,10 e 0,15h-1 foram testados à temperatura de 37ºC. As amostras foram coletadas na fase estacionária de crescimento, para medidas de pH, determinação de açúcares redutores, biomassa, produção de ácido láctico e viabilidade celular. Nestas condições, o consumo de açucares redutores diminuiu de 7,1; 6,4 e 4,8g/L com o aumento respectivo da taxa de diluição. A maior produção de ácido láctico (13,8g/L) foi obtida na taxa de diluição de 0,05h-1, enquanto que D=0,10 e 0,15h-1 suportaram a produção de 10,2 e 7,1g/L, respectivamente. Os valores mais altos de biomassa (1,98g/L) e viabilidade celular (2,03x109UFC/mL) também foram alcançados na menor taxa de diluição (D=0,05h-1). A produção de ácido láctico, por L. curvatus em melaço, previamente tratado com invertase e suplementado, foi mais adequada na menor taxa de diluição.

Utilization of supplemented sugarcane molasses for continuous production of lactic acid by Lactobacillus curvatus / Produção contínua de ácido láctico por Lactobacillus curvatus a partir de melaço de cana-de-açúcar suplementado

Several by-products and raw-materials of food and agricultural industries have been used as culture medium. It is rich nutrients sources, and it is considered low cost and high availability of molasses as a substrate can be used in lactic fermentation. Lactic acid has tradicionally been obtained by chemical process, however, lactic acid production by fermentative process is considered most viable economically. Lactic acid has application in different industries such as pharmaceutical, cosmetic, textiles, leather, chemical and food industries, where about 82% of all world production is used. In this work, lactic acid production by L. curvatus under continuous culture on sugarcane molasses supplemented with yeast extract and peptone was evaluated. Dilution rates tested were 0,05; 0,10 and 0,15h-1 at 37ºC. Samples were taken for pH, reducing sugar, lactic acid, biomass and cellular viability measurements under steady state conditions. When the D was 0,05; 0,10 and 0,15h-1, the reducing sugar consumption was 7,1; 6,4 and 4,8g/L, respectively. Highest lactic acid production (13,8g/L) was obtained when D=0,05h-1 was used, while at D=0,10 and 0,15h-1, lactic acid production reached 10,2 and 7,1g/L, respectively. The highest values of biomass (1,98g/L) and cell viability (2,03x109CFU/mL) were reached at the lowest dilution rate tested (0,05h-1). In conclusion, the results showed that u

Melaço é uma matéria prima de baixo custo e rico em nutrientes para a fermentação láctica. O ácido láctico tem aplicação na indústria farmacêutica, cosmética, têxtil, de couro, química e de alimentos, onde são utilizados cerca de 82% de sua produção mundial. Neste trabalho foi avaliada a produção de ácido láctico por Lactobacillus curvatus em fermentação contínua de melaço de cana-de-açúcar, previamente tratado com invertase e suplementado com extrato de levedura e peptona. Os valores de taxa de diluição D= 0,05; 0,10 e 0,15h-1 foram testados à temperatura de 37ºC. As amostras foram coletadas na fase estacionária de crescimento, para medidas de pH, determinação de açúcares redutores, biomassa, produção de ácido láctico e viabilidade celular. Nestas condições, o consumo de açucares redutores diminuiu de 7,1; 6,4 e 4,8g/L com o aumento respectivo da taxa de diluição. A maior produção de ácido láctico (13,8g/L) foi obtida na taxa de diluição de 0,05h-1, enquanto que D=0,10 e 0,15h-1 suportaram a produção de 10,2 e 7,1g/L, respectivamente. Os valores mais altos de biomassa (1,98g/L) e viabilidade celular (2,03x109UFC/mL) também foram alcançados na menor taxa de diluição (D=0,05h-1). A produção de ácido láctico, por L. curvatus em melaço, previamente tratado com invertase e suplementado, foi mais adequada na menor taxa de diluição.

Lactic acid production by L. curvatus in sugarcane molasses / Produção de ácido lático por Lactobacillus curvatus em melaço de cana-de-açúcar

Lactic acid is important due to its various applications. The bulk of world lactic acid production is used by the food industry and the rest is used in pharmaceutical, textile, leather, cosmetic and chemical industries. In this work, a 33 incomplete factorial design of the response-surface methodology was used to determine the best concentration of sugarcane molasses, yeast extract and peptone in the culture medium for the development of batch lactic fermentation by Lactobacillus curvatus. The fermentation was carried out at 37 ºC for 48 hours without agitation. The mathematical model given by the responsesurface methodology indicated a concentration of 10% (w/v) of sugarcane molasses, 2% (w/v) of yeast extract and 4% (w/v) of peptone as the best conditions for the composition of culture medium for the lactic acid production by L. curvatus. Under these conditions, lactic acid production was 30,5 g/L, comparable with the result obtained in MRS medium, which produced 32,0g/L of lactic acid. Considering the low cost and high availability of the sugarcane molasses, it was concluded that it represented a good culture medium for lactic fermentation. Sugarcane molasses at 10% (w/v) supplemented with yeast extract at 2% (w/v) and peptone at 4% (w/v) was used in the 3L batch lactic fermentation producing 37,5g/L of lactic acid.

A maior parte da produção mundial de ácido lático é utilizada pela indústria de alimentos e o restante em indústrias farmacêutica, têxtil, de couro, cosmética e química. A Metodologia da Superfície de Resposta, planejamento fatorial incompleto 33, foi utilizada para estabelecer as melhores condições, relativas às concentrações, do meio de cultivo contendo melaço de cana-de-açúcar, extrato de levedura e peptona para desenvolvimento da fermentação descontínua por Lactobacillus curvatus. A fermentação se desenvolveu durante 48 horas sob temperatura de 37 ºC. O modelo matemático fornecido pela Metodologia da Superfície de Resposta apontou os valores máximos de concentração testados para melaço de cana-de-açúcar, 10% (m/v); extrato de levedura, 2% (m/v) e peptona, 4% (m/v) como a melhor composição do meio para produção de ácido lático. Na prática, estas condições forneceram uma produção de 30,5g/L de ácido láctico, sendo este resultado comparável ao obtido em meio sintético de Man-Rogosa-Sharpe, o qual produziu 32,0g/L de ácido lático. Considerando-se o baixo custo e alta disponibilidade do melaço, pode-se afirmar que este representa um bom meio de cultivo para a fermentação lática. Fermentação descontínua em escala maior, fermentador de 3L, utilizando-se melaço de cana-de-açúcar 10% (m/v) suplementado com 2% (m/v) de extrato de levedura e 4% (m/v) de peptona produziu 37,5 g/L de ác