Production and characterization of a novel aminopeptidase A from Lactococcus lactis / 生物工程学报

Aminopeptidase A (Pep A) is a metal-dependent enzyme that specifically hydrolyze peptides with the N-terminal amino acids glutamic acid (Glu) and aspartic acid (Asp). A possible application of PepA is the hydrolysis of Glu/Asp-rich food proteins such as wheat gluten and casein, increasing the flavor and solubility of food protein. In the present study, the gene encoding a Pep A from Lactococcus lactis ssp. lactis IL1403 was synthesized and introduced into Pichia pastoris GS115 (His4). Lc-Pep A was successfully expressed and secreted to the culture medium, followed by identification and purification to homogeneity. Characteristics study demonstrated that Lc-Pep A could specifically hydrolyze the substrates Glu-pNA and Asp-pNA with similar catalytic activity, and this was further confirmed by the kinetics parameters measured. Additionally, Lc-Pep A showed a broad thermostability and pH stability with an optimum temperature of 60 ℃ and an optimum pH of 8.0. The enzyme activity of Lc-Pep A was activated by metal ions Co2+, Mn2+, and Zn2+ but was strongly inhibited by Ni2+and Cu2+. The routine proteinase inhibitor had no effect on the activity of Lc-Pep A. However, Lc-Pep A was strongly inhibited by the metallopeptidase inhibitor, EDTA, and disulfide bond-reducing agents. The study may facilitate production and application of Lc-Pep A.

Adherence and internalisation of Lactococcus lactis M4 towards human colorectal cancer cell line, Caco-2

Aims@#Lactococcus lactis is a non-colonizing, generally-regarded as safe (GRAS) lactic acid bacteria that has been frequently studied as a potential vector for bactofection. To mediate bactofection, a series of interaction between the bacteria and the host cell needs to occur. This study aims to investigate the in vitro bacterial-cell interaction between a locally-isolated L. lactis M4 strain with human colorectal cancer line, Caco-2.@*Methodology and results@#Bacterial interaction was evaluated via adherence and internalisation assays. A 250:1 ratio of bacteria to cancer cell was selected as the optimum multiplicity of infection for all assays. After 2 h, L. lactis M4 was able to adhere to and internalise into Caco-2 cells at comparable rates to commercial strains L. lactis NZ9000 and MG1363. @*Conclusion, significance and impact of study@#Findings from this study showed that this strain has similar interaction properties with the commercial strains and would make a promising candidate for future bactofection studies and development of bacteria-mediated DNA vaccination against various diseases.

Ação in vivo de Lactococcus lactis subsp. lactis com potencial probiótico na estabilização de células cancerígenas no epitélio colorretal / In vivo action of Lactococcus lactis subsp. lactis with probiotic potential in the stabilization of cancer cells in the colorectal epithelium

Na última década, a ciência contribuiu significativamente para inúmeros avanços em relação ao tratamento e prevenção do câncer colorretal (CCR), porém, a prevalência global e a taxa de mortalidade permanecem elevadas. Há relatos sobre efeitos benéficos de espécies de Bifidobacterium e Lactobacillus com potencial probiótico na prevenção de CCR. No entanto, a bactéria probiótica Lactococcus lactis subps. lactis é comumente utilizada para fins industriais, não havendo comprovações in vivo sobre seu potencial anticarcinogênico. Visto o interesse emergente dos efeitos benéficos dos probióticos a fim de prevenir ou tratar o CCR, o presente estudo objetivou explorar os efeitos de L. lactis subsp. lactis sobre o CCR. Ratos Wistar receberam doses subcutâneas de 1,2 dimetilhidrazina (DMH) e suspensão de L. lactis subsp. lactis por via oral. Após 20 semanas, os tecidos intestinais foram analisados e de acordo com o resultado, o isolado demonstrou potencial anticarcinogênico contra CCR.

Effects of heme oxygenase-1 recombinant Lactococcus lactis on the intestinal barrier of hemorrhagic shock rats

This study aimed to investigate the effects of heme oxygenase-1 recombinant Lactococcus lactis (LL-HO-1) on the intestinal barrier of rats with hemorrhagic shock. One hundred Sprague-Dawley male rats (280–320 g) were randomly divided into healthy control group (N group) and hemorrhagic shock group (H group). Each group was subdivided into HO1t, HO2t, HO3t, PBS and LL groups in which rats were intragastrically injected with LL-HO-1 once, twice and three times, PBS and L. lactis (LL), respectively. The mortality, intestinal myeloperoxidase (MPO) activity, intestinal contents of TNF-α, IL-10 and HO-1, and intestinal Chiu's score were determined. Results showed that in N group, the HO-1 content increased after LL-HO-1 treatment, and significant difference was observed in HO1t group and HO2t group (P<0.05). In H groups, MPO activity and Chiu's score decreased, but IL-10 content increased in LL-HO-1-treated groups when compared with PBS and LL groups (P<0.05). When compared with N group, the MPO activity reduced dramatically in LL-HO-1-treated groups. Thus, in healthy rats (N group), intragastrical LL-HO-1 treatment may increase the intestinal HO-1 expression, but has no influence on the intestinal barrier. In hemorrhagic shock rats, LL-HO-1 may significantly protect the intestinal barrier, and repeating the intragastrical LL-HO-1 treatments twice has the most obvious protection.

Produção biotecnológica de biossurfactante por Lactococcus lactis CECT-4434 a partir de resíduos agroindustriais e avaliação de suas propriedades / Biotechnological production of biosurfactant by Lactococcus lactis CECT- 4434 from agroindustrial residues and evaluation of its properties

A bactéria Lactococcus lactis subsp. lactis CECT-4434 foi empregada para investigar o efeito da composição do meio de cultivo na produção biotecnológica de biossurfactante e, adicionalmente bacteriocina. Utilizou-se resíduos agroindustriais, tais como soro de leite e vinhaça de uva, para formular meios de cultivos mais econômicos e naturais, suplementados sacarose e extrato de levedura. Um planejamento fatorial fracionado 24, com adição de três ensaios nos pontos centrais foi empregado para avaliar a influência destas variáveis. A produção de biossurfactante foi influenciada positivamente pela concentração soro de leite, onde 15 % deste demonstrou melhor resultado reduzindo a tensão superficial em cerca de 18,1 mN/m, alcançando produção máxima de biossurfactante equivalente em surfactina de 11,02 mg/L. Em relação à síntese de bacteriocina, a fonte de carbono adicional (sacarose) interferiu de forma antagonista, ou seja, quanto menor a concentração de sacarose, maior a síntese de bacteriocina (com aumento da zona de inibição em 14,2% contra Staphylococcus aureus CECT-239). Observou-se que o ensaio conduzido em biorreator, sob microaeração com 5% de oxigênio dissolvido, promoveu maior produção de biossurfactante (11,6 mg/L) quando comparados aos estudos conduzidos com maior concentração de oxigênio entre 30 a 100%, com produção em média de 2,3 mg/mL. Destaca-se que nenhum estudo da influência do oxigênio dissolvido, principalmente em microaerofilia, para a produção de biossurfactante por bactérias láticas já havia sido realizado. Ademais, o biossurfactante produzido se mostrou altamente estável frente a valores extremos de pH e temperatura, além de demonstrar notável propriedade antimicrobiana e antiadesiva, inibindo Listeria monocytogenes NADC 2045 e Salmonella entérica CECT-724 em mais de 90%.

Lactococcus lactis subsp. lactis CECT-4434 was used to investigate the effect of the composition of the culture media on the biotechnological production of biosurfactant and bacteriocin additionally. Agroindustrial residues, such as whey and grape vinasse, were used to formulate more economical and natural culture media, supplemented with sucrose and yeast extract. A fractional factorial design 24, with addition of three runs at the central points was used to evaluate the influence of these variables. The biosurfactant production was positively influenced by the concentration of whey, where 15% showed a better result reducing the surface tension by 18.1 mN/m, reaching a maximum production of biosurfactant equivalent in surfactin of 11.02 mg/L. In relation to bacteriocin synthesis, the sucrose interfered in an antagonistic way, that is, the lower the sucrose concentration, the greater the bacteriocin synthesis (with an increase in the zone of inhibition in 14.2% against Staphylococcus aureus CECT-239). It was observed that the bioreactor conducted under microaeration with 5% dissolved oxygen promoted a higher biosurfactant production (11.6 mg/L) when compared to studies conducted with a higher concentration of oxygen between 30 and 100%, with production on average 2.3 mg/mL. It is noteworthy that no study of the influence of dissolved oxygen, mainly in microaerophilic, for the biosurfactant production by lactic acid bacteria had already been carried out. In addition, the biosurfactant produced proved to be highly stable against extreme values of pH and temperature, and demonstrated remarkable antimicrobial and antiadhesive properties, inhibiting Listeria monocytogenes NADC 2045 and Salmonella entérica CECT-724 in more than 90%.

A Case of Septic Shock Following Catheter-related Infection Caused by Lactococcus lactis subsp. lactis in an Adult

Lactococcus lactis is a gram-positive cocci used extensively in the dairy industry, but considered an unusual pathogen in humans. Among its five subspecies, L. lactis subsp. lactis in particular has rarely been reported as a pathogen. We report a case of septic shock caused by L. lactis subsp. lactis in an adult patient. A 64-yr-old male patient was admitted to outpatient clinics, with chief complaints of fever and chills for one week after convalescent hospital admission. He had severe ileus requiring surgery. He had a peripherally inserted central catheter from convalescent hospital, which was immediately removed. From two sets of blood and catheter tip cultures, we identified L. lactis subsp. lactis using the Vitek 2 system (bioMerieux Inc., USA), and confirmed this result by 16S rRNA sequencing. The patient was empirically treated with ciprofloxacin, and he recovered and was discharged.

Efeito da composição de bactérias láticas e da suplementação do soro de leite na cinética de acidificação, crescimento celular e produção de nisina / Effect of lactic acid bacteria composition and the supplementation of milk whey on acidification kinetics, cell growth and nisin production

As bactérias ácido-láticas (BAL) são micro-organismos que auxiliam nas características organolépticas, funcionais e de bioconservação de produtos fermentados. A utilização do soro de leite como meio de cultivo natural enaltece o conceito da produção de biomoléculas de alto valor agregado, como bacteriocinas, já que é um subproduto gerado por indústrias de laticínios e considerado um agente poluidor. A inulina é um ingrediente prebiótico que promove seletivamente o crescimento de culturas probióticas. Nesse âmbito, o objetivo deste estudo foi avaliar o efeito da composição da cultura de Lactococcus lactis (LL) em cocultura com Streptococcus thermophilus (ST) e da suplementação da base de soro de leite com inulina: (i) nos parâmetros cinéticos de acidificacão, (ii) no crescimento celular, (iii) na viscosidade do produto e (iv) na atividade antimicrobiana da nisina. A fermentação do soro de leite com Lactococcus lactis em cocultura com Streptococcus thermophilus proporcionou a maior taxa de acidificação (Vmax=7,93x10-3 upH/min), assim como apresentou o menor tempo para atingir a velocidade máxima de acidificação (Tvmax=1,13 h). A adição de 2% de inulina ao soro de leite fermentado pela cocultura binária fez com que o tempo para completar o cultivo fosse o mais curto (TpH4,5=4,43 h) quando comparado aos demais ensaios. Quanto ao crescimento celular, pode-se observar que a inulina não afetou significativamente a contagem microbiológica, quando as cepas ST e LL foram utilizadas separadamente no processo fermentativo. Em particular, a adição de 4% de inulina reduziu em 1,2 LogUFC/mL e 0,92 LogUFC/mL a contagem de ST e LL (em monocultura), respectivamente. Por outro lado, em coculturas binárias (ST-LL), percebeu-se ganho na contagem microbiológica nos ensaios que receberam suplementação do ingrediente prebiótico, ou seja, quando adicionados 2% e 4% de inulina, houve aumento de 1 LogUFC/mL e de 1,34 LogUFC/mL na contagem de ST, respectivamente. No caso da cepa LL em cocultura com ST, a suplementação de 2% e 4% do prebiótico aumentou em 0,31 LogUFC/mL e 0,75 LogUFC/mL, respectivamente. A concentração de ácido lático também foi mais elevada nos cultivos realizados com a cocultura binária, sendo 4,56 g/L (na ausência de inulina), 5,28 g/L (com adição de 2% de inulina) e 5,71 g/L (com suplementação de 4% de inulina). A viscosidade foi influenciada tanto pela adição de inulina como pelo efeito sinérgico da cocultura, sendo que o maior valor (7,38 mPas) foi obtido pela cocultura ST-LL e pela adição de 4% do ingrediente prebiótico. Quanto à produção de nisina, observou-se que, no cultivo em cocultura (ST-LL), a concentração de 2% de inulina aumentou em 102% a atividade antimicrobiana quando comparada com a cultura pura LL. Vale ressaltar que ambas as cepas satisfizeram os requisitos tecnológicos relativos à produção de laticínios funcionais

Lactic acid bacteria (LAB) are microorganisms that help in the organoleptic and functional characteristics and in the biopreservation of fermented products. The use of milk whey as a culture medium extols the concept of the production of high value-added biomolecules, such as bacteriocins, since it is a by-product generated by the dairy industry and considered a pollutant. Inulin is a prebiotic ingredient that promotes selectively the growth of probiotic cultures. In this context, the aim of this study was to evaluate the effect of culture composition Lactococcus lactis (LL) in co-culture with Streptococcus thermophilus (ST) and the supplementation of milk whey with inulin on: (i) the acidification kinetic parameters, (ii) the cell growth, (iii) the product viscosity, and (iv) the antimicrobial activity of nisin. The fermentation of milk whey by Lactococcus lactis in coculture with Streptococcus thermophilus provided the highest acidification rate (Vmax = 7.93x10-3 upH/min) and the shortest time to reach the maximum acidification rate ( TVmax = 1.13 h). The addition of 2% inulin in the binary coculture binary led to the shorter time to complete the fermentation (TpH4,5 = 4.43) compared to the other tests. With regard to cell growth, it can be observed that the addition of inulin did not affect the microbiological count of pure cultures of ST and LL strains in the fermentation process. In particular, the addition of 4% inulin reduced by 1.2 Log CFU/mL and 0.92 Log CFU/mL the counts of ST and LL (monoculture), respectively. In the other hand, the binary co-cultures cultivations (ST-LL) with the addition of 2% and 4% inulin increased by 1 LogCFU/mL and 1.34 Log CFU/mL in the case of the ST counts and 0.31 log CFU/mL and 0.75 log CFU/mL the counts of LL, respectively. Lactic acid concentration was higher in cultivations carried out by binary cocultures, thus being 4.56 g/L (in the absence of inulin), 5.28 g/L (with addition of 2% inulin) and 5.71g/L (supplemented with 4% inulin). The viscosity was influenced by the addition of prebiotic ingredient and by the synergistic effect of binary coculture, being the highest value (7.38 mPas) obtained by the addition of 4% inulin. Finally, as regards the production of nisin noted that in the binary coculture cultivations (ST-LL), the concentration of 2% inulin increased at 102% the antimicrobial activity when compared to the pure culture LL. It is worth mentioning that both strains met the technological requirements as regards the production of functional dairy products

Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Application in the control of Listeria monocytogenes in fresh Minas-type goat cheese

Listeria monocytogenes is a pathogen frequently found in dairy products. Its control in fresh cheeses is difficult, due to the psychrotrophic properties and salt tolerance. Bacteriocinogenic lactic acid bacteria (LAB) with proven in vitro antilisterial activity can be an innovative technological approach but their application needs to be evaluated by means of in situ tests. In this study, a novel bacteriocinogenic Lactococcus lactis strain (Lc. lactis DF4Mi), isolated from raw goat milk, was tested for control of growth of L. monocytogenes in artificially contaminated fresh Minas type goat cheese during storage under refrigeration. A bacteriostatic effect was achieved, and counts after 10 days were 3 log lower than in control cheeses with no added LAB. However, this effect did not differ significantly from that obtained with a non-bacteriocinogenic Lc. lactis strain. Addition of nisin (12.5 mg/kg) caused a rapid decrease in the number of viable L. monocytogenes in the cheeses, suggesting that further studies with the purified bacteriocin DF4Mi may open new possibilities for this strain as biopreservative in dairy products.

Metabolitos de bacterias lácticas antagónicos al crecimiento de microorganismos cariogénicos / Antagonistic activity of lactic acid bacteria metabolites toward cariogenic microorganisms growth

Aislar, purificar y conservar cepas de Streptococcus spp. y lactobacillus spp. de la cavidad bucal y enfrentarlas "in vitro" a bacterias lácticas. Materiales y métodos: se seleccionaron individuos con caries y se recolectaron muestras de saliva. Para recuperar Streptococcus spp. se empleó el medio Mitis Salivarius (Difco, Detroit, MI, Estados Unidos) y para Lactobacillus spp. se usó Rogosa (Blokar Diagnostics, Beauvais, Francia). Como cepas productoras de bacteriocinas se utilizaron 7 cepas de Lactococcus lactis subsp. lactis, 2 de Leuconostoc mesenteroides y 1 de Lactococcus lactis subsp. diacetylactis. La actividad antagónica de las bacterias lácticas al crecimiento in vitro de bacterias cariogénicas se determinó con el método de difusión en agar. Resultado: el desarrollo y la multiplicación de las cepas de Streptococcus spp. de origen bucal ensayadas se vieron afectados por la presencia de metabolitos generados por las cepas de Lactococcus lactis subsp. lactis. Conclusión: el crecimiento de las cepas de Streptococcus subsp. fue inhibido por efecto de L. lactis subsp. lactis...

Bioencapsulation of probiotic Lactococcus lactis subsp. lactis on Artemia franciscana nauplii: Effects of encapsulation media on Nauplii survival and probiotic recovery

Aims: This study aimed to investigate the suitability and efficacy of various encapsulation media in bioencapsulating the probiotic Lactococcus lactis subsp. lactis in Artemia franciscana nauplii. The impact of the encapsulation media on nauplii survival and probiotic recovery was also determined. Methodology and results: Various encapsulation media (sodium alginate, palm oil, starch, gum Arabic and gelatin) were prepared by dissolving the respective media in artificial sea water. Each media was prepared in four different concentrations: 0.25, 0.5, 1.0 and 2.0 g/L. To determine the suitability of encapsulation media on the survivability of A. franciscana, survival rate (SR) of Artemia nauplii was determined after 8 hours post-encapsulation. Instar II stage Artemia nauplii at 1 nauplii per mL was used for each replicate. The result revealed that A. franciscana reached 100 % SR in the encapsulation media at ≤ 0.5 g/L. All media enabled > 23 % recovery of L. lactis subsp. lactis from encapsulated A. franciscana, which is similar (p > 0.05) to the recovery of free-cells (non-encapsulated) of L. lactis subsp. lactis. Noticeably in sodium alginate (E1) treatment, the total counts of L. lactis subsp. lactis in bioencapsulated A. franciscana were the highest among others, accounting for 2.44 × 107 CFU/mL per A. franciscana tissue homogenate. Conclusion, significance and impact of study: Artemia nauplii bioencapsulated with L. lactis subsp. lactis using 0.5 g/L sodium alginate as encapsulation medium has the highest SR for nauplii and bioencapsulation efficiency, respectively. This result provides a basic guideline for Artemia bioencapsulation in fish/shrimp larval culture.

Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: characterization of the bacteriocin

Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its antimicrobial activity. The bacteriocin presented a broad spectrum of activity, was sensitive to proteolytic enzymes, resistant to heat and pH extremes, and not affected by the presence of SDS, Tween 20, Tween 80, EDTA or NaCl. Bacteriocin production was dependent on the components of the culture media, especially nitrogen source and salts. When tested by PCR, the bacteriocin gene presented 100% homology to nisin Z gene. These properties indicate that this L. lactis subsp. lactis DF4Mi can be used for enhancement of dairy foods safety and quality.

Bacteriocinogenic Lactococcus lactis subsp: lactis DF04Mi isolated from goat milk: Evaluation of the probiotic potential

Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its probiotic potential. Lc. lactis DF4Mi was resistant to acidic pH and oxbile, presented co-aggregation with Listeria monocytogenes, and was not affected by several drugs from different generic groups, being sensitive to most tested antibiotics. These properties indicate that this Lc. lactis strain can be used for enhancement of dairy foods safety and quality, in combination with potential probiotic properties.

Expression and biological activity of porcine interleukin-18 in recombinant Lactococcus lactis / 生物工程学报

To obtain active protein of pIL-18 expression in Lactococcus lactis, and to observe its biological activity, the total RNA was extracted as template from peripheral blood mononuclear cells. Porcine interleukin 18 (pIL-18) was amplified by RT-PCR. The resulting fragment was cloned into pAMJ399 L. lactis vector, and then transformed to L. lactis MG1363 cells by electroporation. Expression of pIL-18 protein was detected by SDS-PAGE and Western-blotting. Bioactivity of the product was tested by pig spleen lymphocyte proliferation test and cytopathogenic effect inhibition assay. The result of Western blotting and bioactivity test shows that the molecular weight of pIL-18 protein was 19 kDa. The react line was observed in both supernatant and precipitated of the recombinant bacteria pAMJ399-pIL18/MG1363. The expressed pIL-18 can promote the proliferation of pig spleen lymphocyte, and significantly inhibit virus multiplication. As conclusion, porcine interleukin-18 was successfully expressed in L. lactis, and the product was biologically active.

Polymicrobial Peritonitis with Lactococcus lactis in a Peritoneal Dialysis Patient / 전남의대학술지

Lactococcus lactis (L. lactis) is an important gram-positive bacterium in dairy products. It is a rare cause of opportunistic infections with only four cases of Lactococcus peritoneal dialysis (PD) peritonitis reported in the literature. In Korea, L. lactis infection was first reported in a liver abscess patient in 2010; however, PD peritonitis with Lactococcus has not been reported in Korea. Recently, we experienced a case of Lactococcus-associated polymicrobial PD peritonitis. The patient was initially managed with broad-coverage antibiotics; however, owing to a poor response, the PD catheter was removed and the patient was switched to hemodialysis. We discuss this case and review the literature.

Polymicrobial Peritonitis with Lactococcus lactis in a Peritoneal Dialysis Patient / 전남의대학술지

Lactococcus lactis (L. lactis) is an important gram-positive bacterium in dairy products. It is a rare cause of opportunistic infections with only four cases of Lactococcus peritoneal dialysis (PD) peritonitis reported in the literature. In Korea, L. lactis infection was first reported in a liver abscess patient in 2010; however, PD peritonitis with Lactococcus has not been reported in Korea. Recently, we experienced a case of Lactococcus-associated polymicrobial PD peritonitis. The patient was initially managed with broad-coverage antibiotics; however, owing to a poor response, the PD catheter was removed and the patient was switched to hemodialysis. We discuss this case and review the literature.

Obtención de microcápsulas de tamaño controlado conteniendo Lactococcus lactis, utilizando mezclas de gelanos de alto y bajo acilo / Obtaining size-controlled microcapsules by ionic gelation with high and low acyl gellans containing Lactococcus lactis

Control the diameter of microcapsules obtained with functional biopolymer is a crucial parameter in the success of food applications, since it affects the protection of microencapsulated microorganism and also in the texture of the final product. The aim of this study was to assess the obtaining of controlled size microcapsules containing Lactococcus lactis, using mixtures of high acyl gellan (HA) and low acyl gellan (LA). A concentration of 0.2% (w/w) gellan was employed using a simple design, generating the following mixtures: 100HA/0.0LA, 0.0HA/100LA, 25HA/75LA, 50HA/50LA and 75HA/25LA. The diameter of the microcapsules, efficiency of microencapsulation and viability of the microencapsulated microorganism were studied in function of the speed of agitation (400-800 rpm) and surfactant concentration (sorbitan monooleate) (0.0-0.2%)v/v. The results indicated that mixtures with concentration equal or greater than 50% of HA gellan are not efficient for obtaining microcapsules, only the LA gellan and the mixture 25HA/75LA gave acceptable results. The viability of the microorganism and the efficiency of microencapsulation were descending function of the stirring speed and surfactant concentration. The microcapsules obtained had diameters not greater than 80 µm when the highest concentrations of surfactant (0.2% v/v) and stirring speed (800 rpm) were used, suggesting that the ionic gelation can be used to obtain microcapsules of controlled size (15-75 µm) containing Lactococcus lactis with high viability (83.32%) and high efficiency of microencapsulation (82.4%), which makes it feasible for use in food applications.

Controlar el diámetro de microcápsulas obtenidas con biopolímeros funcionales es un parámetro crucial en el éxito de aplicaciones alimentarias, ya que influye en la protección del microorganismo microencapsulado y también en la textura del producto final. El objetivo de este trabajo fue evaluar la obtención de microcápsulas de tamaño controlado conteniendo Lactococcus lactis, utilizando mezclas de gelana de alto (HA) y bajo acilo (LA). Se empleó una concentración de gelana de 0.2% p/p usando un diseño de mezclas simple, generando las siguientes mezclas, 100HA/0.0LA, 0.0HA/100LA, 25HA/75LA, 50HA/50LA, 75HA/25LA. El diámetro de las microcápsulas, la eficiencia de microencapsulación y la viabilidad del microorganismo microencapsulado fueron estudiadas en función de la velocidad de agitación (400-800 rpm) y concentración de surfactante (sorbitan monooleate) (0.0-0.2%)v/v. Los resultaron indicaron que las mezclas con concentración igual o superior al 50% de gelana de HA, no son eficientes para obtener microcápsulas; solamente dieron resultados aceptables la gelana de LA y la mezcla 25HA/75LA. La viabilidad del microorganismo y la eficiencia de microencapsulación variaron en función descendente de la velocidad de agitación y concentración de surfactante. Las microcápsulas obtenidas no presentaron diámetros superiores a 80 µm cuando se emplearon las mayores concentraciones de surfactante (0.2%) y velocidad de agitación (800 rpm), sugiriendo que la gelación iónica puede ser utilizada para obtener microcápsulas de tamaño controlado (15-75 µm) conteniendo Lactococcus lactis con alta viabilidad (83.32%) y eficiencia de microencapsulación (82.4%), cuando se utiliza la mezcla 25HA/75LA a 800 rpm y 0.2% v/v de surfactante, lo cual la hace factible para su uso en aplicaciones alimentarias.

Antimicrobial activity of the Nisin Z producer Lactococcus lactis subsp. lactis Lc08 against Listeria monocytogenes in skim milk / Atividade antimicrobiana de Lactococcus lactis subsp. lactics Lc08 produtor de Nisina Z contra Listeria monocytogenes em leite desnatado

The presented study aimed to verify the effect of different pH values, enzyme solutions and heat treatments on the antimicrobial activity of the bacteriocinogenic strain Lactococcus lactis subsp. lactis Lc08 and to test their antimicrobial activity against Listeria monocytogenes in reconstituted skim milk at refrigeration temperatures. This strain was previously described as a nisin Z producer and capable of inhibiting L. monocytogenes growth in in vitro tests. The antimicrobial activity of the bacteriocin cell-free supernatant of Lc08 was sensitive to enzyme treatments (except papain). The pH values and heating (65ºC for 30min, 75ºC for 15s) had no apparent effect on the antimicrobial activity of the bacteriocin produced by Lc08. Only treatment at autoclave conditions result in loss of their antimicrobial activity. Lc08 presented antimicrobial activity against L. monocytogenes in the milk system after 12h at 25ºC. No effect was found at 7ºC. The results show the application viability of the Lc08 in food systems as a biopreservative against L. monocytogenes.

O presente estudo teve como objetivo verificar o efeito de diferentes valores de pH, soluções enzimáticas e tratamentos térmicos na atividade antimicrobiana da cepa bacteriocinogênica Lactococcus lactis subsp. lactis Lc08 e testar sua atividade antagonista contra Listeria monocytogenes em leite desnatado reconstituído em diferentes temperaturas de estocagem. Essa cepa já foi descrita como produtora de nisina Z e capaz de inibir o desenvolvimento de L. monocytogenes em testes in vitro. A atividade antimicrobiana do sobrenadante de Lc08 contendo a bacteriocina produzida e livre de células foi sensível ao tratamento pelas enzimas testadas (exceto papaína). A aplicação de diferentes valores de pH e o tratamento térmico (65ºC por 30 min, 75ºC por 15s) não influenciaram na atividade antimicrobiana da bacteriocina produzida por Lc08. Apenas o tratamento em autoclave resultou em perda da sua capacidade em inibir o desenvolvimento de L. monocytogenes. A cepa Lc08 apresentou atividade antagonista contra L. monocytogenes em leite após período de estocagem de 12h a 25ºC. Não foi observado efeito a 7ºC. Os resultados mostram a viabilidade de aplicação da cultura Lc08 ou de sua bacteriocina em produtos lácteos como bioconservador contra L. monocytogenes.

Effect of co-expression of nicotinic acid phosphoribosyl transferase and pyruvate carboxylase on succinic acid production in Escherichia coli BA002 / 生物工程学报

Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate was accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. Results in 3 L fermentor showed that OD600 is 4.64 and BA016 consumed 35.00 g/L glucose and produced 25.09 g/L succinate after 112 h under anaerobic conditions. Overexpression of pncB and pyc in BA016, the accumulation of pyruvic acid was further decreased, and the formation of succinic acid was further increased.

Phenotypic and genotypic characterization of lactic acid bacteria isolated from cow, ewe and goat dairy artisanal farmhouses

Lactic acid bacteria collected from artisanal farmhouses were characterized using a polyphasic approach. Phenotypic methods including biochemical assays, ribosomal DNA restriction analysis and 16S rDNA sequence analysis were performed. This approach provides accuracy for identification, and helps to avoid the loss of natural biodiversity including potentially valuable strains.

Suitability of MRS-bile agar for the selective enumeration of mixed probiotic bacteria in presence of mesophilic lactic acid cultures and yoghurt bacteria

Measuring the viability of probiotic microorganisms in food products using plate count methodology is a common practice due to the simplicity [ease of performance], inexpensive and routine testing characters of this method. In present study, the suitability of de man rogosa and sharpe agar [MRS] bile agar medium for the selective enumeration of mixed probiotic bacteria [Lactobacillus acidophilus LA-5, L. casei 431 and Bifidobacterium lactis BB-12] in presence of mesophilic lactic cultures [Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. Cremoris] and yoghurt bacteria [Streptococcus thermophilus and Lactobacillus delbrueckii ssp. Bulgaricus] was investigated. Yoghurt bacteria did not grow neither in presence of 0.15% nor 0.30% of bile salts, as was expected. Mesophilic lactic starters could grow at both concentrations of bile salts at all incubation temperatures except 37°C. According to these results, MRS-bile agar [0.15 bile salts] could be successfully used for selective enumeration of mixed probiotic cultures in presence of mesophilic culture and/or yoghurt bacteria when plates were incubated at 37°C for 72 h