Anti-Listerial Activity of Bacteriocin-like Inhibitory Substance Produced by Enterococcus lactis LBM BT2 Using Alternative Medium with Sugarcane Molasses.

Listeria monocytogenes is a foodborne pathogen that contaminates food-processing environments and persists within biofilms on equipment, thus reaching final products by cross-contamination. With the growing demand for clean-label products, the search for natural antimicrobials as biopreservants, such as bacteriocins, has shown promising potential. In this context, this study aimed to evaluate the anti-listerial action of bacteriocins produced by Enterococcus lactis LBM BT2 in an alternative medium containing sugarcane molasses (SCM). Molecular analyses were carried out to characterize the strain, including the presence of bacteriocin-related genes. In the kinetic study on SCM medium E. lactis, LBM BT2 showed biomass and bacteriocin productions similar to those observed on a sucrose-based medium (control), highlighting the potential of the sugarcane molasses as a low-cost substrate. Stability tests revealed that the molecule remained active in wide ranges of pH (4-10) and temperature (60-100 °C). Furthermore, the proteolytic treatment reduced the biomolecule's antimicrobial activity, highlighting its proteinaceous nature. After primary purification by salting out and tangential flow filtration, the bacteriocin-like inhibitory substance (BLIS) showed bacteriostatic activity on suspended L. monocytogenes cells and against biofilm formation at a concentration of 0.625 mg/mL. These results demonstrate the potential of the produced BLIS as a biopreservative in the food industry.

Dual antibacterial mechanism of [K4K15]CZS-1 against Salmonella Typhimurium: a membrane active and intracellular-targeting antimicrobial peptide.

Salmonella genus is a leading cause of food-borne infections with strong public health impact and economic ramifications. The development of antimicrobial resistance added complexity to this scenario and turned the antibiotic drug discovery into a highly important challenge. The screening of peptides has served as a successful discovery platform to design new antibiotic candidates. Motivated by this, the antimicrobial and cytotoxic properties of three cruzioseptins against Salmonella Typhimurium and RAW 264.7 murine macrophage cells, respectively, were investigated. [K4K15]CZS-1 was the most potent antimicrobial peptide identified in the screening step with a minimum inhibitory concentration (MIC) of 16 µg/mL (7.26 µM) and moderate cytotoxicity. From a structural point of view, in vitro and in silico techniques evidenced that [K4K15]CZS-1 is a α-helical cationic antimicrobial peptide. In order to capture mechanistic details and fully decipher their antibacterial action, we adopted a multidimensional approach, including spectroscopy, electron microscopy and omics analysis. In general lines, [K4K15]CZS-1 caused membrane damage, intracellular alterations in Salmonella and modulated metabolic pathways, such as the tricarboxylic acid (TCA) cycle, fatty acid biosynthesis, and lipid metabolism. Overall, these findings provide deeper insights into the antibacterial properties and multidimensional mode of action of [K4K15]CZS-1 against Salmonella Typhimurium. In summary, this study represents a first step toward the screening of membrane-acting and intracellular-targeting peptides as potential bio-preservatives to prevent foodborne outbreaks caused by Salmonella.

Homo- and heterofermentative lactobacilli are distinctly affected by furanic compounds.

PURPOSE: Second generation (2G) ethanol is produced using lignocellulosic biomass. However, the pre-treatment processes generate a variety of molecules (furanic compounds, phenolic compounds, and organic acids) that act as inhibitors of microbial metabolism, and thus, reduce the efficiency of the fermentation step in this process. In this context, the present study aimed to investigate the effect of furanic compounds on the physiology of lactic acid bacteria (LAB) strains that are potential contaminants in ethanol production. METHODOLOGY: Homofermentative and heterofermentative strains of laboratory LAB, and isolated from first generation ethanol fermentation, were used. LAB strains were challenged to grow in the presence of furfural and 5-hydroxymethyl furfural (HMF). RESULTS: We determined that the effect of HMF and furfural on the growth rate of LAB is dependent on the metabolic type, and the growth kinetics in the presence of these compounds is enhanced for heterofermentative LAB, whereas they are inhibitory to homofermentative LAB. Sugar consumption and product formation were also enhanced in the presence of furanic compounds for heterofermentative LAB, who displayed effective depletion kinetics when compared to the homofermentative LAB. CONCLUSION: Homo- and heterofermentative LAB are affected differently by furanic compounds, in a way that the latter type is more resistant to the toxic effects of these inhibitors. This knowledge is important to understand the potential effects of bacterial contamination in 2G bioprocesses.

Ionic liquids as protein stabilizers for biological and biomedical applications: A review.

Biotechnology has revolutionized science and health care by providing new biomolecules with biological and medical applications. However, the low stability of several life-saving bioproducts still hinders their transport, storage, and application. Hence, protein-based bioproducts instability and high costs are the main bottlenecks limiting access to biopharmaceuticals in low-income countries and communities. Aiming to improve the stability of protein-based products, researchers have studied ionic liquids (ILs) as protein stabilizers due to their unique properties and ability to enhance the solubility and stability of a wide range of biomolecules. Although different classes of ILs have the potential to improve protein stability, their effects are dependent on several variables, such as the complex and intrinsic properties of proteins, the nature and concentration of ILs, and environmental conditions (e.g., temperature, pH). For medical applications, the biocompatibility of ILs can also limit their biological use. Therefore, the current state-of-the-art on ILs applications for non-enzymatic protein stabilization was carefully analyzed and discussed, considering protein properties, ILs classes, and IL solutions concentrations. Lastly, a critical perspective regarding ILs applications as protein stabilizers was presented, highlighting the current lacunas in the field while guiding future studies to answer the existing paradigms.

Effect of Polydextrose on the Growth of Pediococcus pentosaceus as Well as Lactic Acid and Bacteriocin-like Inhibitory Substances (BLIS) Production.

Pediococcus pentosaceus was cultivated in MRS medium supplemented or not with polydextrose under different conditions in order to evaluate its effect on cell growth, lactic acid and bacteriocin-like inhibitory substance (BLIS) production. Independent variables were pH (4.0, 5.0, 6.0), rotational speed (50, 100, 150 rpm), polydextrose concentration (0.5, 1.0, 1.5%) and temperature (25, 30, 35 °C), while cell concentration and productivity after 24 h, maximum specific growth rate, specific rate of substrate (glucose) consumption, volumetric and specific lactic acid productivities, yields of biomass and lactic acid on consumed substrate were the dependent. The maximum cell concentration (10.24 ± 0.16 gX L-1) and productivity (0.42 ± 0.01 gX L-1 h-1) were achieved at pH 6.0, 35 °C, 150 rpm using 1.5% polydextrose, while the maximum specific growth rate (0.99 ± 0.01 h-1) and yield of biomass (2.96 ± 0.34 gX gS-1) were achieved at the same pH and polydextrose concentration, but at 25 °C and 50 rpm. The specific substrate consumption rate (0.09 ± 0.02 gS gX-1 h-1) and the volumetric lactic acid productivity (0.44 ± 0.02 gP L-1 h-1) were maximized at pH 6.0, 35 °C, 50 rpm and 0.5% polydextrose. BLIS produced in this last run displayed the highest antibacterial activity against Escherichia coli, while the same activity was displayed against Enterococcus faecium using 1.5% polydextrose. These results appear to be quite promising in view of possible production of this BLIS as an antibacterial agent in the food industry.

Evaluating the potential of Pediococcus pentosaceus as a biocontrol agent against tenuazonic acid-producing Alternaria alternata on livestock feeds.

AIMS: This study aims to demonstrate the potential of the lactic acid bacteria (LAB) Pediococcus pentosaceus LBM18 against the mycotoxin-producing Alternaria alternata TEF-1A and highlight its application as an effective grain silage inoculant to control mycotoxin contamination. METHODS AND RESULTS: The antifungal properties of Ped. pentosaceus lyophilized (PPL) were assessed by evaluating its effect on A. alternata TEF-1A grown in a corn silage-based medium, which included morphological changes by Scanning Electron Microscopy (SEM) observations, growth rate, conidia production assays, and inhibition of Tenuazonic acid (TeA) production by high-performance liquid chromatography (HPLC-MS/MS) analyses. Furthermore, TeA biosynthesis was monitored for changes at the molecular level by PKS gene expression. The growth and sporulation processes of A. alternata TEF-1A were affected by Ped. pentosaceus LBM18 in a concentration-dependent manner. Moreover, a significant inhibition of TeA production (74.3%) and the transcription level of the PKS gene (42.9%) was observed. CONCLUSIONS: Ped. pentosaceus is one of the promising LAB to be applied as an inoculant for corn silage preservation, aiming to inhibit mycotoxigenic fungi growth and their mycotoxin production. SIGNIFICANCE AND IMPACT OF THE STUDY: Ped. pentosaceus could be used as an inoculant to reduce fungal and mycotoxins contamination in grain silage production.

Pediocin PA-1 production by Pediococcus pentosaceus ET34 using non-detoxified hemicellulose hydrolysate obtained from hydrothermal pretreatment of sugarcane bagasse.

Listeria monocytogenes is one of the foodborne pathogens of most concern for food safety. To limit its presence in foods, bacteriocins have been proposed as natural bio-preservatives. Herein, a bacteriocin was produced on hemicellulose hydrolysate of sugarcane bagasse by Pediococcus pentosaceous ET34, whose genome sequencing revealed an operon with 100% similarity to that of pediocin PA-1. ET34 grown on hydrolysate-containing medium led to an increase in the expression of PA-1 genes and a non-optimized purification step sequence resulted in a yield of 0.8 mg·L-1 of pure pediocin (purity > 95%). Culture conditions were optimized according to a central composite design using temperature and hydrolysate % as independent variables and validated in 3-L Erlenmeyers. Finally, a process for scaled-up implementation by sugar-ethanol industry was proposed, considering green chemistry and biorefinery concepts. This work stands up as an approach addressing a future proper sugarcane bagasse valorisation for pediocin production.

Bioprospecting of probiotics with antimicrobial activities against Salmonella Heidelberg and that produce B-complex vitamins as potential supplements in poultry nutrition.

The demand for animal protein for human consumption has been risen exponentially. Modern animal production practices are associated with the regular use of antibiotics, potentially increasing the emerging multi-resistant bacteria, which may have a negative impact on public health. In poultry production, substances capable of maximizing the animals' performance and displaying an antimicrobial activity against pathogens are very well desirable features. Probiotic can be an efficient solution for such a task. In the present work, lactic acid bacteria (LAB) were isolated from chicken cecum and screened for their antagonistic effect towards many pathogens. Their capacity of producing the B-complex vitamins folate and riboflavin were also evaluated. From 314 isolates, three (C43, C175 and C195) produced Bacteriocin-Like Inhibitory Substances (BLIS) against Staphylococcus aureus (inhibition zones of 18.9, 21.5, 19.5 mm, respectively) and also inhibited the growth of Salmonella Heidelberg. The isolate C43 was identified as Enterococcus faecium, while C173 and C195 were both identified as Lactococcus lactis subsp. lactis. Moreover, the isolates L. lactis subsp. lactis strains C173 and C195 demonstrated high potential to be used as probiotic in poultry feed, in addition to their advantage of producing folate (58.0 and 595.5 ng/mL, respectively) and riboflavin (223.3 and 175.0 ng/mL, respectively).

Ligninolytic Enzymes of Endospore-Forming Bacillus aryabhattai BA03.

In this work we investigated the enzymes produced by Bacillus aryabhattai BA03, particularly in those involved in ligninolytic activities such as laccases (Lac), lignin peroxidases (LiP) and Mn-dependent peroxidase (MnD-P). In this way, the maximal production of Lac (0.069 ± 0.000 U/mL) was obtained at pH 9, and 37 ºC after 72 h. LiP expressed the highest activity at 96 h in acid medium at 37 ºC (0.741 ± 0.029 U/mL). Meanwhile, the strain produced MnD-P (1.052 ± 0.001 U/mL) at the highest temperature assayed (44 ºC) and pH 7 at 72 h. In addition, this microorganism produced resistant endospores able to germinate after the sterilization program (121 ºC, 15 min) showing a high enzymatic activity. Using the heat-treated culture as inoculum, the percentage of decolorization of 150 mg/L of Coomassie Brillant Blue reached 89.42 ± 0.11% in only 24 h. These results open the use of these enzymes and endospores in bioremediation processes carried out under different temperatures and pH values.

Antimicrobial activity of bacteriocin-like inhibitory substance produced by Pediococcus pentosaceus: from shake flasks to bioreactor.

Bacteriocins are peptides produced by various species of bacteria, especially lactic acid bacteria (LAB), which have a large spectrum of action against spoilage bacteria and foodborne pathogens. However, when not entirely characterized, they are alternatively called bacteriocin-like inhibitory substances (BLIS). Pediococcus pentosaceus ATCC 43200 grew and produced BLIS optimally when cultivated anaerobically in bioreactor for 24 h at 30 °C and 200 rpm in De Man, Rogosa and Sharp (MRS) medium supplemented with 1.5% peptone. Under such optimal conditions, the cell mass concentration (3.41 g/L) was 66% higher, the generation time (1.28 h) 38% shorter and the BLIS activity against different indicator strains significantly higher than in MRS medium without any supplement taken as a control, and the exponential phase started 4 h before. The agar diffusion method showed BLIS inhibition halos against LAB strains with diameter in the range 11.0-19.5 mm and specific areas between 377.1 and 2654.6 mm2/mL, while BLIS activity against Listeria strains was better quantified by the liquid medium assay that showed, for the fermented broth without any dilution, 100 and 50% inhibition of Listeria innocua and Listeria seeligeri growth, respectively. These results highlight the potential of P. pentosaceus BLIS as a natural antimicrobial for application in the food industry.

Importance of the agar-media in the evaluation of bacteriocin activity against the same test-microorganisms

Abstract Bacteriocins are peptides produced by various species of bacteria, especially lactic acid bacteria, which exhibit a large spectrum of action against spoilage bacteria and foodborne pathogens. Successful application of techniques for quantitative or qualitative bacteriocin determination relies not only on the sensitivity of the test-microorganisms, but also on the agar-medium employed. Cell free supernatants are routinely used to preliminary screen for antimicrobial activity of bacteria by means of the agar well diffusion method, but the supernatant may also include other molecules (such as medium components and/or intracellular compounds) accidentally released during cell free supernatant preparation, which may interfere with the assay. Reproducibility of bacteriocin activity against the same test-microorganisms is an important factor to be considered. Unfortunately, no specific information about bioassays standardization to determine bacteriocin activity is available in the literature. In this work, growth inhibition by means of the agar well diffusion assays were carried out on different agar-media showing a strong dependence on the agar-medium used, indicating that the inhibitory effects could also depend on the diffusion of exudates that are included in the cell-free supernatant. The results presented in this communication show that selection of the agar-medium is crucial for the bioassay response.

Production of bacteriocin-like inhibitory substance by Bifidobacterium lactis in skim milk supplemented with additives.

Bacteriocins are natural compounds used as food biopreservatives instead of chemical preservatives. Bifidobacterium animalis subsp. lactis (Bifid. lactis) was shown to produce a bacteriocin-like inhibitory substance (BLIS) able to inhibit the growth of Listeria monocytogenes selected as an indicator microorganism. To enhance this production by the strain Bifid. lactis BL 04, skim milk (SM) was used as a fermentation medium either in the presence or in the absence of yeast extract, Tween 80 or inulin as stimulating additives, and the results in terms of bacterial growth and BLIS production were compared with those obtained in a traditional high cost complex medium such as Man, Rogosa and Sharpe (MRS). To this purpose, all the cultivations were carried out in flasks at 200 rpm under anaerobic conditions ensured by a nitrogen flowrate of 1.0 L/min for 48 h, and BLIS production was quantified by means of a modified agar diffusion assay at low values of both temperature and concentration of List. monocytogenes. Although all these ingredients were shown to exert positive influence on BLIS production in both media, yeast extract and SM were by far the best ingredient and the best medium, respectively, allowing for a BLIS production at the late exponential phase of 2000 AU/ml.

Overview of Lactobacillus plantarum as a promising bacteriocin producer among lactic acid bacteria.

Chemical preservatives have been traditionally used during the manufacturing of processed products. However, the continuous growing interest of consumers for fresh and natural products makes it necessary to search for alternative compounds. In this context, food industries have been widely using lactic acid bacteria (LAB) as natural preservatives, due to their ability to produce antibacterial compounds such as bacteriocins. Similarly, pharmaceutical industries have improved the use of these bacterial peptides, with antibacterial activity, trying to reduce the indiscriminate use of antibiotics in food products for human and animal consumption. Among LAB, Lactobacillus plantarum can be adapted to various niches thanks to its ability to ferment a wide range of carbohydrates. Additionally, it can be used as starter culture in food fermentations and as an ingredient for probiotic foods, contributing to the organoleptic characteristics of foods at the same time prolonging the shelf-life and safety of these products. The amount of valuable substances obtained from L. plantarum species isolated from different ecological niches is also worth noting, thus proving it to be one of the most important and versatile species among LAB.

Response of Saccharomyces cerevisiae to cadmium and nickel stress: the use of the sugar cane vinasse as a potential mitigator.

Most of the metals released from industrial activity, among them are cadmium (Cd) and nickel (Ni), inhibit the productivity of cultures and affect microbial metabolism. In this context, the aim of this work was to investigate the capacity of sugar cane vinasse to mitigate the adverse effects of Cd and Ni on cell growth, viability, budding rate and trehalose content of Saccharomyces cerevisiae, likely because of adsorption and chelating action. For this purpose, the yeast was grown batch-wise in YED medium supplemented with selected amounts of vinasse and Cd or Ni. The negative effects of Cd and Ni on S. cerevisiae growth and the mitigating one of sugar cane vinasse were quantified by an exponential model. Without vinasse, the addition of increasing levels of Cd and Ni reduced the specific growth rate, whereas in its presence no reduction was observed. Consistently with the well-proved toxicity of both metals, cell viability and budding rate progressively decreased with increasing their concentration, but in the presence of vinasse the situation was remarkably improved. The trehalose content of S. cerevisiae cells followed the same qualitative behavior as cell viability, even though the negative effect of both metals on this parameter was stronger. These results demonstrate the ability of sugar cane vinasse to mitigate the toxic effects of Cd and Ni.

O efeito da composição de cultura e da suplementação do leite no crescimento, na taxa de acidificação, na sobrevivência e no metabolismo de bactérias probióticas em leite fermentado / The effect of culture composition and the implementation of milk on the growth, acidification rate, survival and metabolism of probiotic bacteria in fermented milk

Os produtos lácteos probióticos e/ou simbióticos são líderes dentro do mercado de alimentos funcionais e têm prioridade de pesquisa em diversos países. Os resultados deste trabalho mostraram que a qualidade do leite fermentado foi fortemente influenciada tanto pela composição das co-culturas probióticas quanto por diferentes prebióticos, como oligofrutose, polidextrose, maltodextrina e inulina. A cinética de acidificação foi influenciada pela composição das culturas probióticas e pelos ingredientes prebióticos no leite fermentado. A suplementação do leite com a inulina reduziu o tempo de fermentação das co-culturas Streptococcus thermophilus + Lactobacillus acidophilus (St-La); Streptococcus thermophilus + Lactobacillus rhamnosus (St-Lr) e Streptococcus thermophilus + Bifidobacterium lactis (St-Bl), além de melhorar a firmeza do leite fermentado probiótico. Foram também relatadas que as quantidades de ácido linoléico conjugado (CLA) aumentaram no leite fermentado pela co-cultura de S. thermophilus + L. acidophilus, suplementado com maltodextrina. No que diz respeito às contagens, a adição de inulina favorece a viabilidade das bactérias probióticas durante o armazenamento a 4°C além de causar um efeito bifidogênico, in vitro, estimulando o crescimento de B. lactis. Em relação ao estudo metabólico entre as co-culturas homofermentativas (St-La e St-Lb), feitas neste presente trabalho, pode-se dizer que a lactose foi apenas parcialmente fermentada a ácido lático, a galactose foi metabolizada em certa medida, e se formaram diacetil e acetoína em níveis apreciáveis. A acetoína e o diacetil provavelmente foram produzidos pelas atividades da α-acetolactato sintase e da α-acetolactato descarboxilase de S. thermophilus.

Probiotics dairy products and/or symbiotic are leaders in the functional foods market and have the research priority in several countries. The results of this study showed that the quality of fermented milk was strongly influenced by composition of probiotic co-cultures and different prebiotics, such as oligofructose, polydextrose, maltodextrin and inulin. The acidification kinetics was influenced by the composition of the probiotic co-cultures and prebiotic ingredients in the fermented milk. The milk supplementation with inulin reduced the fermentation time of the co-cultures of Streptococcus thermophilus + Lactobacillus acidophilus (St-La); Streptococcus thermophilus + Lactobacillus rhamnosus (St-Lr) and Streptococcus thermophilus + Bifidobacterium lactis (St-Bl), and improved the firmness of the probiotic fermented milk. It has also been observed that the amount of conjugated linoleic acid (CLA) increased in the milk fermented by the co-culture S. thermophilus + L. acidophilus supplemented with maltodextrin. As far as the bacterial counts are concerned, the inulin addition promoted the viability of probiotic bacteria during storage at 4°C and led to a bifidogenic effect, in vitro, stimulating the growth of B. lactis. As regards to the metabolic studies of the homofermentative co-cultures (St-La and St-Lb), studied in this work, it can be said that lactose was only partially fermented to lactic acid, galactose was metabolized to some extent, diacetyl and acetoin formed at appreciable levels. The acetoin and diacetyl were probably produced by the activities of α-acetolactate synthase and α-acetolactate decarboxylase of S. thermophilus.

Condições microbiológicas e avaliação da pasteurização em amostras de leite comercializadas no município de Piracicaba, SP / Condition microbiological and evaluation of pasteurization in milk samples commercialized in Piracicaba, SP

Foram submetidas à enumeração e bactérias aeróbias mesófilas, termófilas e psicrotróficas, Staphylococcus coagulase positiva, determinação do Número Mais Provável (NMP) de coliformes totais e fecais e pesquisa de Salmonella spp. 27 amostras de leite comercializado no município de Piracicaba-SP. Com base na legislação do DIPOA (Brasil, 2002) os resultados mostraram que 44,4 por cento das amostras de leite tipo A apresentaram-se fora do padrão microbiológico para aeróbios mesófilos. Para coliformes totais e fecais, o mesmo leite apresentou 66,7 por cento e 55,6 por cento da amostras respectivamente, em desacordo com a legislação vigente. No leite tipo B, nenhuma amostra esteve fora do padrão microbiológico em relação a aeróbios mesófilos. Já para coliformes totais e fecais, 33,3 por cento da amostras estiveram em desacordo com a legislação em vigor. O leite tipo C foi o que apresentou resultado mais satisfatório, pois ao contrário do que se esperava, nenhuma amostra esteve fora do padrão em relação às análises microbiológicas realizadas no presente trabalho.