Pediococcus acidilactici strains as silage inoculants for improving the fermentation quality, nutritive value and in vitro ruminal digestibility in different forages.

AIMS: To examine the characteristics of three isolated Pediococcus acidilactici strains (LTG7, LOG9 and LH9) and evaluate their effects on silage quality, nutritive value and in vitro ruminal digestibility in a variety of forages. METHODS AND RESULTS: One commercial inoculant Lactobacillus plantarum MTD-1 (G) and three isolated lactic acid bacteria (LAB) strains were measured by morphological, physiological and biochemical tests. All the LAB strains were added to Italian ryegrass (Lolium multiflorum Lam.), tall fescue (Festuca arundinacea Schred.) and oat (Avena sativa L.) for ensiling 30 days in laboratory silos (1 l) respectively. Isolated strains could grow normally at 5-20°C, pH 3·5-7·0 and NaCl (3·0, 6·5%), and were identified as P. acidilactici by sequencing 16S rDNA. In Italian ryegrass and oat silages, all inoculants obviously (P < 0·05) increased lactic acid (LA) contents, LAB numbers and in vitro dry matter digestibility (IVDMD), and decreased pH, undesirable micro-organism numbers, butyric acid and ammonia nitrogen (NH3 -N) contents compared with the corresponding controls. LTG7, LOG9 and G silages in Italian ryegrass and oat had markedly (P < 0·05) higher LA content and IVDMD, and lower pH and NH3 -N contents than LH9 silages. In tall fescue silage, LAB inoculants had no obvious (P > 0·05) effect on fermentation quality, while markedly (P < 0·05) enhanced IVDMD. CONCLUSIONS: Based on our results, strains LTG7 and LOG9 had similar potential with the commercial inoculant G in silage making. SIGNIFICANCE AND IMPACT OF THE STUDY: Few studies involved inoculation of silage with P. acidilactici in different forage types. Analysis of effects of LAB strains with their physiological and biochemical characteristics help understand how LAB inoculants affect the digestibility.

Encapsulation of Lactobacillus plantarum ATCC 8014 and Pediococcus acidilactici ATCC 8042 in a freeze-dried alginate-gum arabic system and its in vitro testing under gastrointestinal conditions.

The aim of this study was to investigate the viability of Pediococcus acidilactici ATCC 8042 and Lactobacillus plantarum ATCC 8014 in a freeze-dried capsules system prepared with sodium alginate and gum arabic using the extrusion technique. The capsules made with alginate 2% (w/v)/gum arabic 2% (w/v) showed higher hardness (7.12 ± 0.71 N), with highly cohesive (0.81 ± 0.02) and elastic (0.99 ± 0.00) features on the Texture Profile Analysis (TPA), as well as higher sphericity with 1.75 ± 0.12 mm y 1.73 ± 0.13 mm diameter axes and regularity in their surface by Scanning Electron Microscopy (SEM). The use of skimmed milk at 10% as a cryoprotector in the freeze-drying process allowed the obtention of high viability percentages (88% a 96%) for both strains. Best results of viability for P. acidilactici encapsulated was with the use of alginate 2% (w/v)/gum arabic 2% (w/v) (92%±2.65), and L. plantarum with the use of alginate 2% (w/v) (84.71%±10.33) during the gastrointestinal environment challenge.

Antimicrobial Activity of Supernatant of Lactobacillus plantarum against Pathogenic Microorganisms.

We studied ntimicrobial activity of L. plantarum strain against different pathogens: Escherichia coli, Pseudomonas aeruginosa, Streptococcus pyogenes, Staphylococcus aureus. It was shown that supernatant of 48-h L. plantarum culture in liquid nutrient medium exhibits inhibitory activity against gram-positive and gram-negative pathogenic microorganisms. Supernatant of 24-h culture exhibited lower activity, while supernatant of 72-h culture produced no inhibitory effect. Boiling and proteinase K treatment did not affect activity of the preparation, i.e. antimicrobial activity of the supernatant was not associated with protein or peptide component. These data were confirmed by the results observed after ultrafiltration of the preparation: the growth of E. coli, P. aeruginosa, and S. aureus was inhibited by the low-molecular-weight fraction, but not high-molecular-weight fraction of the supernatant. On the other hand, the high-molecular-weight fraction suppressed the growth of streptococcus by 3 times. We hypothesized that L. plantarum supernatant obtained in our experiments contained at least two antimicrobial components with different molecular weights.

Development of Probiotic Formulation for the Treatment of Iron Deficiency Anemia.

Probiotics are increasingly more present both as functional foods, and in pharmaceutical preparations with multiple levels of action that contribute to human health. Probiotics realize their positive effects with a proper dose, and by maintaining a declared number of probiotics cells by the expiration date. Important precondition for developing a probiotic product is the right choice of clinically proven probiotic strain, the choice of other active components, as well as, the optimization of the quantity of active component of probiotic per product dose. This scientific paper describes the optimization of the number of probiotics cells in the formulation of dietary supplement that contains probiotic culture Lactobacillus plantarum 299v, iron and vitamin C. Variations of the quantity of active component were analyzed in development batches of the encapsulated probiotic product categorized as dietary supplement with the following ingredients: probiotic culture, sucrosomal form of iron and vitamin C. Optimal quantity of active component L. plantarum of 50 mg, was selected. The purpose of this scientific paper is to select the optimal formulation of probiotic culture in a dietary supplement that contains iron and vitamin C, and to also determine its expiration date by the analysis of the number of viable probiotic cells.

Hepatoprotective Effects of Lactobacillus on Carbon Tetrachloride-Induced Acute Liver Injury in Mice.

The aim of this study was to investigate and compare the effects of heat-killed and live Lactobacillus on carbon tetrachloride (CCl4)-induced acute liver injury mice. The indexes evaluated included liver pathological changes, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) in the serum, related gene expression (IL-1ß, TNF-α, Bcl-2, and Bax), and related proteins levels (Bax, Bcl-2, Caspase 3, and NF-κB p65). Compared with the model group, the results indicated that the levels of ALT, AST, and MDA in the serum, the expression levels of IL-1ß, TNF-α, and Bax, and the protein levels of Bax, Caspase 3, and NF-κB p65 significantly decreased, and the pathologic damage degree all significantly reduced after live Lactobacillus fermentum (L-LF) and live Lactobacillus plantarum (L-LP) treatment. Additionally, the levels of SOD and GSH in the serum, the gene expression of Bcl-2, and the protein level of Bcl-2 significantly increased after L-LF and L-LP treatment. Although HK-LF and HK-LP could also have obvious regulating effects on some of the evaluated indexes (ALT, AST, the expression levels of TNF-α and Bax, and the protein level of Bcl-2) and play an important role in weakening liver damage, the regulating effects of L-LF or L-LP on these indexes were all better compared with the corresponding heat-killed Lactobacillus fermentum (HK-LF) and heat-killed Lactobacillus plantarum (HK-LP). Therefore, these results suggested that LF and LP have an important role in liver disease.

One-pot conjugated linoleic acid production from castor oil by Rhizopus oryzae lipase and resting cells of Lactobacillus plantarum.

Conjugated linoleic acid (CLA) has attracted as novel type of fatty acids having unusual health-promoting properties such as anticarcinogenic and antiobesitic effects. The present work employed castor oil as substrate for one-pot production of CLA using washed cells of Lactobacillus plantarum (L. plantarum) and lipases as catalysts. Among the screened lipases, the lipase Rhizopus oryzae (ROL) greatly assisted resting cells to produce CLA. Mass spectral analysis of the product showed that two major isomers of CLA were produced in the reaction mixture i.e. cis-9, trans-11 56.55% and trans-10, cis-12 43.45%. Optimum factors for CLA synthesis were found as substrate concentration (8 mg/mL), pH (6.5), washed cell concentration (12% w/v), and incubation time of 20 h. Hence, the combination of ROL with L. plantarum offers one pot production of CLA selectively using castor oil as a cost-effective substrate.

Study of surface damage on cell envelope assessed by AFM and flow cytometry of Lactobacillus plantarum exposed to ethanol and dehydration.

AIMS: In this work, we evaluated freeze-drying damage at the surface level of oenological strain Lactobacillus plantarum UNQLp155, as well as its ability to grow in a synthetic wine with and without pre-acclimation. METHODS AND RESULTS: Damage on cell surface was studied by flow cytometry, zeta potential and atomic force microscopy, and cell survival was analysed by plate count. Results showed that beside cells acclimated at lower ethanol concentration (6% v/v) became more susceptible to drying than nonacclimated ones, after rehydration they maintain their increased ability to grow in a synthetic wine. Acclimation at a higher ethanol concentration (10% v/v) produces several damages on the cell surface losing its ability to grow in a synthetic wine. CONCLUSIONS: In this work, we showed for the first time that sublethal alterations on bacterial surface induced by a pre-acclimation with a low ethanol concentration (6%), upon a freeze-drying process, result in a better bacterial adaptation to the stress conditions of wine-like medium, as well as to the preservation process. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the adaptation to ethanol of oenological strains and their effects on the preservation process has a strong impact on winemaking process and allows to define the most appropriate conditions to obtain malolactic starters cultures.

Effect of acclimation medium on cell viability, membrane integrity and ability to consume malic acid in synthetic wine by oenological Lactobacillus plantarum strains.

AIMS: The aim of this work was to evaluate the effect of acclimation on the viability, membrane integrity and the ability to consume malic acid of three oenological strains of Lactobacillus plantarum. METHODS AND RESULTS: Cultures in the stationary phase were inoculated in an acclimation medium (Accl.) containing 0, 6 or 10% v/v ethanol and incubated 48 h at 28°C. After incubation, cells were harvested by centrifugation and inoculated in a synthetic wine, containing 14% v/v ethanol and pH 3.5 at 28°C. Viability and membrane integrity were determined by flow cytometry (FC) using carboxyfluorescein diacetate (cFDA) and propidium iodide. Bacterial growth and malic acid consumption were monitored in a synthetic wine during 15 days. In nonacclimated strains, the damage of bacterial membranes produced a dramatic decrease in microbial viability in synthetic wine. In contrast, survival of strains previously acclimated in Accl. with 6 and 10% v/v ethanol was noticeable higher. Therefore, acclimation with ethanol increased the cultivability in synthetic wine and consequently, the consumption of l-malic acid after 15 days of growth. CONCLUSION: Acclimation of oenological strains in media containing ethanol prior to wine inoculation significantly decreases the membrane damage and improves viability in the harsh wine conditions. The role of membrane integrity is crucial to warrant the degradation of l-malic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: The efficiency of multiparametric FC in monitoring viability and membrane damage along with the malic acid consumption has a strong impact on winemaking because it represents a useful tool for a quick and highly reliable evaluation of oenological parameters.

Permeabilized probiotic Lactobacillus plantarum as a source of ß-galactosidase for the synthesis of prebiotic galactooligosaccharides.

Permeabilized probiotic Lactobacillus plantarum was used as a source of ß-galactosidase for the synthesis of galactooligosaccharides (GOS) from lactose. ß-galactosidase activity was highest when galactose (1,724 Miller Units) was used as a carbon source compared to lactose, sucrose or glucose at 37 °C, 18 h. Permeabilized cells had the highest transgalactosylation activity resulting in 34 % (w/w) GOS synthesis from 40 % (w/v) lactose at 50 °C over 12 h. HPLC revealed that the GOS were composed of 13 % disaccharides (non-lactose), 17 % trisaccharides and 4 % tetrasaccharides that were further confirmed by ESI­MS.

Lactobacillus plantarum CECT 7527, 7528 and 7529: probiotic candidates to reduce cholesterol levels.

BACKGROUND: Hypercholesterolaemia plays a key role in the development and progression of coronary artery disease. There are numerous drugs available to treat this condition but they are often expensive and can have unwanted side effects. Therefore, a screening of lactic acid bacteria to select candidate probiotic strains to reduce cholesterol levels was carried out. Three Lactobacillus plantarum strains (CECT 7527, 7528 and 7529) were selected as potential probiotics to reduce cholesterol levels after conducting several in vitro tests for demonstrating the functionalities of the strains according to international guidelines. RESULTS: The three strains showed a high ability to survive under gastrointestinal tract conditions and to adhere to intestinal cells. Regarding lipid metabolism, the strains showed a great production of bile salt hydrolase, especially when combined. Moreover, the strains assimilated cholesterol directly from the medium. Part of the cholesterol present in the medium was removed via binding onto the bacterial cellular surface. Finally, the three strains, especially CECT 7529, produce large quantities of propionic and butyric acids. CONCLUSION: Combined, these characteristics suggest that these strains could be excellent candidates for reducing high blood cholesterol levels.

Extracellular electron transfer across bacterial cell membranes via a cytocompatible redox-active polymer.

A redox-active phospholipid polymer with a phospholipid-mimicking structure (2-methacryloyloxyethyl phosphorylcholine; MPC) was synthesized to construct a biocompatible electron mediator between bacteria and an electrode. In this study, a copolymer of MPC and vinylferrocene [VF; poly(MPC-co-VF)] (PMF) is synthesized. When PMF is added to cultures of the bacterial species Escherichia coli (Gram negative) and Lactobacillus plantarum (Gram positive), which have different cell wall structures, a catalytic current mediated by PMF is observed. In addition, growth curves and live/dead assays indicate that PMF does not decrease metabolic activity or cell viability. These results indicate that PMF mediates extracellular electron transfer across bacterial cell membranes without associated cytotoxicity.

Mutants of Lactobacillus plantarum ML11-11 deficient in co-aggregation with yeast exhibited reduced activities of mixed-species biofilm formation.

Lactic acid bacteria (LAB) mutants deficient in inter-species co-aggregation with yeast were spontaneously derived from Lactobacillus plantarum ML11-11, a significant mixed-species biofilm former in static co-cultures with budding yeasts. These non-co-aggregative mutants also showed significant decreases in mixed-species biofilm formation. These results suggest the important role of co-aggregation between LAB and yeast in mixed-species biofilm formation. Cell surface proteins obtained by 5 M LiCl extraction from the wild-type cells and non-co-aggregative mutant cells were analyzed by SDS-PAGE. There was an obvious difference in protein profiles. The protein band at 30 kDa was present abundantly in the wild-type cell surface fraction but was significantly decreased in the mutant cells. This band assuredly corresponded to the LAB surface factors that contribute to co-aggregation with yeasts.

Microencapsulation of Lactobacillus plantarum (mtcc 5422) by spray-freeze-drying method and evaluation of survival in simulated gastrointestinal conditions.

Spray-drying (SD) and freeze-drying (FD) are widely used methods for microencapsulation of heat-sensitive materials like probiotics for long-term preservation and transport. Spray-freeze-drying (SFD) is relatively a new technique that involves spraying a solution into a cold medium and removal of solvent (water) by conventional vacuum FD method. In this study, the SFD microencapsulated Lactobacillus plantarum powder (1:1 and 1:1.5 core-to-wall ratios of whey protein) is compared with the microencapsulated powders produced by FD and SD methods. The SFD and FD processed microencapsulated powder show 20% higher cell viability than the SD samples. In simulated gastrointestinal conditions, the SFD and FD cells show up to 4 h better tolerance than SD samples and unencapsulated cells in acidic and pepsin condition. The morphology of SFD samples shows particles almost in spherical shape with numerous fine pores, which in turn results in good rehydration behaviour of the powdered product.

Increase of acidification of synthetic brines by ultrasound-treated Lactiplantibacillus plantarum strains isolated from olives.

This paper focused on the evaluation of Ultrasound effect on the growth patterns (3-6% of salt and 45 °C), acidification (pH-decrease), interactions with microorganisms, and membrane permeability of nine strains of Lactiplantibacillus plantarum. Ultrasound treatment was applied at 20% of net power by modulating duration (2-10 min) and pulses (2-10 s). Viable count (7.15-8.16 log CFU/mL) was never affected by Ultrasound, while the treatment increased the extent of pH decrease of at least three strains (109, 162 and c19). L. plantarum c19 was the best performer, as a low intensity treatment was able to increase its acidification, without affecting its growth. The effects could be attributed to an increased permeability of the cellular membrane, as suggested by the increase of released intracellular components. Other factors should be further assessed (e.g. possible changes in the metabolism) and the performances of Ultrasound-treated strains in real brines.

Dissecting signal molecule AI-2 mediated biofilm formation and environmental tolerance in Lactobacillus plantarum.

Quorum sensing (QS) exists in bacteria to communicate with each other and regulate group behaviors in a cell density-dependent manner, which uses signal molecule autoinducer-2 (AI-2) to intra- and inter-species communication. Effects of exogenous AI-2 on biofilm formation and environmental tolerance in Lactobacillus plantarum are the focus of this review. The responses to the exogenous AI-2 cross multiple physiological metabolic behaviors involving the bacteria growth, morphological characterization, biofilm development, extracellular polysaccharides (EPS) amount and related genes expression as well as the environmental stresses tolerance. The cell surface was smoother in the AI-2 supplemented treatments than without AI-2. Meanwhile, AI-2 had ability to promote the growth and formation of biofilm by increasing the yield of EPS, the main components of biofilm. The changes in lamC and ftsH gene expression point to altered regulation for hydrolysis process of polysaccharides as well as the potential for enhanced biofilm formation. The presence of AI-2 also significantly improved (p < 0.01) the tolerance of bile salts in L. plantarum, but the same results did not appear in acid tolerance. In conclusion, AI-2 supplementation could improve the biofilm formation and bile salts tolerance in L. plantarum, and this effect was likely modulated by facilitating EPS production and suppression polysaccharides hydrolysis.

The applications of Lactobacillus plantarum-derived extracellular vesicles as a novel natural antibacterial agent for improving quality and safety in tuna fish.

Bacteria release membrane vesicles into the extracellular environment but which activity is unclear. We investigated the applications of extracellular vesicles (EVs) isolated from probiotic Lactobacillus plantarum to protect tuna fish against spoilage and quality loss in this study. A significant difference was found in EVs size obtained from L. plantarum after 8, 24, and 48 hr incubation. The L. plantarum-derived EVs were collected and used to confirm the anti-bacterial activity versus Shewanella putrefaciens. Finally, the tuna fish was stored at 4 °C for 5 days after coating with EVs or sodium erythorbate, and the quality indexes were assayed. Results indicated that EVs markedly inhibited oxidation reaction, total volatile base nitrogen (TVBN), peroxide value (PV), malondialdehyde (MDA), and bacteria levels. These results finding out that EVs from L. plantarum may have potential for application in food storage technology. Overall, we indicated this new material may be developed as an anti-bacterial agent for prolonging the shelf life of tuna fish.

Effect of Extracellular Vesicles Derived From Lactobacillus plantarum Q7 on Gut Microbiota and Ulcerative Colitis in Mice.

Probiotics plays an important role in regulating gut microbiota and maintaining intestinal homeostasis. Extracellular vesicles (EVs) derived from probiotics have emerged as potential mediators of host immune response and anti-inflammatory effect. However, the anti-inflammatory effect and mechanism of probiotics derived EVs on inflammatory bowel disease (IBD) remains unclear. In this study, the effect of Lactobacillus plantarum Q7-derived extracellular vesicles (Q7-EVs) on gut microbiota and intestinal inflammation was investigated in C57BL/6J mice. The results showed that Q7-EVs alleviated DSS-induced colitis symptoms, including colon shortening, bleeding, and body weight loss. Consumption of Q7-EVs reduced the degree of histological damage. DSS-upregulated proinflammatory cytokine levels including IL-6, IL-1ß, IL-2 and TNF-α were reduced significantly by Q7-EVs (p < 0.05). 16S rRNA sequencing results showed that Q7-EVs improved the dysregulation of gut microbiota and promoted the diversity of gut microbiota. It was observed that the pro-inflammatory bacteria (Proteobacteria) were reduced and the anti-inflammatory bacteria (Bifidobacteria and Muribaculaceae) were increased. These findings indicated that Q7-EVs might alleviate DSS-induced ulcerative colitis by regulating the gut microbiota.

Improving the freeze-drying survival rate of Lactobacillus plantarum LIP-1 by increasing biofilm formation based on adjusting the composition of buffer salts in medium.

Lactic acid bacteria can improve their resistance to adverse environments through the formation of biofilm. This study found that adding different buffer salts in culture medium had a great impact on the freeze-drying survival rate of the Lactobacillus plantarum LIP-1, which could be linked to biofilm formation. Transcriptome data showed that potassium ions in buffer salt increased the expression of the luxS gene in the LuxS/autoinducer-2 (AI-2) quorum sensing system and increase synthesis of the quorum sensing signal AI-2. The AI-2 signal molecules up-regulated the cysE gene, which helps to promote biofilm formation. By adding a biofilm inhibitor, d-galactose, and performing a real-time quantitative polymerase chain reaction experiment, we found that d-galactose could down-regulated the luxS and cysE genes, reduced biofilm formation, and decreased the freeze-drying survival rate. The results of this study showed that promoting biofilm formation using appropriate buffer salts may lead to better freeze-drying survival rates.

Comparison of components and synthesis genes of cell wall teichoic acid among Lactobacillus plantarum strains.

The contents, components, and synthesis genes of cell wall teichoic acid (WTA) in 18 strains of Lactobacillus plantarum were compared. The WTA of each strain was classified by its components as being either the glycerol- or the ribitol-type. The different strains in the WTA type showed marked differences also in two gene regions, tagD1-tagF2 and lp_1816-tagB2, as for the presence or absence, nucleotide sequences, and transcriptional activities. Our results clearly showed that the tagD1-tagF2 and lp_1816-tagB2 regions contained the synthesis genes of the WTA backbone of L. plantarum. We verified that the genes in the tagD1-tagF2 region were involved in the synthesis of the glycerol-type backbone. Furthermore, we propose that the genes in the lp_1816-tagB2 region were tarI, tarJ, tarK, and tarL, which are involved in the synthesis of the ribitol-type backbone.

Characterization of γ-aminobutyric acid (GABA)-producing Saccharomyces cerevisiae and coculture with Lactobacillus plantarum for mulberry beverage brewing.

One elite γ-aminobutyric acid (GABA) producing strain of Saccharomyces cerevisiae SC125 was isolated and identified from Chinese traditional paocai. S. cerevisiae SC125 and Lactobacillus plantarum BC114 were used as cooperative species to ferment mulberry (Morus alba L.) and produce a novel beverage enriched with GABA. The GABA, organic acids and volatile compounds in different fermentation stages were determined by high-performance liquid chromatography (HPLC) and headspace solid-phase microextraction/gas chromatography-mass spectrometry (HS-SPME/GC-MS). It was noted that coculture changed the profiles of flavor compounds in mulberry beverage. The tartaric and succinic acid contents increased to 1.34 g/L and 0.39 g/L, respectively. Lactic, malic, citric, and oxalic acid levels ranged between 0.92 and 2.56 g/L, and ethanol and glycerol were produced at 2.66 g/L and 1.81 g/L, respectively. More volatile compounds were detected in the coculture with significantly enhanced concentrations of fruity esters including ethyl caproate, ethyl propionate, butyl acetate, ethyl butyrate, ethyl caprylate and ethyl caprate, and alcohols of phenylethyl alcohol, 1-pentanol and 2-amino-1,3-propanediol. Also, a yield of 2.42 g/L GABA was achieved in the coculture. In conclusion, S. cerevisiae SC125 and L. plantarum BC114 coculture promotes the production of flavor compounds and GABA in mulberry beverage brewing.