Phenyllactic Acid Research Articles

Phenyllactic acid modulates the gut microbiota, enhances intestinal health, and alleviates physical frailty in aging mice

Phenyllactic acid (PLA) is a natural antibiotic-like compound derived from certain foods and probiotics. PLA levels have been associated with age-related sarcopenia and provide benefits to metabolic health when derived from probiotics. However, the specific regulatory effects of PLA in aging remain largely unexplored. In this study, aging mice were administered PLA via gavage, followed by fecal 16S rRNA sequencing, measurements of targeted metabolites, glucose metabolism monitoring, and physical performance assessments. Our results indicate that PLA administration significantly altered gut microbiota composition, increased the abundance of short-chain fatty acids (SCFAs) and succinate producing microbiota, and enhanced gut integrity in aging mice. Furthermore, PLA treatment raised fasting blood glucose levels and improved physical activity. Mechanistically, PLA intake elevated the levels of circulating SCFAs and succinate, promoting glycogen metabolic homeostasis and maintaining skeletal muscle oxidative capacity. This study provides evidence that PLA modulates the gut microbiota in aging mice, supports intestinal health, promotes glucose homeostasis, and enhances physical activity.

Antifungal activities of Pediococcus pentosaceus LWQ1 and Lactiplantibacillus plantarum LWQ17 isolated from sourdough and their efficacies on preventing spoilage of Chinese steamed bread

The short shelf life of Chinese steamed bread (CSB) at room temperature due to moldy contamination restricts its popularity. This study aimed to evaluate two selected lactic acid bacteria strains, namely Lactiplantibacillus plantarum LWQ17 (LWQ17) and Pediococcus pentosaceus LWQ1 (LWQ1), on their antifungal activities and potential applications in CSB making. The results showed that both strains, especially LWQ17, displayed remarkable inhibition against Aspergillus niger and Penicillium polonicum, the representative species found in CSB spoilage. LWQ17 and LWQ1 destroyed the cell membranes of the fungi and caused a loss of intracellular substances. When applied in CSB making, both strains could delay the germination of mold on the surface of CSB in a season-dependent way. LWQ17 delayed the germination of mold by 4 d in Spring and Winter, while 1 d in Summer and Autumn. Further study revealed that their antifungal activities were greatly influenced by temperatures and humidity. Both strains could completely inhibit the germination of mold during two weeks of storage under 37 °C but with different humidity levels. Moreover, our results demonstrated that acidic substances were crucial in inhibiting the fungi, while proteinic substances and hydrogen peroxide played an auxiliary role. Analysis of their acids by targeted metabolomics showed that LWQ17 produced more kinds of acids, and the amount of phenyllactic acid produced by LWQ17 was 60 times higher than that produced by LWQ1, which may explain its better antifungal efficacy. This study provides a reference for employing lactic acid bacteria in CSB preservation under different conditions.

Antibacterial and antibiofilm activity of human gut lactic acid bacteria.

The present study focused on the antibacterial and antibiofilm activity of novel lactic acid bacterial (LAB) strains isolated from the healthy human volunteers of different age groups and their consortium (LABCON), against the enteropathogenic bacteria. From the study, methanolic extract of LAB isolates and their consortia were found to have promising antibacterial activity and antibiofilm activity against Escherichia coli (ATCC 35218) and Staphylococcus aureus (ATCC 25923). The antimicrobial compounds including the DL-3 phenyllactic acid, DL-p-hydroxyphenyllactic acid, and Succinic acid produced by the LAB could be considered to inhibit the growth and biofilm formation by E. coli (ATCC 35218) and S. aureus (ATCC 25923). Detailed insight into the antibiofilm activity could also be demonstrated by Confocal Raman microscopy attached with AFM and Fluorescent microscope. From the results of the study, the consortium LABCON was superior in antimicrobial and antibiofilm activity and can be considered to have promising application in infection control.

Microbiological Safety and Functional Properties of a Fermented Nut-Based Product

Fermented nut-based products, obtained after soaking and fermentation, are gaining increasing interest as animal food substitutes because of ethical, environmental and health reasons. In these products, Lactic Acid Bacteria (LAB) perform the fermentation, leading to matrix acidification and contributing to controlling spoilage and pathogenic microbiota. In this work, LAB strains isolated from an artisanal product and combined with a commercial strain were added as starter cultures during nut soaking to produce a cheese-like fermented plant-based product. Three different LAB consortia were used in challenge tests at laboratory scale against Listeria monocytogenes, Escherichia coli or Salmonella Enteritidis, inoculated in nuts at 5 log CFU/g, and monitored for pathogen survival and matrix acidification. The combination of Lactiplantibacillus plantarum 82 and Leuc. carnosum 4010 resulted in faster acidification (pH value < 4.4 after 18 h instead of 48 h) and the reduction of target pathogens; L. monocytogenes was already absent after seven days from production, and the counts of E. coli or S. Enteritidis were lower with respect to other samples. Thus, this microbial consortium was used for a pilot-scale production in which, beyond safety, the fermented plant-based product was also characterized for aroma profile and phenolic compounds, parameters that are known to be affected by LAB fermentation. The results showed an enhancement of the aroma profile, with an accumulation of molecules able to confer cheese-like notes (i.e., acetoin and diacetyl) and higher phenolic content, as well as the presence of compounds (i.e., phenyllactic acid and hydroxyphenyllactic acid) that could exert antimicrobial activity. This study allowed us to set up a guided fermentation for a cheese-like vegan product, guaranteeing safety and improving aromatic and functional features.

A novel high-level phenyllactic acid fungal producer, Kodamaea ohmeri w5 screened from fermented broad bean-chili-paste

Phenyllactic acid (PLA) is a broad-spectrum and efficient antimicrobial phenolic acid with potential applications in the food industry. Previous studies have demonstrated that fungi may be ideal producers of PLA. In this study, 15 fungi screened from Doubanjiang with the ability to produce PLA were first reported, including Wickerhamomyces anomalus, Candida etchellsii, Candida parasitosis, Pichia kudriavzevii, Pichia membranifaciens and Kodamaea ohmeri. Among them, K. ohmeri w5 had the highest PLA yield, producing up to 7160 mg/L PLA in shake flask fermentation with phenylalanine as substrate, which was more than ten times higher than the PLA produced by wild-type LAB under the similar conditions. In addition, K. ohmeri w5 was able to grow under extreme hypertonic conditions of 20 % NaCl (w/v) and 50 % glucose (w/v) as well as produce 57.12 ± 0.42 and 1609.22 ± 36.26 mg/L of PLA, respectively. Furthermore, the fermentation supernatant of K. ohmeri w5 demonstrated direct inhibitory effects against foodborne pathogenic microorganisms, Aspergillus flavus and Bacillus cereus. However, the inhibitory effect was weaker than that of the PLA standard at the same concentration. Further, 12,497,932 bp of w5 genome-wide information was obtained by sequencing and assembling. And its gene model was predicted based on transcriptomic evidence, which showed that a total of 7 genes related to PLA synthesis were identified in the w5 genome. Based on qRT-PCR, structure prediction, and molecular docking, a potentially key genetic resource from K. ohmeri w5 for PLA production was uncovered. The results will provide novel producers of PLA and its potential genetic resources.

Optimization of phenyllactic acid biosynthesis and separation by machine learning with neural network and overlay sampling uniform design

Machine learning methodology with neural network models was developed using the datasets based on the overlay sampling uniform design (OSUD) for optimization of phenyllactic acid biosynthesis and separation processes by cryogels. Compared with the multiple regression, the machine learning models exhibited a significant improvement of predictive accuracy of phenyllactic acid biosynthesis, in which the radial basis function neural network (RBFNN) model had the best predictive performance with the accuracy increased by 65.2 %. The combination of RBFNN and OSUD was further employed to optimize the chromatographic separation of phenyllactic acid from crude fermentation broth using two poly(hydroxyethyl methacrylate) based anion-exchange cryogel packed-beds (grafted with (vinylbenzyl)trimethylammonium chloride and N,N-dimethylaminoethyl methacrylate). After optimizing the three critical separation parameters: sample volume (5.3–31.8 mL), flow velocity (1.0–6.0 cm/min), and elution salt concentration (0.05–0.3 mol/L), it was found that the models provided excellent predictions. The optimized recovery rates for the two packed-beds were determined to be 76.5 % and 83.0 %, and the optimal adsorption capacities were 0.26 mg/mL and 0.39 mg/mL from the fermentation broth, respectively. This study provides a reliable integrated approach for optimizing the synthesis and separation processes of high-value bioproducts like phenyllactic acid from crude feedstocks.

Antimicrobial metabolites from Probiotics, Pleurotus ostreatus and their co-cultures against foodborne pathogens isolated from ready-to-eat foods

BackgroundThe incidence of foodborne pathogens in ready- to-eat (RTE) can be attributed to various foodborne diseases. Most of the isolated microorganisms from RTE foods are resistant to common antibiotics and thus, resulted to treatment failure when commercially available antibiotics are administered. However, the secondary metabolites secreted by microorganisms can serve as alternative therapy that are reliable and safe. Secondary metabolites obtained from mono- and co-culture microorganisms can inhibit the growth of antibiotic-resistant microorganisms. Bioactive compounds in the secreted metabolites can be identified and utilized as sources of new antibiotics. In this study, antimicrobial activity of secondary metabolites from Lactobacillus fermentum, Saccharomyces cerevisiae, Pleurotus ostreatus, and their co-cultures were tested against foodborne pathogens isolated from RTE foods using agar well diffusion. The bioactive compounds in the metabolites were identified using gas chromatography-mass spectrometry.ResultsFrom a total of 100 RTE foods examined, Salmonella enterica, Shigella dysenteriae, Escherichia coli, Klebsiella pneumoniae (subsp ozaenae), Pseudomonas fluorescens, Clostridium perfringes, Bacillus cereus, Listeria monocytogens, and Staphylococcus aureus, Penicillium chrysogenum, Aspergillus flavus, and Aspergillus niger were isolated and displayed multiple antibiotic resistance. The secondary metabolites secreted by co-culture of L. fermentum + P. ostreatus + S. cerevisiae, and co-culture of P. ostreatus + S. cerevisiae have the highest (P ≤ 0.05) zones of inhibition (23.70 mm) and (21.10 mm) against E. coli, respectively. Metabolites from mono-cultured L. fermentum, P. ostreatus, and S. cerevisiae showed zones of inhibition against indicator microorganisms with values ranging from 8.80 to 11.70 mm, 9.00 to 14.30 mm, and 9.30 to 13.00 mm, respectively. Some of the bioactive compounds found in the metabolites of co-cultured microorganisms were alpha-linolenic acid (25.71%), acetic acid 3-methylbutyl ester (13.83%), trans-squalene (12.39%), pentadecylic acid (11.68%), 3- phenyllactic acid (30.13%), linolelaidic acid methyl ester (15.63%), and 4-O-methylmannose (53.74%).ConclusionRTE foods contain multiple antibiotics resistance pathogens. The pronounced antimicrobial activity of the secondary metabolites against microorganisms from RTE foods could be attributed to the presence of bioactive compounds in the metabolites. These metabolites can be exploited as alternative food preservatives, biopharmaceuticals and can be used towards better health delivering systems.

One-pot enantioselective synthesis of chiral phenyllactic acids by combining stereocomplementary d- and l-lactate dehydrogenases with multi-enzyme expression fine-tuning

Chiral phenyllactic acid (PLA) is a new type of antiseptic agent and a valuable precursor for active ingredients in pharmaceuticals and agrochemicals. In this study, we designed a multi-enzyme cascade that combined stereocomplementary d- and l-lactate dehydrogenases with threonine aldolase, phenylserine dehydratase, and formate dehydrogenase for the one-pot conversion of achiral glycine and benzaldehyde to synthesize d-PLA and l-PLA. To overcome the imbalance of multi-enzymes in a single cell, two enzyme modules, overexpressing four enzymes, were assembled in Escherichia coli cells to construct whole-cell catalysis systems (WCCSs). Furthermore, by optimizing reaction conditions and components, recombinant E. coli (WCCS 26) was able to produce 100 mM d-PLA with >99 % ee using a fed-batch strategy, while E. coli (WCCS 60) produced 47.2 mM l-PLA with >99 % ee. This study presents a sustainable and efficient method for synthesizing chiral PLAs from food-grade achiral starting materials.

Metabolomic Profiling of Tumor Tissues Unveils Metabolic Shifts in Non-Small Cell Lung Cancer Patients with Concurrent Diabetes Mellitus.

A comprehensive understanding of the exact influence of type 2 diabetes mellitus (T2DM) on the metabolic status of non-small cell lung cancer (NSCLC) is still lacking. This study explores metabolic alterations in tumor tissues among patients with coexisting NSCLC and T2DM in comparison with NSCLC patients. A combined approach of clinical analysis and metabolomics was employed, including 20 NSCLC patients and 20 NSCLC+T2DM patients. Targeted metabolomics analysis was performed on tumor tissues using the liquid chromatography-mass spectrometry (LC-MS) approach. A clear segregation was observed between NSCLC+T2DM and matched NSCLC tissue samples in Orthogonal Partial Least Squares Discrimination Analysis (OPLS-DA). Furthermore, the levels of 7 metabolites are found to be significantly different between diabetes/nondiabetes tumor tissue samples. The related pathways included arginine biosynthesis, glutathione metabolism, arginine and proline metabolism, purine metabolism, biotin metabolism, and histidine metabolism. 3-Phenyllactic acid, carnitine-C5, carnitine-C12, and serotonin showed a positive linear correlation with fasting blood glucose levels in NSCLC patients. Uridine, pipecolic acid, cytosine, and fasting blood glucose levels were found to have a negative correlation. Our results suggest that NSCLC patients with concurrent T2DM exhibit distinct metabolic shifts in tumor tissues compared to those of solely NSCLC patients.

Stereoselective Crystallization of Chiral Pharmaceuticals Aided by Cellulose Derivatives through Helical Pattern Matching.

Stereoselective inhibition aided by "tailor-made" polymeric additives is an efficient approach to obtain enantiopure compounds through conglomerate crystallization. The chemical and configurational match between the side groups of polymers and the molecules of undesired enantiomer is considered to be a necessary condition for successful stereoseparation. Whereas in this contribution, we present an effective resolution of chiral pharmaceuticals by using cellulose acetates as the additives, which stereoselectively reside on the specific crystal faces of one enantiomer and inhibit its crystal nucleation and growth through helical pattern and supramolecular interaction complementarity. An investigation of nimodipine serves as a case study to highlight the novelty of this strategy wherein R-crystals exhibiting an impressive enantiomeric excess value of 97 % can be attained by employing a mere 0.01 wt % cellulose acetate. Guaifenesin and phenyl lactic acid are also well-resolved by utilizing this methodology. Our work not only brings about a brand-new design strategy for "tailor-made" additives, but will also promote the further exploration of the endless potential for utilizing natural biomolecules in chiral recognition and resolution.

Microbial Contamination of Food: Probiotics and Postbiotics as Potential Biopreservatives.

Microbial contamination of food and alimentary toxoinfection/intoxication in humans are commonly caused by bacteria such as Salmonella spp., Escherichia coli, Yersinia spp., Campylobacter spp., Listeria monocytogenes, and fungi (Aspergillus, Fusarium). The addition of probiotic cultures (bacterial strains Lactobacillus and Bifidobacterium and the yeast Saccharomyces cerevisiae var. boulardii) to food contributes primarily to food enrichment and obtaining a functional product, but also to food preservation. Reducing the number of viable pathogenic microorganisms and eliminating or neutralizing their toxins in food is achieved by probiotic-produced antimicrobial substances such as organic acids (lactic acid, acetic acid, propionic acid, phenylacetic acid, and phenyllactic acid), fatty acids (linoleic acid, butyric acid, caproic acid, and caprylic acid), aromatic compounds (diacetyl, acetaldehyde, reuterin), hydrogen peroxide, cyclic dipeptides, bacteriocins, and salivabactin. This review summarizes the basic facts on microbial contamination and preservation of food and the potential of different probiotic strains and their metabolites (postbiotics), including the mechanisms of their antimicrobial action against various foodborne pathogens. Literature data on this topic over the last three decades was searched in the PubMed, Scopus, and Google Scholar databases, systematically presented, and critically discussed, with particular attention to the advantages and disadvantages of using probiotics and postbiotics as food biopreservatives.

Characterization of lactic acid bacteria isolated from human breast milk and their bioactive metabolites with potential application as a probiotic food supplement.

The probiotic properties of twenty-five lactic acid bacteria (LAB) isolated from human breast milk were investigated considering their resistance to gastrointestinal conditions and proteolytic activity. Seven LAB were identified and assessed for auto- and co-aggregation capacity, antibiotic resistance, and behavior during in vitro gastrointestinal digestion. Three Lacticaseibacillus strains were further evaluated for antifungal activity, metabolite production (HPLC-Q-TOF-MS/MS and GC-MS/MS) and proteolytic profiles (SDS-PAGE and HPLC-DAD) in fermented milk, whey, and soy beverage. All strains resisted in vitro gastrointestinal digestion with viable counts higher than 7.9 log10 CFU mL-1 after the colonic phase. Remarkable proteolytic activity was observed for 18/25 strains. Bacterial auto- and co-aggregation of 7 selected strains reached values up to 23 and 20%, respectively. L. rhamnosus B5H2, L. rhamnosus B9H2 and L. paracasei B10L2 inhibited P. verrucosum, F. verticillioides and F. graminearum fungal growth, highlighting L. rhamnosus B5H2. Several metabolites were identified, including antifungal compounds such as phenylacetic acid and 3-phenyllactic acid, and volatile organic compounds produced in fermented milk, whey, and soy beverage. SDS-PAGE demonstrated bacterial hydrolysis of the main milk (caseins) and soy (glycines and beta-conglycines) proteins, with no apparent hydrolysis of whey proteins. However, HPLC-DAD revealed alpha-lactoglobulin reduction up to 82% and 54% in milk and whey, respectively, with L. rhamnosus B5H2 showing the highest proteolytic activity. Overall, the three selected Lacticaseibacillus strains demonstrated probiotic capacity highlighting L. rhamnosus B5H2 with remarkable potential for generating bioactive metabolites and peptides which are capable of promoting human health.

Analysis and identification of the main antimicrobial metabolites of Lactobacillus plantarum LPZN19

BackgroundLactobacillus plantarum can produce many secondary metabolites, some of which have antibacterial effects. This study aimed to explore the main antimicrobial metabolites of Lactobacillus plantarum LPZN19. ResultsThe results of antibacterial activity after fermentation for different durations showed that the metabolites from the LPZN19 cell-free supernatant (LCFS) after 24 h had the strongest antibacterial activity, which was confirmed by the highest contents of organic acids and fatty acids in the LCFS after 24 h. Lactic acid, phenyllactic acid, malic acid, aspartic acid, dodecanoic acid and propionic acid were the main differentially abundant metabolites. LCFS was separated by semi-preparative liquid chromatography to obtain 4 antibacterial parts, mainly organic acids such as lactic acid, glycolic acid, and citric acid, and fatty acids such as stearic acid, palmitic acid, and octanoic acid. In addition, fatty glycerides and amino acids with antimicrobial activity were included. ConclusionsOur findings indicate that the main antimicrobial metabolites of L. plantarum LPZN19 include organic acids, fatty acids, fatty glycerides and some amino acids with antimicrobial activity, which not only clarifies the main antimicrobial metabolites of L. plantarum LPZN19 but also provides an effective method for rapid screening of antimicrobial substances.How to cite: Wang Y, Xu Y. Analysis and identification of the main antimicrobial metabolites of Lactobacillus plantarum LPZN19. Electron J Biotechnol 2024;71. https://doi.org/10.1016/j.ejbt.2024.05.005.

Variation of microbiological and small molecule metabolite profiles of Nuodeng ham during ripening by high-throughput sequencing and GC-TOF-MS

The internal microbial diversity and small molecular metabolites of Nuodeng ham in different processing years (the first, second and third year sample) were analyzed by high-throughput sequencing technology and gas chromatography-time of flight mass spectrography (GC-TOF-MS) to study the effects of microorganisms and small molecular metabolites on the quality of ham in different processing years. The results showed that the dominant bacteria phyla of Nuodeng ham in different processing years were Proteobacteria and Firmicutes, the dominant fungi phyla were Ascomycota and Basidiomycota, while Staphylococcus and Aspergillus were the dominant bacteria and fungi of Nuodeng ham, respectively.Totally, 252 kinds of small molecular metabolites were identified from Nuodeng ham in different processing years, and 12 different metabolites were screened through multivariate statistical analysis. Further metabolic pathway analysis showed that 23 metabolic pathways were related to ham fermentation, of which 8 metabolic pathways had significant effects on ham fermentation (Impact > 0.01, P < 0.05). The content of L-proline, phenyllactic acid, L-lysine, carnosine, taurine, D-proline, betaine and creatine were significantly positively correlated with the relative abundance of Staphylococcus and Serratia, but negatively correlated with the relative abundance of Halomonas, Aspergillus and Yamadazyma.

Facemask acne attenuation through modulation of indirect microbiome interactions

During the COVID-19 pandemic, facemasks played a pivotal role in preventing person-person droplet transmission of viral particles. However, prolonged facemask wearing causes skin irritations colloquially referred to as ‘maskne’ (mask + acne), which manifests as acne and contact dermatitis and is mostly caused by pathogenic skin microbes. Previous studies revealed that the putative causal microbes were anaerobic bacteria, but the pathogenesis of facemask-associated skin conditions remains poorly defined. We therefore characterized the role of the facemask-associated skin microbiota in the development of maskne using culture-dependent and -independent methodologies. Metagenomic analysis revealed that the majority of the facemask microbiota were anaerobic bacteria that originated from the skin rather than saliva. Previous work demonstrated direct interaction between pathogenic bacteria and antagonistic strains in the microbiome. We expanded this analysis to include indirect interaction between pathogenic bacteria and other indigenous bacteria classified as either ‘pathogen helper (PH)’ or ‘pathogen inhibitor (PIn)’ strains. In vitro screening of bacteria isolated from facemasks identified both strains that antagonized and promoted pathogen growth. These data were validated using a mouse skin infection model, where we observed attenuation of symptoms following pathogen infection. Moreover, the inhibitor of pathogen helper (IPH) strain, which did not directly attenuate pathogen growth in vitro and in vivo, functioned to suppress symptom development and pathogen growth indirectly through PH inhibitory antibacterial products such as phenyl lactic acid. Taken together, our study is the first to define a mechanism by which indirect microbiota interactions under facemasks can control symptoms of maskne by suppressing a skin pathogen.

A Snapshot, Using a Multi-Omic Approach, of the Metabolic Cross-Talk and the Dynamics of the Resident Microbiota in Ripening Cheese Inoculated with Listeria innocua.

Raw milk cheeses harbor complex microbial communities. Some of these microorganisms are technologically essential, but undesirable microorganisms can also be present. While most of the microbial dynamics and cross-talking studies involving interaction between food-derived bacteria have been carried out on agar plates in laboratory-controlled conditions, the present study evaluated the modulation of the resident microbiota and the changes of metabolite production directly in ripening raw milk cheese inoculated with Listeria innocua strains. Using a proxy of the pathogenic Listeria monocytogenes, we aimed to establish the key microbiota players and chemical signals that characterize Latteria raw milk cheese over 60 days of ripening time. The microbiota of both the control and Listeria-inoculated cheeses was analyzed using 16S rRNA targeted amplicon sequencing, while direct analysis in real time mass spectrometry (DART-HRMS) was applied to investigate the differences in the metabolic profiles of the cheeses. The diversity analysis showed the same microbial diversity trend in both the control cheese and the inoculated cheese, while the taxonomic analysis highlighted the most representative genera of bacteria in both the control and inoculated cheese: Lactobacillus and Streptococcus. On the other hand, the metabolic fingerprints revealed that the complex interactions between resident microbiota and L. innocua were governed by continuously changing chemical signals. Changes in the amounts of small organic acids, hydroxyl fatty acids, and antimicrobial compounds, including pyroglutamic acid, hydroxy-isocaproic acid, malic acid, phenyllactic acid, and lactic acid, were observed over time in the L. innocua-inoculated cheese. In cheese that was inoculated with L. innocua, Streptococcus was significantly correlated with the volatile compounds carboxylbenzaldheyde and cyclohexanecarboxylic acid, while Lactobacillus was positively correlated with some volatile and flavor compounds (cyclohexanecarboxylic acid, pyroxidal acid, aminobenzoic acid, and vanillic acid). Therefore, we determined the metabolic markers that characterize a raw milk cheese inoculated with L. innocua, the changes in these markers with the ripening time, and the positive correlation of flavor and volatile compounds with the resident microbiota. This multi-omics approach could suggest innovative food safety strategies based on the enhanced management of undesirable microorganisms by means of strain selection in raw matrices and the addition of specific antimicrobial metabolites to prevent the growth of undesirable microorganisms.

Phenyllactic acid affects cell wall thickness by targeting the synthesis of peptidoglycan in Listeria monocytogenes

Phenyllactic acid (PLA) exhibits antibacterial activity against Listeria monocytogenes which is a significant foodborne pathogen. In this study, effects of PLA on the cell survival, cell morphology, cell wall thickness, extraction of cell wall peptidoglycan and expression levels of genes involved in the synthesis of cell wall peptidoglycan were investigated using L. monocytogenes 10403S, which were either PLA shocked for 30 min (PLA shock) or grown in medium with PLA (PLA growth). Flow cytometry analysis demonstrated that at the same concentration, PLA shock resulted in a higher proportion of dead and injured cells than those from PLA growth. Images of field emission scanning electron microscopy showed that PLA growth had almost no effect on cell morphology, while damages on L. monocytogenes cells were observed after PLA shock. Results from transmission electron microscopy showed that the cell walls became thinner after PLA shock while PLA growth resulted in thicker cell walls. Coincidently, lower extraction yields were obtained after PLA shock while PLA growth led to higher extraction yields compared to the corresponding control group. PLA shock inhibited expression of genes associated with the production of lipid II and polymerization of peptidoglycan, while PLA growth induced expression of genes responsible for the production and translocation of lipid II. Our results suggest that L. monocytogenes responds differently to PLA shock and PLA growth. PLA affects the cell wall thickness of L. monocytogenes through regulating the synthesis of peptidoglycan.

The use of organic peroxyacids for the inactivation of calcium-mediated biofilm formation by Bacillus licheniformis

Biofilms formed by Bacillus licheniformis usually serve as a continuous bacterial contamination in the dairy industry. The purpose of this study was to explore how 1% organic acids (citric acid, phenyllactic acid, malic acid, and butyric acid) and their 1% organic peroxyacids (peroxycitric acid, peroxyphenyllactic acid, peroxymalic acid, and peroxybutyric acid) can inactive the planktonic and biofilm cells of B. licheniformis. Results demonstrated that the number of B. licheniformis planktonic cells was increased from 4 to 6.46 Log CFU mL−1 when treated with water. Organic peroxyacids, especially peroxyphenyllactic acid (PPA), obviously inactivated planktonic cells of B. licheniformis by a range of 3.32–4.14 Log CFU mL−1. Moreover, organic peroxyacids could also decrease the cell surface hydrophobicity of bacteria, and effectively control the Ca2+-mediated biofilm formation by B. licheniformis in 96-well plates (reduction of OD values by 0.14–0.33) and on stainless steel (reduction of cell counts by 1.20–2.49 Log CFU cm−2). The low minimum biofilm eradication concentration of PPA (0.125%) also suggests its strong biofilm inactivation activity, which could serve as a novel and environmentally friendly biofilm control agent in the dairy industry.

Inactivation mechanism of phenyllactic acid against Bacillus cereus spores and its application in milk beverage

Phenyllactic acid (PLA) as a natural phenolic acid exhibits antibacterial activity against non-spore-forming bacteria, while the inhibitory effect against bacterial spore remained unknown. Herein, this study investigated the inactivation effect of PLA against Bacillus cereus spores. The results revealed that the minimum inhibitory concentration of PLA was 1.25 mg/mL. PLA inhibited the outgrowth of germinated spores into vegetative cells rather than germination of spores. PLA disrupted the spore coat, and damaged the permeability and integrity of inner membrane. Moreover, PLA disturbed the establishment of membrane potential due to the inhibition of oxidative metabolism. SEM observations further visualized the morphological changes and structural disruption caused by PLA. Besides, PLA caused the degradation of DNA of germinated spores. Finally, PLA was applied in milk beverage, and showed promising inhibitory effect against B. cereus spores. This finding could provide scientific basis for the application of PLA against spore-forming bacteria in food industry.

Antifungal mechanism of phenyllactic acid against Mucor investigated through proteomic analysis

The primary inhibitory targets of phenyllactic acid (PLA, including D-PLA and L-PLA) on Mucor were investigated using Mucor racemosus LD3.0026 isolated from naturally spoiled cherry, as an indicator fungi. The results demonstrated that the minimum inhibitory concentration (MIC) of PLA against Mucor was 12.5 mmol·L−1. Results showed that the growing cells at the tip of the Mucor were not visibly deformed, and there was no damage to the cell wall following PLA treatment; however, PLA damaged the cell membrane and internal structure. The results of isobaric tags for relative and absolute quantification (iTRAQ) indicated that the Mucor mitochondrial respiratory chain may be the target of PLA, potentially inhibiting the energy supply of Mucor. These results indicate that the antifungal mechanism of PLA against mold is independent of its molecular configuration. The growth of Mucor is suppressed by PLA, which destroys the organelle structure in the mycelium and inhibits energy metabolism.