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Growing concern over antimicrobial resistance in chronic wound patients necessitates the exploration of alternative treatments from natural sources. This study suggests that honey's phenolic compounds may offer antimicrobial benefits, warranting further investigation for therapeutic development. The main aim of this study was to investigate the antimicrobial activity of phenolic compounds and to determine the effects of their sub-inhibitory concentrations against Escherichia coli (E. coli). 3-phenyllactic acid (PLA), p-coumaric acid (PCA), and phloretin were tested against the bacterial strain of E. coli ATCC 25922. Comparison of the antimicrobial activity of honey constituents in vitro was performed using a broth culture assay. Measurement of the inhibitory properties of constituents in vitro was conducted using disc and well diffusion assays. The effects of sub-inhibitory concentrations of PCA on the susceptibility of E. coli ATCC 25922 to penicillin-streptomycin were tested. The results demonstrated that PLA was the most efficient antimicrobial agent, followed by PCA, whereas phloretin, at lower (2 mg/mL) concentrations, led to an increase in the growth of E. coli. Various modifications of the agar diffusion assay did not reveal the antibacterial properties of the studied phytochemicals. The enhancing effect of a sub-inhibitory concentration of PCA in cooperation with penicillin-streptomycin was shown. These findings might be helpful for the further investigation and development of new antimicrobial agents for the treatment of skin infections and wounds.

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Diastase is used internationally as a quality monitor for excessive heat treatment and prolonged storage of honey; honey must contain an activity of at least 8 diastase numbers (DN) for it to be considered export quality. Freshly harvested manuka honey can have diastase activity close to the export threshold of 8 DN without excess heating, increasing susceptibility for export failure. This research investigated the effect of compounds unique to or high in concentration in manuka honey on diastase activity. Investigation of the effect of methylglyoxal, dihydroxyacetone, 2-methoxybenzoic acid, 3-phenyllatic acid, 4-hydroxyphenyllactic acid and 2'-methoxyacetophenone on diastase activity was carried out. Manuka honey was stored at 20 and 27 °C and clover honey spiked with compounds of interest were stored at 20, 27 and 34 °C and monitored overtime. Methylglyoxal and 3-phenyllactic acid were found to accelerate the loss of diastase above the loss normally observed with time and elevated temperature.

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The function of the aminotransferase Aat (GenBank Protein WP_159211138) from Pediococcus acidilactici FAM 18098 was studied in vivo. For this purpose, the gene was replaced with an erythromycin resistance gene using the temperature-sensitive Escherichia coli-Pediococcus shuttle plasmid pSET4T_Δaat. The knockout was verified by PCR and genome sequencing. Subsequently, the differences between the metabolism of the knockout and of the wild-type strain were investigated by determining the free amino acids and organic acids in culture supernatants. It was found that the knockout mutant no longer synthesized 3-phenyllactic acid (PLA) and 4-hydroxyphenyllactic acid (HPLA). Additionally, the mutant strain no longer catabolized phenylalanine. Metabolic pathway analysis using the KEGG database indicate that P. acidilactici cannot synthesize α-ketoglutarate that is a predominant amino-group acceptor in many transamination reactions. To study the transfer of the amino group of phenylalanine, the wild-type strain was incubated with [15N] phenylalanine. Mass spectrometry showed that during fermentation, [15N] alanine was formed, indicating that pyruvic acid is an amino group acceptor in P. acidilactici. The present study shows that Aat plays a crucial role in PLA/HPLA biosynthesis and pyruvic acid is an amino acceptor in transamination reactions in P. acidilactici.

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Manuka honey is known for its unique antibacterial activity, which is due to methylglyoxal (MGO). After establishing a suitable assay for measuring the bacteriostatic effect in a liquid culture with a time dependent and continuous measurement of the optical density, we were able to show that honey differs in its growth retardingeffect on Bacillus subtilis despite the same content of MGO, indicating the presence of potentially synergistic compounds. In model studies using artificial honey with varying amounts of MGO and 3-phenyllactic acid (3-PLA), it was shown that 3-PLA in concentrations above 500 mg/kg enhances the bacteriostatic effect of the model honeys containing 250 mg/kg MGO or more. It has been shown that the effect correlates with the contents of 3-PLA and polyphenols in commercial manuka honey samples. Additionally, yet unknown substances further enhance the antibacterial effect of MGO in manuka honey. The results contribute to the understanding of the antibacterial effect of MGO in honey.

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In the present study, the effect of PLA on a periodontic pathogen, Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), the biofilm, and virulence-related genes was investigated. We confirmed that two lactic acid bacteria (LAB) strains isolated from plant sources, Lactiplantibacillus plantarum MSC-C2 and Pediococcus pentosaceus K40, secrete PLA into the de Man, Rogosa & Sharpe (MRS) broth when supplemented with phenyl pyruvic acid (PPA) as a precursor to PLA. Moreover, PLA was generated in the fermentation broths of two medicinal plant extracts, Paeonia lactiflora Pall (PR) and Carthamus tinctorius (CT), when used by each LAB strain and each extract supplemented with PPA. We determined that the minimum inhibitory concentration (MIC) of PLA against A. actinomycetemcomitans was 20 mM. PLA significantly decreased biofilm formation and suppressed the transcription of pgA, ltxA, and cdtB genes, which encode the poly-N-acetylglucosamine (PGA) polysaccharide of biofilm matrix and exotoxins leukotoxin and cytolethal distending toxin (CDT), respectively. The PLA produced by the MSC-C2 and K40 strains was increased several times by the addition of PPA to the MRS broth. The anti-biofilm effect of the extracts from the fermentation broth was proportional to the increasing PLA concentration, while a cumulatively higher effect than that of PLA alone suggested a combinational effect of PLA and the other metabolites, such as lactic acid (LA). Among the two medicinal plants, PLA, produced after the addition of PPA, was higher in PR extract in case of both the LAB strains. PLA production by the MSC-C2 strain in the PR extract reached 4.8 ± 0.23 mM, which was obviously higher than that in the MRS broth (3.88 ± 0.12 mM) supplemented with 1 mg/ml PPA. The activity to inhibit biofilm formation in the fermented PR extract was clearly high. PLA formed in the fermented PR extract downregulated the dispersin B encoding the dspB gene together with pgA, ltxA, and cdtB. In conclusion, this study shows a promising activity of PLA against the A. actinomycetemcomitans biofilm and virulence genes. In addition, the combinational effect of PLA and the medicinal plant extract can be achieved by fermentation with a specific plant-derived LAB strain.

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3-Phenyllactic acid (PLA) is a common secondary product of Lactobacillus sp. and promotes adventitious-root formation in Azuki beans (Vigna angularis). Root promotion activity of PLA is synergistically enhanced by tryptophan (Trp). In this study, stereoisomers of PLA and Trp amide conjugates and their alkyl esters were synthesized to investigate the structure-activity relationships on root-promotion activity. The rooting activity of D-PLA-L-Trp conjugate shows more than 40 times higher than that of the mixture of D-PLA and L-Trp. Modification of PLA-Trp with ethyl ester showed the highest activity at 3,400 times of a mixture of D-PLA and L-Trp. However, L-or D-PLA-D-Trp conjugate and the isopropyl ester of PLA-Trp conjugates, both lost the root promotion activity and implicated that a requirement for steric structure for PLA related root promotion mechanism. Unlike auxin substances, which are commonly used as rooting agents that displayed high activity in low concentrations, PLA-Trp ethyl ester exhibited far less phytotoxicity at high concentration of 1 mM, despite its high rooting activity. Innovation of PLA-Trp ethyl ester may be expected for agricultural aspects with low environmental impact.

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Many microorganisms have been reported to produce compounds that promote plant growth and are thought to be involved in the establishment and maintenance of symbiotic relationships. 3-Phenyllactic acid (PLA) produced by lactic acid bacteria was previously shown to promote root growth in adzuki cuttings. However, the mode of action of PLA as a root-promoting substance had not been clarified. The present study therefore investigated the relationship between PLA and auxin. PLA was found to inhibit primary root elongation and to increase lateral root density in wild-type Arabidopsis, but not in an auxin signaling mutant. In addition, PLA induced IAA19 promoter fused ß-glucuronidase gene expression, suggesting that PLA exhibits auxin-like activity. The inability of PLA to promote degradation of Auxin/Indole-3-Acetic Acid protein in a yeast heterologous reconstitution system indicated that PLA may not a ligand of auxin receptor. Using of a synthetic PLA labeled with stable isotope showed that exogenously applied PLA was converted to phenylacetic acid (PAA), an endogenous auxin, in both adzuki and Arabidopsis. Taken together, these results suggest that exogenous PLA promotes auxin signaling by conversion to PAA, thereby regulating root growth in plants.

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3-phenyllactic acid (PLA) has broad anti-fungal activity, however, target sites of PLA on fungal cells and its anti-fungal mechanism of action have been poorly studied. In this study, we explored the inhibition mechanism of Rhizopus oryzae (R. oryzae) on rotten lily bulbs by PLA. The minimum inhibitory concentration value of PLA against R. oryzae was 8 mg/mL. We observed the ultrastructure of R. oryzae by scanning electron microscopy and transmission electron microscopy which indicated that PLA did not damage the cell membrane, but destroyed the mitochondria and other organelles. Tandem mass tag proteomes showed that PLA significantly down-regulated (P < 0.05) the expression of hexokinase (HK), phosphofructokinase (PFK), a-ketoglutarate dehydrogenase (a-KGDH), adenylate kinase (ADK1), Cytochrome C oxidase and NADH dehydrogenase, and up-regulated (P < 0.05) the expression of mitochondrial ADP/ATP carrier proteins (AAC) and subunit IV (CCIO IV) in glycolysis, tricarboxylic acid cycle or oxidative phosphorylation metabolism. Following these findings, down-regulated HK and a-KGDH activity of aforementioned pathways was shown by enzyme activity assay, and regulated gene expression of ADK1, AAC, CCIO IV and NADH dehydrogenase was further confirmed by real-time quantitative PCR. Central carbon metabolomics showed that citric acid, cis-Aconitic acid, isocitric acid, alpha-Ketoglutaric acid, succinate, fumarate and malic acid of the tricarboxylic acid cycle metabolites were significantly down-regulated (P < 0.05), and ATP production by oxidative phosphorylation was also significantly reduced (P = 0.02), resulting in insufficient energy production. Thus, ROS levels increased by 141% of the control values and cytochrome C was released, resulting in gradual cell apoptosis. All data indicated that energy metabolism was the target of PLA against R. oryzae. This was the first study to show that energy metabolism could be the target of PLA against R. oryzae, which could provide a theoretical basis to study the mechanism of fungal inhibition.

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G protein-coupled receptors (GPCRs) are key regulatory proteins of immune cell function inducing signaling in response to extracellular (pathogenic) stimuli. Although unrelated, hydroxycarboxylic acid receptor 3 (HCA3) and GPR84 share signaling via Gαi/o proteins and the agonist 3-hydroxydecanoic acid (3HDec). Both receptors are abundantly expressed in monocytes, macrophages and neutrophils but have opposing functions in these innate immune cells. Detailed insights into the molecular mechanisms and signaling components involved in immune cell regulation by GPR84 and HCA3 are still lacking. Here, we report that GPR84-mediated pro-inflammatory signaling depends on coupling to the hematopoietic cell-specific Gα15 protein in human macrophages, while HCA3 exclusively couples to Gαi protein. We show that activated GPR84 induces Gα15-dependent ERK activation, increases intracellular Ca2+ and IP3 levels as well as ROS production. In contrast, HCA3 activation shifts macrophage metabolism to a less glycolytic phenotype, which is associated with anti-inflammatory responses. This is supported by an increased release of anti-inflammatory IL-10 and a decreased secretion of pro-inflammatory IL-1ß. In primary human neutrophils, stimulation with HCA3 agonists counteracts the GPR84-induced neutrophil activation. Our analyses reveal that 3HDec acts solely through GPR84 but not HCA3 activation in macrophages. In summary, this study shows that HCA3 mediates hyporesponsiveness in response to metabolites derived from dietary lactic acid bacteria and uncovers that GPR84, which is already targeted in clinical trials, promotes pro-inflammatory signaling via Gα15 protein in macrophages.

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Hydroxycarboxylic acid receptor 3 (HCA3) was recently identified in the genomes of humans and other hominids but not in other mammals. We examined the production of HCA3 ligands by Bifidobacterium spp. In addition to 4-hydroxyphenyllactic acid, phenyllactic acid (PLA), and indole-3-lactic acid (ILA), we found that LeuA was produced by Bifidobacterium as an HCA3 ligand. The four ligands produced were the mixtures of enantiomers, and D-ILA, D-PLA, and D-LeuA showed stronger activity of the HCA3 ligand than their respective L-isomers. However, there was no difference in AhR activity between the two ILA enantiomers. These results provide new insights into the HCA3 ligands produced by Bifidobacterium and suggest the importance of investigating the absolute stereo structures of these metabolites.

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Lactic acid bacteria (LAB) have been used for various food fermentations for thousands of years. Recently, LAB are receiving increased attention due to their great potential as probiotics for man and animals, and also as cell factories for producing enzymes, antibodies, vitamins, exopolysaccharides, and various feedstocks. LAB are safe organisms with GRAS (generally recognized as safe) status and possess relatively simple metabolic pathways easily subjected to modifications. However, relatively few studies have been carried out on LAB inhabiting plants compared to dairy LAB. Kimchi is a Korean traditional fermented vegetable, and its fermentation is carried out by LAB inhabiting plant raw materials of kimchi. Kimchi represents a model food with low pH and is fermented at low temperatures and in anaerobic environments. LAB have been adjusting to kimchi environments, and produce various metabolites such as bacteriocins, γ-aminobutyric acid, ornithine, exopolysaccharides, mannitol, etc. as products of metabolic efforts to adjust to the environments. The metabolites also contribute to the known health-promoting effects of kimchi. Due to the recent progress in multi-omics technologies, identification of genes and gene products responsible for the synthesis of functional metabolites becomes easier than before. With the aid of tools of metabolic engineering and synthetic biology, it can be envisioned that LAB strains producing valuable metabolites in large quantities will be constructed and used as starters for foods and probiotics for improving human health. Such LAB strains can also be useful as production hosts for value-added products for food, feed, and pharmaceutical industries. In this review, recent findings on the selected metabolites produced by kimchi LAB are discussed, and the potentials of metabolites will be mentioned.

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The modification of the mesoporous carbon sorbent with 3-phenylpropanoic acid was carried out in order to create preparations of complex, prolonged action, exhibiting detoxifying, antibacterial, and antifungal properties due to the applied modifier, which is capable of migrating into the solution and exhibiting its own biospecific properties. A technique was developed for fixing 3-phenylpropionic acid (PhPA) on a carbon support by its adsorption from solution. Three types of sorbents with various content of the modifier (PhPA) and the sorbent without modifier were studied. The sorption activity of new sorbents was studied using liquid-liquid extraction and gas chromatography-mass spectrometry methods on model experiments with plasma and aqueous additives of hydroxylated phenyl-containing acids (PhCAs) in various concentrations. The specific surface area was significantly changed for sorbent, modified with 1 × 10-3 mol/L of PhPA solution, and was 25% less than the area of unmodified sorbent. Potentially toxic biologically active hydroxylated PhCAs were used to create model solutions. The degrees of sorption of these compounds were close to 100%, except phenyllactic acid (over 80%). The sorbent without modifier and two sorbents with the lowest content of the modifier are considered to be more effective for the purification of the plasma from the hydroxylated PhCAs than the sorbent with the highest concentration of the modifier. Simultaneous adsorption of toxic metabolites from the bloodstream and desorption of beneficial ones can be used for a more subtle correction of the patient's condition.

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Nisin produced by certain Lactococcus lactis strains is commercially used in meat and dairy industries because of its effective antibacterial activity and food safety characteristics. It has been proved that the antibacterial activity could be enhanced when combined with other antimicrobial agents. In this study, we demonstrated that nisin and 3-phenyllactic acid (PLA) in combination displayed excellent combinational antibacterial activity against foodborne pathogens including S. xylosus and M. luteus. The potential application in food preservation was further verified via microbial analysis during the storage of meat and milk, and determination of strawberry rot rate. Scanning electron microscopy observation indicated a distinct mode of PLA with nisin, which may target at the dividing cell, contributing to their combinational antibacterial effect of nisin and PLA. Considering the positive results, a nisin-PLA co-producing strain was constructed based on the food-grade strain L. lactis F44, a nisin Z producer. By the knockout of two L-lactate dehydrogenase (LDH) and overexpression of D-LDH Y25A, the yield of PLA was significantly increased 1.77-fold in comparison with the wild type. Anti-bacterial assays demonstrated that the fermentation product of the recombinant strain performed highly effective antibacterial activity. These results provided a promising prospect for the nisin-PLA co-expressing L. lactis in food preservation on account of its considerable antibacterial activity and cost-effective performance.

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3-Phenyllactic acid (PLA) is a novel and natural antimicrobial compound. However, the concentration of PLA produced by native microbes was rather low. To enhance the production of PLA of Lactobacillus plantarum AB-1, the microcapsules of L. plantarum AB-1 cells with a high quorum-sensing capacity was established and investigated. In addition, the relation between PLA production and quorum sensing was further investigated and confirmed by adding the exogenous 4,5-dihydroxy-2,3-pentanedione (DPD, AI-2 precursor). The results indicated that the PLA production of L. plantarum AB-1 in microencapsulated cells (MC cells) was higher than that of the free cells, and the lactate dehydrogenase activity, autoinducer-2 (AI-2) levels and the relative expression of the luxS gene were also significantly increased in MC cells (P < 0.05). In addition, the cell growth, AI-2 levels and PLA production of L. plantarum AB-1 were also significantly promoted after adding 24 µM exogenous DPD. The results suggest that the PLA production of L. plantarum was partly regulated by the AI-2/LuxS system, and microencapsulation can increase the local AI-2 level and enhance QS capacity, which are beneficial to PLA production. The results may provide a new insight and experimental basis for the industrial production of PLA.

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Social insects are frequently observed in symbiotic association with bacteria that produce antimicrobial natural products as a defense mechanism. There is a lack of studies on the microbiota associated with stingless bees and their antimicrobial compounds. To the best of our knowledge, this study is the first to report the isolation of Paenibacillus polymyxa ALLI-03-01 from the larval food of the stingless bee Melipona scutellaris. The bacterial strain was cultured under different conditions and produced (L)-(-)-3-phenyllactic acid and fusaricidins, which were active against entomopathogenic fungi and Paenibacillus larvae. Our results indicate that such natural products could be related to colony protection, suggesting a defense symbiosis between P. polymyxa ALLI-03-01 and Melipona scutellaris.

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In this work, we explored the potential of 25 Lactobacillus plantarum strains isolated from cereals and milk-based products, testing characteristics related to antifungal activity and to nutritional quality. The tested strains demonstrated interesting beneficial traits, such as the ability to utilize fructo-oligosaccharides, prebiotic substances that help probiotic microorganisms to grow in the human gut, and to reduce phytate, an antinutrient present in cereal sector. Regarding mould inhibition, we highlighted the ability of the strains to inhibit Penicillium roqueforti, Mucor circinelloides and mycotoxinogenic moulds associated with cereal grains as Aspergillus flavus, A. niger, Fusarium verticillioides. Moreover, a moderate reduction of the bioavailability of aflatoxin AFB1 was detected. The selected L. plantarum strain ITEM 17215, showed a strong inhibitory ability towards fungal growth and was able to produce 1,2-dihydroxybenzene, benzoic acid, p-hydroxyphenyllactic acid and 3-phenyllactic acid. The latter compound, already described as efficient antifungal inhibitor, was the most abundant and its concentration was further increased by adding phenylalanine and phenylpyruvic acid in the growth medium. The metabolites produced by strain ITEM 17215 could also be related to the ability of the strain to induce cereal germination and promote plant growth. This aspect, not yet investigated in L. plantarum, could have interesting applications in the agro-food sector.

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Phenyllactic acid (PLA) is a novel antimicrobial compound. A novel NADH-dependent d-lactate dehydrogenase (d-LDH), named as LF-d-LDH0653, with high phenylpyruvate (PPA) reducing activity was isolated from Lactobacillus fermentum JN248. Its optimum pH and temperature were 8.0 and 50 °C, respectively. The Michaelis-Menten constant (Km ), turnover number (kcat ), and catalytic efficiency (kcat /Km ) for NADH were 1.20 mmol/L, 67.39 s-1 , and 56.16 (mmol/L)-1 s-1 , respectively. The (Km ), (kcat ), and (kcat /Km ) for phenylpyruvate were 1.68 mmol/L, 122.66 s-1 , and 73.01 (mmol/L)-1 s-1 , respectively. This enzyme can catalyze phenylpyruvate and the product presented excellent optical purity (enantioselectivity >99%). The results suggest that LF-d-LDH0653 is a promising biocatalyst for the efficient synthesis of optically pure d-PLA. PRACTICAL APPLICATION: A novel d-LDH with phenylpyruvate reducing activity has been isolated and identified. It could be used as a reference for improving the production of optically pure d-PLA. d-PLA has a potential for application as antimicrobial an agent in dairy industry and baking industry, pharmaceutical agent in medicine and cosmetics.

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3-Phenyllactic acid is an antimicrobial compound with broad-spectrum activity against various bacteria and fungus. The observed difference in pharmacological activity between optical isomeric 3-phenyllactic acid necessitates a method for enantioseparation. Chiral ligand exchange countercurrent chromatography was investigated for the enantioseparation of 3-phenyllactic acid with a synthesized chiral ligand. A two-phase solvent system was composed of n-butanol/hexane/water (0.4:0.6:1, v/v/v) to which N-n-dodecyl-l-hydroxyproline was added to the organic phase as chiral ligand and cupric acetate was added in the aqueous phase as a transitional metal ion. The influence factors were optimized by enantioselective liquid-liquid extraction. Baseline enantioseparation of racemic 3-phenyllactic acid by analytical high-speed countercurrent chromatography was achieved. The optical purities of enantiomeric 3-phenyllactic acid reached 99.0%, as determined by chiral high-performance liquid chromatography.

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3-Phenyllactic acid (PLA) is a broad-spectrum antimicrobial compound, produced by a wide range of lactic acid bacteria. A novel lactic acid bacteria strain with high PLA-producing ability, Pediococcus pentosaceus SK25, was isolated from traditional Chinese pickles. When grown in de Man, Rogosa, Sharpe broth at 30°C for 36h, this strain produced 135.6mg/L of PLA. Using this strain as starter for milk fermentation, 47.2mg/L of PLA was produced after fermentation for 12h. The PLA production was significantly improved by phenylalanine supplement, but was completely inhibited by tyrosine supplement.

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Thistle (Galactites tomentosa Moench.) honey organic extracts were obtained by headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE) and analyzed by gas chromatography (GC-FID and GC-MS) for the first time. Most abundant headspace compounds were terpenes, particularly linalool derivatives (hotrienol was predominant with a range of 38.6-57.5%). 3-Phenyllactic acid dominated in the solvent extracts (77.4-86.4%) followed by minor percentages of other shikimate pathway derivatives. After determination of an adequate enantioseparation protocol on Chirallica PST-4 column, the honey solvent extracts were analyzed by high-performance liquid chromatography (HPLC). The chiral analysis revealed high enantiomeric excess (>95%) of (-)-3-phenyllactic acid in all samples. Therefore, previous findings of chemical markers of thistle honey were extended, providing new potential for advanced chemical fingerprinting (optical pure chemical marker).

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