Antifungal activity of phenyllactic acid against molds isolated from bakery products

Phenyllactic acid (PLA) has recently been found in cultures of Lactobacillus plantarum that show antifungal activity in sourdough breads. The fungicidal activity of PLA and growth inhibition by PLA were evaluated by using a microdilution test and 23 fungal strains belonging to 14 species of Aspergillus, Penicillium, and Fusarium that were isolated from bakery products, flours, or cereals. Less than 7.5 mg of PLA ml(-1) was required to obtain 90% growth inhibition for all strains, while fungicidal activity against 19 strains was shown by PLA at levels of < or = 10 mg ml(-1). Levels of growth inhibition of 50 to 92.4% were observed for all fungal strains after incubation for 3 days in the presence of 7.5 mg of PLA ml(-1) in buffered medium at pH 4, which is a condition more similar to those in real food systems. Under these experimental conditions PLA caused an unpredictable delaying effect that was more than 2 days long for 12 strains, including some mycotoxigenic strains of Penicillium verrucosum and Penicillium citrinum and a strain of Penicillium roqueforti (the most widespread contaminant of bakery products); a growth delay of about 2 days was observed for seven other strains. The effect of pH on the inhibitory activity of PLA and the combined effects of the major organic acids produced by lactic acid bacteria isolated from sourdough bread (PLA, lactic acid, and acetic acid) were also investigated. The ability of PLA to act as a fungicide and delay the growth of a variety of fungal contaminants provides new perspectives for possibly using this natural antimicrobial compound to control fungal contaminants and extend the shelf lives of foods and/or feedstuffs.     

Antifungal Activity of Phenyllactic Acid against Molds Isolated from Bakery Products

Phenyllactic acid (PLA) has recently been found in cultures of Lactobacillus plantarum that show antifungal activity in sourdough breads. The fungicidal activity of PLA and growth inhibition by PLA were evaluated by using a microdilution test and 23 fungal strains belonging to 14 species of Aspergillus, Penicillium, and Fusarium that were isolated from bakery products, flours, or cereals. Less than 7.5 mg of PLA ml⁻¹ was required to obtain 90% growth inhibition for all strains, while fungicidal activity against 19 strains was shown by PLA at levels of ≤10 mg ml⁻¹. Levels of growth inhibition of 50 to 92.4% were observed for all fungal strains after incubation for 3 days in the presence of 7.5 mg of PLA ml⁻¹ in buffered medium at pH 4, which is a condition more similar to those in real food systems. Under these experimental conditions PLA caused an unpredictable delaying effect that was more than 2 days long for 12 strains, including some mycotoxigenic strains of Penicillium verrucosum and Penicillium citrinum and a strain of Penicillium roqueforti (the most widespread contaminant of bakery products); a growth delay of about 2 days was observed for seven other strains. The effect of pH on the inhibitory activity of PLA and the combined effects of the major organic acids produced by lactic acid bacteria isolated from sourdough bread (PLA, lactic acid, and acetic acid) were also investigated. The ability of PLA to act as a fungicide and delay the growth of a variety of fungal contaminants provides new perspectives for possibly using this natural antimicrobial compound to control fungal contaminants and extend the shelf lives of foods and/or feedstuffs.     

青海湖裸鲤肠道乳酸菌多样性与抑菌活性

【目的】通过生理生化特性,结合16S r RNA基因序列分析研究青海湖裸鲤肠道乳酸菌分离株的多样性,并对这些代表株的抑菌活性进行初步探讨,以期筛选具有高效抑菌活性的鱼源益生菌。【方法】对分离的47株乳酸菌代表株进行p H、温度生长范围、耐盐性等生理生化特征检测,结合16S r RNA基因序列对已分离到的乳酸菌进行基因分型和菌种鉴定,采用牛津杯双层平板法检测乳酸菌代表株的抑菌活性。【结果】鉴定结果显示:23株为Lactobacillus fuchuensis(48.94%),12株为Lactobacillus curvatus(25.53%),3株为Leuconostoc fallax(6.38%),2株为Lactobacillus sakei(4.26%),2株为Weissella ceti(4.26%);2株为Lactococcus cremoris(4.26%),1株为Leuconostoc lactis(2.13%),1株为Weissella minor(2.13%),1株为Enterococcus devriesei(2.13%)。qz1217、qz1196、qz1220所在的A、B、C三组乳酸菌在5-50°C的温度范围内生长良好,qz1196、qz1220所在的B、C组在pH 3.0-10.0的范围内生长良好,几乎所有乳酸菌都具有耐6.5%盐浓度特性。13株乳酸菌菌株对6种病原菌都具有抑制作用。通过排除酸、过氧化氢实验,发现上清液仍然具有抑菌活性。对qz1251发酵液进行蛋白酶处理,抑菌活性消失,确定其抑菌物质属于蛋白类物质,是一种细菌素。【结论】青海湖裸鲤肠道附着乳酸菌的多样性为益生性乳酸菌的筛选提供优质资源及数据参考。     

Use of poly(lactic acid) amendments to promote the bacterial fixation of metals in zinc smelter tailings

The ability of poly(lactic acid) (PLA) to serve as a long-term source of lactic acid for bacterial sulfate reduction activity in zinc smelter tailings was investigated. Solid PLA polymers mixed in water hydrolyzed abiotically to release lactic acid into solution over an extended period of time. The addition of both PLA and gypsum was required for indigenous bacteria to lower redox potential, raise pH, and stimulate sulfate reduction activity in highly oxidized smelter tailings after one year of treatment. Bioavailable cadmium, copper, lead and zinc were all lowered significantly in PLA/gypsum treated soil, but PLA amendments alone increased the bioavailability of lead, nickel and zinc. Similar PLA amendments may be useful in constructed wetlands and reactive barrier walls for the passive treatment of mine drainage, where enhanced rates of bacterial sulfate reduction are desirable.     

Insight into the bovine milk peptide LPcin‐YK3 selection in the proteolytic system of Lactobacillus species

Antimicrobial peptides are class of small, positively charged peptides known for their broad‐spectrum antimicrobial activity. Antimicrobial activities for most antimicrobial peptides have largely remained elusive, particularly in the lactic acid bacteria. However, recently our investigation using LPcin‐YK3, an antimicrobial peptide from bovine milk, suggests that in vitro antimicrobial activity was reduced over 100‐fold compared with pathogenic bacteria. Additionally, for the structural study of how antimicrobial peptide undergoes its reaction at the proteolytic pathway of lactic acid bacteria based on degradation assay and propidium iodide staining, we performed molecular docking for interaction between oligopeptide‐binding protein A and LPcin‐YK3 peptide. Given that degradation related to the LPcin‐YK3 peptide in lactic acid bacteria proteolytic system, the inhibitory inactivity of LPcin‐YK3 against beneficial lactic acid bacteria strains may be one of the primary pharmacological properties of recombinant peptide discovered in bovine milk. These results provide structural and functional insights into the proteolytic mechanism and possibility as a putative substrate of oligopeptide‐binding protein A in respect of LPcin‐YK3 peptide.     

Safety and probiotic functionality of isolated goat milk lactic acid bacteria

Purpose

Lactic acid bacteria (LAB) are traditionally employed in the food industry. LAB strains from goat milk may also present probiotic potential, and it is fundamental to study the safety and functionality aspects which are desirable for their use in food. The objective of this study was to verify the probiotic potential of lactic bacteria isolated from goat milk.

Methods

The presence of safety-related virulence factors (hemolytic activity, gelatinase production, coagulase, and sensitivity to antibiotics) as well as functionality (exopolysaccharide (EPS) production, proteolytic activity, autoaggregation, gas production, survival in the gastrointestinal tract, and antimicrobial activity against bacteria that impair oral health) were determined.

Result

The selected LAB strains are safe against the evaluated parameters and have characteristics of possible probiotic candidates. Especially L. plantarum (DF60Mi) and Lactococcus lactis (DF04Mi) have potential to be added to foods because they have better resistance to simulated gastrointestinal conditions. In addition, they are isolated with already proven antimicrobial activity against Listeria monocytogenes, an important food-borne pathogen. DF60Mi was able to produce EPS (exopolysaccharides). LS2 and DF4Mi strains, both Lactococcus lactis subsp. lactis, demonstrated antimicrobial activity against S. mutans ATCC 25175, a recurrent microorganism in oral pathologies, mainly caries.

Conclusion

This study provides subsidies for future exploration of the potentialities of these LAB strains for both the development of new functional foods and for application in oral health.

     

Native and heterologous production of bacteriocins from gram-positive microorganisms

In nature, microorganisms can present several mechanisms for setting intercommunication and defense. One of these mechanisms is related to the production of bacteriocins, which are peptides with antimicrobial activity. Bacteriocins can be found in Gram-positive and Gram-negative bacteria. Nevertheless, bacteriocins produced by Gram-positive bacteria are of particular interest due to the industrial use of several strains that belong to this group, especially lactic acid bacteria (LAB), which have the status of generally recognized as safe (GRAS) microorganisms. In this work, we will review recent tendencies in the field of invention and state of art related to bacteriocin production by Gram-positive microorganism. Hundred-eight patents related to Gram-positive bacteriocin producers have been disclosed since 1965, from which 57% are related bacteriocins derived from Lactococcus, Lactobacillus, Streptococcus, and Pediococcus strains. Surprisingly, patents regarding heterologous bacteriocins production were mainly presented just in the last decade. Although the major application of bacteriocins is concerned to food industry to control spoilage and foodborne bacteria, during the last years bacteriocin applications have been displacing to the diagnosis and treatment of cancer, and plant disease resistance and growth promotion.     

Synergistic antimicrobial effect of nisin whey permeate and lactic acid on microbes isolated from fish

The antimicrobial activity of a combination of lactic acid and whey permeate fermented by a nisin-producing Lactococcus lactis strain was tested by the agar diffusion method using bacteria isolated from fish as test organisms. Lactic acid inhibited all bacterial strains studied, but nisin whey permeate inhibited Gram-positive bacteria only. The combination was more effective than lactic acid alone against Pseudomonas fluorescens and Staphylococcus hominis isolated from fish, and Pseudomonas aeruginosa ATCC9721 and Micrococcus luteus ATCC9341.     

Rapid screening method for the detection of antimicrobial substances

Bioluminescence is phenomenon where living organisms produce light and this production is directly dependent on metabolic activity of the organism. Genes encoding enzymes, luciferases, responsible for light production can be cloned into indicator strains, thus allowing sensitive detection of antimicrobial activity. This study utilized bacterial luciferase genes cloned into Staphylococcus aureus, Escherichia coli and Salmonella enterica serovar Typhimurium indicator strains and showed that the detection of antimicrobial activity can be obtained already in 2 h without laborious plate counting and overnight incubation. Indicator strains used in the study harboured luxAB genes responsible of producing light as well as luxCDE genes for synthesis of long-chain fatty aldehyde as substrate for light production. As a consequence, no exogenous aldehyde addition was needed allowing stable light production. Furthermore, the method was used for the detection of antimicrobial activity from lactic acid bacteria after the effect of organic acids was eliminated.     

The effect of prebiotics on production of antimicrobial compounds, resistance to growth at low pH and in the presence of bile, and adhesion of probiotic cells to intestinal mucus

AIMS: Screening of five bile salt-resistant and low pH-tolerant lactic acid bacteria for inhibitory activity against lactic acid bacteria and bacterial strains isolated from the faeces of children with HIV/AIDS. Determining the effect of prebiotics and soy milk-base on cell viability and adhesion of cells to intestinal mucus. METHODS AND RESULTS: Lactobacillus plantarum 423, Lactobacillus casei LHS, Lactobacillus salivarius 241, Lactobacillus curvatus DF 38 and Pediococcus pentosaceus 34 produced the highest level of antimicrobial activity (12,800 AU ml(-1)) when grown in MRS broth supplemented with 2% (m/v) dextrose. Growth in the presence of Raftilose Synergy1, Raftilose L95 and Raftiline GR did not lead to increased levels of antimicrobial activity. Cells grown in the presence of Raftilose Synergy1 took longer to adhere to intestinal mucus, whilst cells grown in the absence of prebiotics showed a linear rate of binding. CONCLUSIONS: A broad range of gram-positive and gram-negative bacteria were inhibited. Dextrose stimulated the production of antimicrobial compounds. Adhesion to intestinal mucus did not increase with the addition of prebiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: The strains may be incorporated in food supplements for HIV/AIDS patients suffering from gastro-intestinal disorders.     

Vaginal lactic acid bacteria in the mare: evaluation of the probiotic potential of native Lactobacillus spp. and Enterococcus spp. strains

Lactic acid bacteria (LAB) are important members of the human vaginal microbiota and their presence is considered beneficial. However, little is known about native vaginal bacteria in other animal species such as the horse. The aim of this work was to quantify the vaginal lactic acid bacteria and lactobacilli of mares and to establish if selected equine vaginal lactic acid bacteria, particularly Lactobacillus and Enterococcus spp. strains, could exhibit potential as probiotics. The vaginal lactic acid bacteria and lactobacilli of 26 mares were quantified by plate counts. Five strains (three Lactobacillus spp. and two Enterococcus spp.) were characterised and adhesion to vaginal epithelial cells, antimicrobial activity and ability to form biofilms were evaluated. Lactic acid bacteria were recovered from the 26 samples and lactobacilli counts were detected in 18 out of 26 mares (69%). Probiotic properties tested in this study varied among the isolates and showed promising features for their use as equine probiotics.     

Antimicrobial activity against Shigella sonnei and probiotic properties of wild lactobacilli from fermented food

Four lactobacilli strains (Lactobacillus paracasei subp. paracasei M5-L, Lactobacillus rhamnosus J10-L, Lactobacillus casei Q8-L and L. rhamnosus GG (LGG), were systematically assessed for the production of antimicrobial substances active towards Shigella sonnei, Escherichia coli and Salmonella typhimurium. Agar-well assay showed that the four lactobacilli strains displayed strong antibacterial activity towards S. sonnei. The nature of antimicrobial substances was also investigated and shown to be dependent on the production of organic acids, in particular the lactic acid. Time-kill assay showed that the viability of the S. sonnei was decreased by 2.7-3.6logCFU/ml after contact with CFCS (cell-free culture supernatants) of four lactobacilli for 2h, which confirmed the result of the agar-well assay. Further analysis of the organic acid composition in the CFCS revealed that the content of lactic acid range from 227 to 293mM. In addition, the aggregations properties, adherence properties and tolerance to simulated gastrointestinal conditions were also investigated in vitro tests. The result suggested that the M5-L, J10-L and Q8-L strains possess desirable antimicrobial activity towards S. sonnei and probiotic properties as LGG and could be potentially used as novel probiotic strains in the food industry.     

Antimicrobial activity of lactobacilli and streptococci

Of nine strains of lactic acid bacteria commonly used as starter cultures for the dalry industry and ensiling, six (Lactobacillus bulgaricus, L. casei, L. acidophilus CH=5, L. plantarum, Streptococcus latis and Strep. taecium) had antibiotic activity. Gram-positive bacteria were more sensitive than Gram-negative bacteria to the antibiotics. The most sensitive strain of Staphylococcus aureus was used as a target micro-organism for the characterization of the antimicrobial substance. The cultures of Streptococcus faecium and L. plantarum gave the most intense antimicrobial activity. Adding CaCO₃ to the medium (to bind accumulated lactic acid) increased the antibiotic activity of the lactic acid bacteria.The authors are with the Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 41000 Zagreb, Yugoslavia.     

Efficient conversion of phenylpyruvic acid to phenyllactic acid by using whole cells of Bacillus coagulans SDM

Background

Phenyllactic acid (PLA), a novel antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi, can be produced by many microorganisms, especially lactic acid bacteria. However, the concentration and productivity of PLA have been low in previous studies. The enzymes responsible for conversion of phenylpyruvic acid (PPA) into PLA are equivocal.

Methodology/Principal Findings

A novel thermophilic strain, Bacillus coagulans SDM, was isolated for production of PLA. When the solubility and dissolution rate of PPA were enhanced at a high temperature, whole cells of B. coagulans SDM could effectively convert PPA into PLA at a high concentration (37.3 g l⁻¹) and high productivity (2.3 g l⁻¹ h⁻¹) under optimal conditions. Enzyme activity staining and kinetic studies identified NAD-dependent lactate dehydrogenases as the key enzymes that reduced PPA to PLA.

Conclusions/Significance

Taking advantage of the thermophilic character of B. coagulans SDM, a high yield and productivity of PLA were obtained. The enzymes involved in PLA production were identified and characterized, which makes possible the rational design and construction of microorganisms suitable for PLA production with metabolic engineering.     

沙棘根瘤内生细菌中抑菌促生菌株的筛选和鉴定

【背景】植物内生细菌可产生具有抑菌和促生活性的物质,既能抑制植物病原菌对寄主植物的侵染,也能促进植物的生长。沙棘根瘤内生细菌是根瘤内除共生固氮的弗兰克氏菌外,与沙棘共生的一大类微生物。研究具有抑菌和促生活性的植物内生菌,可为微生物菌肥的研究提供理论基础。【目的】筛选具有优良抑菌和促生活性的沙棘根瘤内生细菌,初步研究其抑菌和促生活性,并对菌株进行鉴定。【方法】采用双层琼脂法、琼脂扩散法、双层平板对峙法、牛津杯法进行沙棘根瘤拮抗性内生细菌的筛选。选取抑菌活性较高的内生细菌,分别采用Salkowski比色法、ChromeAzurolS(CAS)平板检测法和钼锑抗比色法进行产吲哚乙酸、铁载体及溶磷能力的测定。采用发酵液灌根法测定沙棘根瘤内生细菌SR308对黄瓜促生作用的盆栽效果。通过形态和培养特征、生理生化试验及16S rRNA基因序列分析法对菌株TT201和SR308进行鉴定。【结果】从131株沙棘根瘤内生细菌中筛选出9株具有较强抑菌活性的内生细菌,其中菌株TT201抑菌性最佳、抑菌谱广;菌株SR308的促生活性最好,其发酵液对黄瓜的生长具有较强的促进作用。对具有较强抑菌和促生活性的菌株TT201和SR308进行鉴定的结果表明,菌株TT201为侧孢短芽孢杆菌(Brevibacilluslaterosporus),菌株SR308为蕈状芽孢杆菌(Bacillusmycoides)。【结论】获得2株具有优良抑菌和促生活性的沙棘根瘤内生细菌,为进一步开发微生物农药及菌肥提供了资源。     

Promotion of polylactide degradation by ammonia under hyperthermophilic anaerobic conditions

The objective of this study was to evaluate the promotion effect of ammonia on the biodegradation of polylactide (PLA) under hyperthermophilic (80 °C) and thermophilic (55 °C) anaerobic condition. The results showed that PLA was transformed to lactic acid under hyperthermophilic conditions, but that the transformation ratio was negligible under thermophilic conditions. The hydrolysis process can be markedly increased with ammonia addition and microorganism activity. The maximum transformation ratios of the two kinds of PLA used in this study were 65.2% and 51.8%, respectively, with ammonia addition of 4 g N/L over 3 days treatment of anaerobic sludge. After the hyperthermophilic pretreatment, the hydrolysis products were converted to methane by methanogens under the thermophilic and anaerobic conditions. The final methane conversion ratios of the two kinds of PLA after 22 days treatment were 81.8% and 77.0%, respectively.     

The antimicrobial activity of lactic acid bacteria from fermented maize (kenkey) and their interactions during fermentation

A total of 241 lactic acid bacteria belonging to Lactobacillus plantarum, Pediococcus pentosaceus, Lactobacillus fermentum/reuteri and Lactobacillus brevis from various processing stages of maize dough fermentation were investigated. Results indicated that each processing stage has its own microenvironment with strong antimicrobial activity. About half of the Lact. plantarum and practically all of the Lact. fermentum/reuteri investigated were shown to inhibit other Gram-positive and Gram-negative bacteria, explaining the elimination of these organisms during the initial processing stages. Further, widespread microbial interactions amounting to 85% to 18% of all combinations tested were demonstrated amongst lactic acid bacteria within the various processing stages, i.e. raw material, steeping, 0 h and 48 h of fermentation, explaining the microbial succession taking place amongst lactic acid bacteria during fermentation. The antimicrobial effect was explained by the combined effect of acids, compounds sensitive to proteolytic enzymes and other compounds with antimicrobial activity with the acid production being the most important factor.
The pattern of antimicrobial factors was not species-specific and the safety and storage stability of fermented maize seem to depend on a mixed population of lactic acid bacteria with different types of antimicrobial characteristics. This means that introduction of pure cultures as starters may impose a risk to the product.     

3-Phenyllactic acid,a root-promoting substance isolated from Bokashi fertilizer,exhibits synergistic effects with tryptophan

Bokashi fertilizer, an organic fertilizer made of plant residue, has been used in Japan not only to fertilize plants but to regulate their growth. Lactic acid bacteria have been found to play an important role in the fermentation process of Bokashi, but the relationship between these bacteria and plant growth activity has not been clarified. Using the adzuki rooting assay, this study identified 3-phenyllactic acid (PLA) produced by lactic acid bacteria as a root promoting compound in Bokashi. PLA showed synergistic effect with tryptophan, but no stem elongation activity. Lactic acid bacteria produced equal quantities of the L- and D-forms of PLA, which have similar root promoting activity. PLA did not significantly affect the amount of endogenous indole-3-acetic acid (IAA), although the chemical structure of PLA is highly similar to that of L-2-aminooxy-3-phenypropionic acid (L-AOPP), which inhibits IAA biosynthesis. These results indicate that the root promoting activity of PLA is not simply due to its increase in the amount of active auxin.     

3-Phenyllactic acid is converted to phenylacetic acid and induces auxin-responsive root growth in Arabidopsis plants

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 β-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.     

Microbial production of lactic acid: the latest development

Lactic acid is an important platform chemical for producing polylactic acid (PLA) and other value-added products. It is naturally produced by a wide spectrum of microbes including bacteria, yeast and filamentous fungi. In general, bacteria ferment C5 and C6 sugars to lactic acid by either homo- or hetero-fermentative mode. Xylose isomerase, phosphoketolase, transaldolase, l- and d-lactate dehydrogenases are the key enzymes that affect the ways of lactic acid production. Metabolic engineering of microbial strains are usually needed to produce lactic acid from unconventional carbon sources. Production of d-LA has attracted much attention due to the demand for producing thermostable PLA, but large scale production of d-LA has not yet been commercialized. Thermophilic Bacillus coagulans strains are able to produce l-lactic acid from lignocellulose sugars homo-fermentatively under non-sterilized conditions, but the lack of genetic tools for metabolically engineering them severely affects their development for industrial applications. Pre-treatment of agriculture biomass to obtain fermentable sugars is a pre-requisite for utilization of the huge amounts of agricultural biomass to produce lactic acid. The major challenge is to obtain quality sugars of high concentrations in a cost effective-way. To avoid or minimize the use of neutralizing agents during fermentation, genetically engineering the strains to make them resist acidic environment and produce lactic acid at low pH would be very helpful for reducing the production cost of lactic acid.