Production and characterization of antifungal compounds produced by Lactobacillus plantarum IMAU10014

Lactobacillus plantarum IMAU10014 was isolated from koumiss that produces a broad spectrum of antifungal compounds, all of which were active against plant pathogenic fungi in an agar plate assay. Two major antifungal compounds were extracted from the cell-free supernatant broth of L. plantarum IMAU10014. 3-phenyllactic acid and Benzeneacetic acid, 2-propenyl ester were carried out by HPLC, LC-MS, GC-MS, NMR analysis. It is the first report that lactic acid bacteria produce antifungal Benzeneacetic acid, 2-propenyl ester. Of these, the antifungal products also have a broad spectrum of antifungal activity, namely against Botrytis cinerea, Glomerella cingulate, Phytophthora drechsleri Tucker, Penicillium citrinum, Penicillium digitatum and Fusarium oxysporum, which was identified by the overlay and well-diffusion assay. F. oxysporum, P. citrinum and P. drechsleri Tucker were the most sensitive among molds.     

植物乳杆菌细菌素的研究与应用

植物乳杆菌细菌素不仅种类多,产生菌在发酵过程中还可产生良好的保健功效,因此成为研究的热点。本文对植物乳杆菌细菌素的种类、分子结构、抑菌机制及遗传控制做了较为详尽的介绍,并简要介绍了植物乳杆菌细菌素在食品、医药、饲料中的应用,为进一步研究植物乳杆菌细菌素提供了参考。     

Characterization of Rhamnosidases from Lactobacillus plantarum and Lactobacillus acidophilus

Lactobacilli are known to use plant materials as a food source. Many such materials are rich in rhamnose-containing polyphenols, and thus it can be anticipated that lactobacilli will contain rhamnosidases. Therefore, genome sequences of food-grade lactobacilli were screened for putative rhamnosidases. In the genome of Lactobacillus plantarum, two putative rhamnosidase genes (ram1_Lp and ram2_Lp) were identified, while in Lactobacillus acidophilus, one rhamnosidase gene was found (ramA_La). Gene products from all three genes were produced after introduction into Escherichia coli and were then tested for their enzymatic properties. Ram1_Lp, Ram2_Lp, and RamA_La were able to efficiently hydrolyze rutin and other rutinosides, while RamA_La was, in addition, able to cleave naringin, a neohesperidoside. Subsequently, the potential application of Lactobacillus rhamnosidases in food processing was investigated using a single matrix, tomato pulp. Recombinant Ram1_Lp and RamA_La enzymes were shown to remove the rhamnose from rutinosides in this material, but efficient conversion required adjustment of the tomato pulp to pH 6. The potential of Ram1_Lp for fermentation of plant flavonoids was further investigated by expression in the food-grade bacterium Lactococcus lactis. This system was used for fermentation of tomato pulp, with the aim of improving the bioavailability of flavonoids in processed tomato products. While import of flavonoids into L. lactis appeared to be a limiting factor, rhamnose removal was confirmed, indicating that rhamnosidase-producing bacteria may find commercial application, depending on the technological properties of the strains and enzymes.Lactobacilli such as Lactobacillus plantarum have been used for centuries to ferment vegetables such as cabbage, cucumber, and soybean (34). Fruit pulps, for instance, those from tomato, have also been used as a substrate for lactobacilli for the production of probiotic juices (38). Recently, the full genomic sequences of several lactobacilli have become available (1, 22). A number of the plant-based substrates for lactobacilli are rich in rhamnose sugars, which are often conjugated to polyphenols, as in the case of cell wall components and certain flavonoid antioxidants. Utilization of these compounds by lactobacilli would involve α-l-rhamnosidases, which catalyze the hydrolytic release of rhamnose. Plant-pathogenic fungi such as Aspergillus species produce the rhamnosidases when cultured in the presence of naringin, a rhamnosilated flavonoid (24, 26). Bacteria such as Bacillus species have also been shown to use similar enzyme activities for metabolizing bacterial biofilms which contain rhamnose (17, 40).In food processing, rhamnosidases have been applied primarily for debittering of citrus juices. Part of the bitter taste of citrus is caused by naringin (Fig. ​(Fig.1),1), which loses its bitter taste upon removal of the rhamnose (32). More recently, application of rhamnosidases for improving the bioavailability of flavonoids has been described. Human intake of flavonoids has been associated with a reduced risk of coronary heart disease in epidemiological studies (19). Food flavonoids need to be absorbed efficiently from what we eat in order to execute any beneficial function. Absorption occurs primarily in the small intestine (12, 37). Unabsorbed flavonoids will arrive in the colon, where they will be catabolized by the microflora, which is then present in huge quantities. Therefore, it would be desirable for flavonoids to be consumed in a form that is already optimal for absorption in the small intestine prior to their potential degradation. For the flavonoid quercetin, it has been demonstrated that the presence of rhamnoside groups inhibits its absorption about fivefold (20). A number of flavonoids which are present in frequently consumed food commodities, such as tomato and citrus products, often carry rutinoside (6-β-l-rhamnosyl-d-glucose) or neohesperidoside (2-β-l-rhamnosyl-d-glucose) residues (Fig. ​(Fig.1).1). Therefore, removal of the rhamnose groups from such flavonoid rutinosides and neohesperidosides prior to consumption could enhance their intestinal absorption. With this aim, studies were recently carried out toward the application of fungal enzyme preparations as a potential means to selectively remove rhamnoside moieties (16, 30).Open in a separate windowFIG. 1.Chemical structures of rhamnose-containing flavonoids from plants. Relevant carbon atoms in glycoside moieties are numbered. (1) Rutin (quercetin-3-glucoside-1→6-rhamnoside); (2) narirutin (naringenin-7-glucoside-1→6-rhamnoside); (3) naringin (naringenin-7-glucoside-1→2-rhamnoside); (4) p-nitrophenol-rhamnose.In view of the frequent occurrence of lactobacilli on decaying plant material and fermented vegetable substrates, one could anticipate that their genomes carry one or more genes encoding enzymes capable of utilizing rhamnosilated compounds. In the work reported here, we describe the identification of three putative rhamnosidase genes in lactobacillus genomes. We expressed these genes in Escherichia coli and characterized their gene products. The activities of all three lactobacillus rhamnosidases on flavonoids naturally present in tomato pulp were then assessed. One of the L. plantarum genes, which encoded the enzyme with the highest activity and stability in E. coli, was then also expressed in Lactococcus lactis, with the aim of investigating the potential use of such a recombinant organism to improve the bioavailability of fruit flavonoids and thus their efficacy in common foodstuffs.     

Characterization of plantaricin KW30, a bacteriocin produced by Lactobacillus plantarum

A protease-sensitive antibacterial substance, produced by a strain of Lactobacillus plantarum isolated from fermented corn, was classified as a bacteriocin and designated plantaricin KW30. The bacteriocin was stable to heat, pH and treatment with surfactants, and unaffected by α-amylase, lipase or lysozyme. Plantaricin KW30 exhibited a bactericidal and non-bacteriolytic mode of action against indicator cells, and inhibitory activity was limited to other lactobacilli. Maximum production was in MRS broth, and coincided with the onset of stationary phase under conditions of low pH and high cell numbers. In a complex medium bacteriocin production was enhanced by the presence of sodium acetate and Tween 80. Curing experiments gave derivatives that no longer produced the bacteriocin but retained immunity to it. These Bac derivatives showed the same plasmid pattern as the parent strain suggesting a chromosomal location for the genes for bacteriocin production.     

Citrate metabolism by Lactobacillus plantarum isolated from orange juice

The behaviour of Strains of Lactobacillus plantarum isolated from fermented orange juice and Lact. plantarum DSM 20174 was studied in the presence of citrate. When used as sole carbon source, citrate scarcely supported the growth of the bacteria. It was shown to enhance the growth of Lact. plantarum in glucose media. Under acid conditions (pH 4·0–5·0), 1 mol of citrate yielded 1·7 mol of acetate as sole major final metabolite with release of CO₂ in the gas phase.     

Characterization and purification of a new bacteriocin with a broad inhibitory spectrum produced by Lactobacillus plantarum lp 31 strain isolated from dry-fermented sausage

Aims:  Characterization and purification of a new bacteriocin produced by Lactobacillus plantarum LP 31 strain, isolated from Argentinian dry-fermented sausage.
Methods and Results:  Lactobacillus plantarum LP 31 strain produces an antimicrobial compound that inhibits the growth of food-borne pathogenic bacteria. It was inactivated by proteolytic enzymes, was stable to heat and catalase and exhibited maximum activity in the pH range from 5·0 to 6·0. Consequently, it was characterized as a bacteriocin. It was purified by RP (reverse-phase) solid-phase extraction, gel filtration chromatography and RP-HPLC. Plantaricin produced by Lact. plantarum LP 31 is a peptide with a molecular weight of 1558·85 Da as determined by Maldi-Tof mass spectrometry and contains 14 amino acid residues. It was shown to have a bactericidal effect against Pseudomonas sp., Staphylococcus aureus , Bacillus cereus and Listeria monocytogenes.
Conclusions:  The bacteriocin produced by Lact. plantarum LP 31 may be considered as a new plantaricin according to its low molecular weight and particular amino acid composition.
Significance and Impact of the Study:  In view of the interesting inhibitory spectrum of this bacteriocin and because of its good technological properties (resistance to heat and activity at acidic pH), this bacteriocin has potential applications as a biopreservative to prevent the growth of food-borne pathogens and food spoilage bacteria in certain food products.     

植物乳杆菌CMCC-P0002代谢产物中抑菌物质的研究

目的通过对一株植物乳杆菌代谢产物的抑菌作用以及该产物的某些物理特性的研究,为进一步发现可替代现有抗生素的抗菌物质奠定基础。方法首先利用低温离心和超滤法对植物乳杆菌培养后的上清液进行初步提取,用打孔法行抑菌试验,明确其对铜绿假单胞菌、大肠埃希菌、肺炎克雷伯菌和金黄色葡萄球菌等临床分离菌株的抑菌活性;并进一步经加热、调整pH及过氧化氢酶等处理上清液后,再验证其活性变化。结果植物乳杆菌的培养上清液对铜绿假单胞菌等多株临床分离菌株均具有一定的抑菌活性,且以pH在6以内时该物质抑菌活性较好,过氧化氢酶处理后或加热至100℃、30min,上清液的抑菌活性依然存在。结论在植物乳杆菌的培养上清液中存在着具有抑菌活性的物质,对从临床标本所分离的部分革兰阴性菌及革兰阳性菌有抑菌作用,该物质对热耐受,其活性受pH变化的影响。     

Characterization of undecaprenol kinase from Lactobacillus plantarum.

A membrane-bound undecaprenol kinase from Lactobacillus has been identified by observing the ATP-dependent phosphorylation of [14C]undercaprenol. The product of this reaction was shown to be [14C]undecaprenyl monophosphate by comparison of its chromatographic mobilities with authentic undecaprenyl monophosphate. It was shown that 32P from [gamma-32P]ATP was incorporated into undecaprenyl monophosphate. The kinase was partially solubilized by a variety of methods utilizing Triton X-100. Both the membrane-associated and solubilized enzymes required Mg2+, Triton X-100 and dimethylsulfoxide for activity. The enzyme preferentially phosphorylated the C34, C50 AND C 55 polyprenols. Geranylgeraniol (C20) and dolichol (C100), however, were utilized only 6% and 13% as well as undecaprenol, respectively. Despite the 8-fold difference in apparent V values, the apparent Km values for dolichol and undecaprenol were both 14 microM. The apparent Km for the nucleotide cosubstrate, ATP, was 2 mM. No other nucleoside triphosphate could substitute for ATP.     

New types of antimicrobial compounds produced by Lactobacillus plantarum

New types of antimicrobial compounds were identified in the culture filtrate of Lactobacillus plantarum VTT E-78076. Activity was detected in the low molecular mass fraction separated by gel chromatography. This fraction totally inhibited the growth of the Gram-negative test organism, Pantoea agglomerans (Enterobacter agglomerans) VTT E-90396. Characteristic compounds from this fraction were identified by GC/MS-analysis and the identification was confirmed using pure commercial reference compounds in identical chromatographs and in antimicrobial tests. The active fraction included benzoic acid (CAS 65-85-0), 5-methyl-2,4-imidazolidinedione (CAS 616-03-5, methylhydantoin), tetrahydro-4-hydroxy-4-methyl-2H- pyran-2-one (CAS 674-26-0, mevalonolactone) and 3-(2-methylpropyl)-2,5-piperazinedione (CAS 5845-67-0, cyclo(glycyl-L-leucyl)). These compounds in concentrations of 10 ppm inhibited growth of the test organism by 10-15% when acting separately, but 100% when all were applied together with 1% lactic acid. The inhibition was 40% by 1% lactic acid alone. The compounds were also active against Fusarium avenaceum (Gibberella avenacea) VTT-D-80147. The inhibition was 10-15% by separate compounds in concentrations of 10 ppm and maximally 20% in combinations. Fungal growth was not inhibited by lactic acid. Inhibition by unfractionated Lact. plantarum culture filtrate was 37% and by the low molecular mass fraction, 27%.     

Activity of plantaricin SA6, a bacteriocin produced by Lactobacillus plantarum SA6 isolated from fermented sausage

N. REKHIF, A ATRIH AND G. LEFEBVRE. 1995. Plantaricin SA6, a bacteriocin produced by Lactobacillus plantarum SA6, exhibited an inhibitory action against several mesophilic lactobacilli. It was stable at 90–100°C at pH 2–4 and it remained stable in the presence of several organic solvents, urea or β-mercaptoethanol. Plantaricin SA6 bound specifically to the cell surface of only plantaricin SA6-sensitive bacteria. The putative receptors are not destroyed by different hydrolytic enzymes added to the phosphate buffer. Plantaricin SA6 acted as a bactericidal agent lysing sensitive strains, that became more permeable to ortho-nitro-phenol-β-galactoside and lost their intracellular K⁺ ions and u.v.-absorbing materials. Both the adsorption and lethal action of plantaricin SA6 were maximal between pH 4 and 7, but the range of temperature tested (5–37βC) had no effect. Ions (of several salts such as MgCl₂) inhibited the binding of plantaricin SA6 and protected cells against bacteriocin action.     

Characterization of undecaprenyl pyrophosphate synthetase from Lactobacillus plantarum

A soluble long-chain polyprenyl pyrophosphate synthetase has been isolated from Lactobacillus plantarum and partially purified by DEAE-cellulose chromotography in 1% Triton X-100. This enzyme catalyzes the synthesis of polyprenyl pyrophosphate from farnesyl pyrophosphate and Δ³-isopentenyl pyrophosphate. The enzyme displays a requirement for farnesyl pyrophosphate and Triton X-series detergents. Treatment of polyprenyl pyrophosphate with C₅₅-isoprenyl pyrophosphate phosphatase (Micrococcus lysodeikticus) yielded polyprenyl monophosphate. Subsequent treatment of this product with a crude phosphatase from baker's yeast resulted in the formation of free polyprenol, which was characterized by thin layer chromatography and exhibited R_fs which corresponded to those of authentic undecaprenol isolated from Lactobacillus plantarum. Reverse phase cochromatography of the enzymically produced polyprenol and authentic undecaprenol indicated that the major enzymic products were undecaprenol and probably a longer chain polyprenol.     

Characterization of Lactobacillus plantarum from wine must by PCR species-specific and RAPD-PCR

AIMS: Physiological and molecular analysis such as PCR species-specific and randomly amplified polymorphic PCR (RAPD-PCR) have been used for typing of Lactobacillus plantarum strains from typical wine must. METHODS AND RESULTS: Phenotypic tests such as API 50CH and evaluation of D-L-lactate production from glucose were used to perform a preliminary characterization of lactobacilli. Furthermore, 18 strains of lactobacilli were analyzed by PCR species-specific oligonucleotides based on short sequences of the recA gene. CONCLUSIONS: Four strains were identified as belonging to the L. plantarum species and were further analysed by RAPD-PCR. The RAPD-PCR profiles were similar in all strains that had positive results for species-specific PCR, suggesting that the four L. plantarum strains were closely related. SIGNIFICANCE AND IMPACT OF THE STUDY: Using PCR species-specific as a preliminary screening test and then RAPD-PCR can be as considered the most reliable method of performing a rapid and correct typing of L. plantarum from wine must.     

不同分离源植物乳杆菌的群体基因组分析

【背景】植物乳杆菌(Lactobacillus plantarum)广泛存在于植物、乳制品、肉制品、哺乳动物和昆虫的肠道等多种生态环境中。【目的】探究不同分离源L. plantarum基因组与其所在环境是否存在潜在的联系。【方法】利用比较基因组学对126株分离自植物、乳制品、肉制品、果蝇及哺乳动物肠道和口腔等部位的L. plantarum菌株基因组进行系统发育分析和功能基因组分析,解析不同分离源菌株间的亲缘关系和进化历程。【结果】果蝇分离株的基因组大小显著高于植物、哺乳动物肠道、肉制品和乳制品分离株(P0.05),植物和哺乳动物肠道、口腔等部位与肉制品分离株的基因组大小和编码基因数量无显著差异(P0.05)。基于单拷贝基因串联和核心基因系统发育树分析均发现,果蝇分离株和乳制品分离株分别集中聚集分布在某一分支中,其余分离源均匀分布在各个分支中。附属基因分析结果与系统发育树分析结果一致。功能基因注释结果发现,果蝇分离株的环境特异性基因参与低聚果糖和几丁质代谢,乳制品分离株的环境特异性基因参与mazEF毒素-抗毒素系统和CRISPR系统。【结论】植物乳杆菌分离株为适应较为独特的果蝇和乳制品生境而发生了适应性进化。本研究为植物乳杆菌适应性进化提供了新见解,同时为解析菌株的进化历程提供了理论基础。     

Multi-spectrometric analyses of lipoteichoic acids isolated from Lactobacillus plantarum

Lipoteichoic acid is a major cell wall virulence factor of gram-positive bacteria. LTAs from various bacteria have differential immunostimulatory potentials due to heterogeneity in their structures. Although recent studies have demonstrated that LTA isolated from Lactobacillus plantarum (pLTA) has anti-inflammatory properties and is less inflammatory than LTAs from pathogenic bacteria, little is known about the structure of pLTA. In this study, high-field NMR spectra of the pLTA were compared with those of LTA from pathogenic bacterium, Staphylococcus aureus (aLTA). The 2D NMR results demonstrated that pLTA possesses α-linked hexose sugar substituents on the poly-glycerophosphate backbone instead of N-acetylglucosamine substituents, and unsaturated fatty acids in its glycolipids. The sugar substituents were revealed as an approximately 29:1 molar ratio of the glucose to galactose by HPAEC-PAD analysis. MALDI-TOF/TOF MS analyses identified the presence of unsaturated fatty acids in the glycolipid moieties of pLTA. In addition, the glycolipid structure was found to be composed of trihexosyl-diacyl- and/or trihexosyl-triacyl-glycerol ceramide units by means of unique fragment ions of the glycolipids. These results enabled us to elucidate the pLTA structure, which is distinctively different from canonical LTA structure, and suggest that the unique immunological property of pLTA might be caused by the pLTA structure.     

Parameters affecting the adsorption of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum 423 isolated from sorghum beer

Plantaricin 423 is bactericidal to logarithmic and stationary-phase cells of Enterococcus sp. HKLHS and L. sakei DSM 20017. Detection of extracellular DNA and beta-galactosidase suggests that the mode of action is most probably by destabilizing of the cell membrane. Adsorption of plantaricin 423 to target cells ranged from 17% for Streptococcus caprinus ATCC 700066 to 67% for Lactobacillus plantarum LMG 13556, Lactobacillus curvatus DF38, Listeria innocua LMG 13568 and Lactobacillus sakei DSM 20017. Treatment of Enterococcus sp. HKLHS and L. sakei DSM 20017 with Triton X-100, Triton X-114 and chloroform increased the adsorption of plantaricin.     

In vitro antifungal,probiotic and antioxidant properties of novel Lactobacillus plantarum K46 isolated from fermented sesame leaf

This study aimed to describe the diversity of antifungal lactic acid bacteria (LAB) in popular traditional Korean fermented food. A total of 22 LAB strains was selected and subjected to a monophasic identical approach using 16S rRNA gene sequence analysis. Antifungal LAB associated with fermented food was identified as Lactobacillus plantarum (9), Lactobacillus graminis (5), Lactobacillus pentosus (4), Lactobacillus sakei (2), Lactobacillus paraplantarum (1), and Leuconostoc mesenteroides subsp. mesenteroides (1). Novel Lactobacillus plantarum strain K46 exhibited comparatively better antifungal activity against several spoilage fungi, and was deposited in the Korean Collection for Type Cultures (KACC91758P). Antifungal substances from the spent medium in which K46 was cultivated were extracted with ethyl acetate. Antifungal activity was assessed using the broth micro dilution technique. Compounds were characterized based on infrared, ¹³C nuclear magnetic resonance (NMR), and ¹H NMR spectral data. The minimum inhibitory concentration (MIC) of the compounds against Aspergillus clavatus, Aspergillus oryzae, Penicillium chrysogenum and Penicillium roqueforti was 2.5 mg/mL and that against Aspergillus fumigatus, Aspergillus niger, Curvularia lunata and Gibberella moniliformis was 5.0 mg/mL. K46 was able to survive gastrointestinal conditions simulating the stomach and the duodenum passage with the highest percentage of hydrophobicity. In addition, its resistance to hydrogen peroxide and highest hydroxyl radical and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities, with inhibition rates of 43.53 % and 56.88 %, respectively, were to its advantage. An antimicrobial susceptibility pattern was an intrinsic feature of this strain, thus consumption does not represent a health risk to humans. The results showed the potential of K46 strain as an antifungal, probiotic and antioxidant culture, and hence it was determined to be suitable for application in functional foods.     

Characterization and partial purification of plantaricin LC74, a bacteriocin produced by Lactobacillus plantarum LC74

Summary Plantaricin LC74, a bacteriocin produced during the growth of Lactobacillus plantarum LC74 isolated from crude goat's milk, inhibited some other mesophilic lactobacilli. It was sensitive to high temperature (95°C) but was relatively stable at 30°C in acidic conditions, and also in the presence of various organic solvents, several detergents, urea or -mercaptoethanol. It was destroyed by proteases. By ultrafiltration plantaricin LC74 showed an apparent molecular mass smaller than 5 kDa. Plantaricin LC74 bounded non specifically to both sensitive and resistant Gram-positive bacteria and displayed a bacteriostatic effect. Its partial purification was obtained by ammonium sulphate precipitation followed by cationic exchange chromatography and hydrophobic interaction chromatography.     

Production of pediocin AcH by Lactobacillus plantarum WHE 92 isolated from cheese.

Among 1,962 bacterial isolates from a smear-surface soft cheese (Munster cheese) screened for activity against Listeria monocytogenes, six produced antilisterial compounds other than organic acids. The bacterial strain WHE 92, which displayed the strongest antilisterial effect, was identified at the DNA level as Lactobacillus plantarum. The proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action of the antilisterial compound produced by this bacterium suggested that it was a bacteriocin. Purification to homogeneity and sequencing of this bacteriocin showed that it was a 4.6-kDa, 44-amino-acid peptide, the primary structure of which was identical to that of pediocin AcH produced by different Pediococcus acidilactici strains. We report the first case of the same bacteriocin appearing naturally with bacteria of different genera. Whereas the production of pediocin AcH from P. acidilactici H was considerably reduced when the final pH of the medium exceeded 5.0, no reduction in the production of pediocin AcH from L. plantarum WHE 92 was observed when the pH of the medium was up to 6.0. This fact is important from an industrial angle. As the pH of dairy products is often higher than 5.0, L. plantarum WHE 92, which develops particularly well in cheeses, could constitute an effective means of biological combat against L. monocytogenes in this type of foodstuff.