Ensiling whole-crop wheat and corn in large containers with Lactobacillus plantarum and Lactobacillus buchneri

The effect of applying Lactobacillus buchneri, alone or in combination with Lactobacillus plantarum, at ensiling, on the aerobic stability of wheat and corn silages was studied in 50-l plastic containers. Treatments comprised control (no additives), L. plantarum, L. buchneri and a combination of L. plantarum+L. buchneri. After 3 months of storage, the wheat silages treated with L. buchneri had higher acetic acid contents than the control or L. plantarum-treated silages, and were free of mold, whereas the top layers of the control or L. plantarum-treated silages were moldy. In an aerobic stability test the L. buchneri-treated silages were stable, whereas those treated with L. plantarum deteriorated. In the corn silages the effects of L. buchneri were not as clear and the top layer was moldy in all silages. However, L. buchneri also improved the aerobic stability of the corn silage, as indicated by lower yeast numbers, less CO₂ production and stable pH. It is concluded that L. buchneri has a potential as a silage additive that protects the silage upon aerobic exposure. The 50-l plastic containers can serve as an appropriate model to test silage additives before conducting full-scale farm experiments. Journal of Industrial Microbiology & Biotechnology (2002) 28, 7–11 DOI: 10.1038/sj/jim/7000207 Received 17 April 2001/ Accepted in revised form 25 August 2001     

Integration and expression of alpha-amylase and endoglucanase genes in the Lactobacillus plantarum chromosome

A commercial grass silage starter strain of Lactobacillus plantarum was transformed by high-frequency electroporation with plasmids containing an alpha-amylase gene from Bacillus stearothermophilus and an endoglucanase gene from Clostridium thermocellum. Both genes were expressed from their native regulatory signals, and active enzymes were found in the supernatant. However, the segregational stability of the transforming plasmids was rather low. Therefore, the transforming genes were inserted in the L. plantarum chromosome by means of single homologous recombination. In the majority of the transformants, this led to extremely stable segregation and expression of the transforming genes, without generating secondary mutations in the host. Increased selective pressure led to tandem amplification of the transforming DNA. The transformed strains demonstrated the ability of L. plantarum to express heterologous gene products; they can be used to detect the inoculum in silage ecology studies; and they demonstrate the feasibility of engineering truly cellulolytic silage starter bacteria.     

Integration and expression of alpha-amylase and endoglucanase genes in the Lactobacillus plantarum chromosome.

A commercial grass silage starter strain of Lactobacillus plantarum was transformed by high-frequency electroporation with plasmids containing an alpha-amylase gene from Bacillus stearothermophilus and an endoglucanase gene from Clostridium thermocellum. Both genes were expressed from their native regulatory signals, and active enzymes were found in the supernatant. However, the segregational stability of the transforming plasmids was rather low. Therefore, the transforming genes were inserted in the L. plantarum chromosome by means of single homologous recombination. In the majority of the transformants, this led to extremely stable segregation and expression of the transforming genes, without generating secondary mutations in the host. Increased selective pressure led to tandem amplification of the transforming DNA. The transformed strains demonstrated the ability of L. plantarum to express heterologous gene products; they can be used to detect the inoculum in silage ecology studies; and they demonstrate the feasibility of engineering truly cellulolytic silage starter bacteria.     

Influence of the inoculum size ofLactobacillus plantarum on the competition withEnterobacter cloacae

Summary The size of the inoculum ofLactobacillus plantarum or its natural density, appears to be of predominant importance in the exclusion ofEnterobacter cloacae in mixed fermentations, such as ensilage. In a liquid medium, simulating adverse silage conditions, an initial density ofL. plantarum at least twice that ofE. cloacae was found necessary in order to obtain a succesful silage.     

Vector-free cloning of a bacterial endo-1,4-β-glucanase in Lactobacillus plantarum and its effect on the acidifying activity in silage: Use of recombinant cellulolytic Lactobacillus plantarum as silage inoculant

In this research, the advantage of use of cellulolytic recombinant Lactobacillus plantarum as microbial inoculants for alfalfa silage fermentation was evaluated. To such purpose, two L. plantarum strains, one (L. plantarum Lp80) currently commercialised and the other (L. plantarum B41) suitable as silage microbial additive, were genetically modified by integration of celA gene, encoding an alkaline endo-1,4--glucanase from Bacillus sp., in the chromosome, by means of a vector-free cloning technique. The heterologous gene was cloned in two fashions: preceded by two promoters (AC1 modification) or in translational coupling with a partial upstream ORF (AC2 modification). Therefore two different genetically modified organisms (GMOs) per each wild-type (WT), producing 43–59 U/l cellulase in 16 h, were examined. Thirty-five micro-ensiling experiments were carried out by inoculating the WT or the derived GMOs. L. plantarum B41AC1 cellulolytic clone exhibited significantly increased acidification capacity in silage samples incubated at 37°C. No advantage of use was evident for the other GMOs.     

不同来源植物乳杆菌基因组结构和功能差异的比较研究

[目的] 本试验研究不同来源植物乳杆菌（Lactobacillus plantarum）基因特点以及在不同环境下其基因多样性,探究2株L.plantarum A8和P9在肠道生境及植物表面适应性的异同,为优良菌株的开发提供理论基础。[方法] 本研究对从动物肠道和植物表面分离获得的L.plantarum A8和L.plantarum P9的基因组进行分析,利用第二代测序技术（NextGeneration Sequencing,NGS）,基于Illumina NovaSeq测序平台,同时利用第三代单分子测序技术,基于PacBio Sequel测序平台,对L.plantarum A8和L.plantarum P9进行测序。采用Carbohydrate-active enzymes（CAZy）、Koyto encyclopedia of genes and genomes（KEGG）和Clusters of orthologous genes（COG）数据库对基因组进行功能注释;采用CGView软件绘制菌株的基因组环形图谱。应用比较基因组学与已经公开发表的其他L.plantarum基因组进行比较分析。[结果] 由研究可知L.plantarum A8和L.plantarum P9基因组大小存在差异,通过构建系统发育树发现2株菌与其他来源的L.plantarum分在同一分支,并且L.plantarum P9与母乳来源的L.plantarum WLPL04菌株距离最近,而L.plantarum A8与L.paraplantarum DSM10667距离最近。通过基因家族分析可知,2株菌共有基因为2643个,其中包括一些抗应激蛋白如热休克蛋白、冷休克蛋白。L.plantarum A8和P9独特基因分别为321和336个,L.plantarum A8中独特基因主要参与DNA复制、ABC转运系统（ABC transfer system）、PTS系统（phosphotransferase system）、磺酸盐转运系统、氨基酸生物合成等代谢通路;L.plantarum P9的独特基因以参与碳水化合物的运输和代谢基因居多,例如rpiA基因、lacZ基因、FruA基因等。[结论] 通过比较基因组学方法解析L.plantarum的基因组信息,发现动物肠道来源的L.plantarum具有较好的氨基酸转运能力,植物表面附着的L.plantarum菌株具有较好碳水化合物利用能力,从而为益生菌的开发与利用提供理论依据。     

Quorum-Sensing Regulation of Constitutive Plantaricin by Lactobacillus plantarum Strains under a Model System for Vegetables and Fruits

This study aimed at investigating the regulatory system of bacteriocin synthesis by Lactobacillus plantarum strains in vegetables and fruits in a model system. Sterile and neutralized cell-free supernatant (CFS) from L. plantarum strains grown in MRS broth showed in vitro antimicrobial activities toward various indicator strains. The highest activity was that of L. plantarum C2. The antimicrobial activity was further assayed on vegetable and fruit agar plates (solid conditions) and in juices (liquid conditions). A regulatory mechanism of bacteriocin synthesis via quorum sensing was hypothesized. The synthesis of antimicrobial compounds seemed to be constitutive under solid conditions of growth on vegetable and fruit agar plates. In contrast, it depended on the size of the inoculum when L. plantarum C2 was grown in carrot juice. Only the inoculum of ca. 9.0 log CFU ml⁻¹ produced detectable activity. The genes plnA, plnEF, plnG, and plnH were found in all L. plantarum strains. The genes plnJK and plnN were detected in only three or four strains. Reverse-phase high-performance liquid chromatography purification and mass spectrometry analysis revealed the presence of a mixture of eight peptides in the most active fraction of the CFS from L. plantarum C2. Active peptides were encrypted into bacteriocin precursors, such as plantaricins PlnJ/K and PlnH and PlnG, which are involved in the ABC transport system. A real-time PCR assay showed an increase in the expression of plnJK and plnG during growth of L. plantarum C2 in carrot juice.     

Assessment of Pediococcus acidilactici as a Potential Silage Inoculant

Eighteen Pediococcus strains were screened for their potential as silage inoculants. Pediococcus acidilactici G24 was found to be the most suitable, exhibiting a short lag phase on both glucose and fructose, a rapid rate of acid production, a high sugar-to-lactate conversion efficiency, no detectable breakdown of proteins or lactic acid, and the ability to grow within a broad range of pH and temperature. When tested in laboratory silos using grass with a water-soluble carbohydrate content of 24 g/kg of aqueous extract, P. acidilactici G24 stimulated the natural Lactobacillus plantarum population and accelerated the rates of lactic acid production and pH decrease. After 6 days of fermentation, the inoculated silage exhibited a 12% decrease in ammonia nitrogen and an 11% increase in crude protein levels compared with uninoculated controls. The use of an L. plantarum inoculant at a rate of 10⁴ bacteria per g of grass in conjunction with P. acidilactici G24 produced no additional beneficial effect. Inoculation of grass with a water-soluble carbohydrate level of 8 g/kg of aqueous extract with P. acidilactici G24 led to no acceleration in the rate of L. plantarum growth or pH decrease. However, after 7 days of fermentation the inoculated silage had a 14% lower ammonia nitrogen protein content than did uninoculated controls. The results suggest that P. acidilactici G24 may be useful as a silage inoculant for crops with a sufficiently high water-soluble carbohydrate level.     

Effects of <Emphasis Type="Italic">Lactobacillus buchneri</Emphasis> and <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> on fermentation,aerobic stability,bacteria diversity and ruminal degradability of alfalfa silage

Silages are important feedstuffs. Homofermentative lactic acid bacterial inoculants are often used to control silage fermentation. However, some research pointed out those homofermentative lactic acid bacteria (LAB) impaired the aerobic stability of wheat, sorghum, and corn silages. Adding heterofermentative LAB can produce more acetic acid, thereby stabilizing silages during aerobic exposure. Alfalfa is difficult to ensile. The present work was to study the effects of L. buchneri (heterofermentative LAB), alone or in combination with L. plantarum (homofermentative LAB) on the fermentation, aerobic stability, bacteria diversity and ruminal degradability of alfalfa silage. After 90 days ensiling, the pH, NH₃-N/TN, butyric acid content and molds counts of control were the highest. The inoculated silages had more lactic acid, acetic acid content and more lactic acid bacteria than the control. Inoculating LAB inhibited harmful microorganisms, such as Enterobacterium and Klebsiella pneumoniae. The L. buchneri + L. plantarum-inoculated silage had more acetic acid and less yeasts than other three treatments (P < 0.05), and lower NH₃-N/TN than control (P < 0.05). The CO₂ production of L. buchneri + L. plantarum-inoculated silage was less than that of L. plantarum-inoculated silage (P < 0.05). Inoculating LAB in alfalfa silages can decrease pH, increase the production of lactic and acetic acids, reduce the number of yeasts and molds, and inhibit Enterobacterium and K. pneumoniae. Inoculating with L. buchneri or L. buchneri + L. plantarum can improve aerobic stability of alfalfa silages. A combination of L. buchneri and L. plantarum is preferable because it enhanced alfalfa silage quality and aerobic stability.     

Cloning and characterization of the lactate dehydrogenase genes from<Emphasis Type="Italic">Lactobacillus</Emphasis> sp. RKY2

Lactic acid is an environmentally benign organic acid that could be used as a raw material for biodegradable plastics if it can be inexpensively produced by fermentation. Two genes (IdhL andIdhD) encoding the L-(+) and D-(−) lactate dehydrogenases (L-LDH and D-LDH) were cloned fromLactobacillus sp., RKY2, which is a lactic acid hyper-producing bacterium isolated from Kimchi. Open reading frames ofIdhL for andIdhD for the L and D-LDH genes were 962 and 998 bp, respectively. Both the L(+)- and D(−)-LDH proteins showed the highest degree of homology with the L- and D-lactate dehydrogenase genes ofLactobacillus plantarum. The conserved residues in the catalytic activity and substrate binding of both LDHs were identified in both enzymes.     

High level expression of a heterologous protein in Lactobacillus plantarum and its effect on the persistence of the recombinant strain in silage

Summary This paper reports the high level expression of the Staphylococcus aureus cat gene in Lactobacillus plantarum using the expression vector pMTL500F. When the recombinant strain of L. plantarum was grown in pure culture, CAT contributed 1.4% of the total soluble cell protein. The recombinant strain of L. plantarum continued to express a high level of CAT when inoculated into silage, the heterologous protein constituting up to 1.75% of the total soluble cell protein. The recombinant L. plantarum strain was still able to survive and proliferate when inoculated into silage, despite its additional metabolic load.     

Expression of Bacillus subtilis levanase gene in Lactobacilus plantarum and Lactobacillus casei

Two Lactobacillus-Escherichia coli shuttle vectors, harbouring the levanase gene from Bacillus subtilis under the control of its own promoter (pLPEW1) or behind the E.coli tac promoter (pESIEW2), were constructed. Lactobacillus plantarum showed the same growth characteristics on selective plates and in liquid media containing inulin, after transformation with either pLPEW1 or pESIEW2. L. plantarum transformed with pLPEW1 could be selected on inulin plates, indicating that levanase expression can be used as a food-grade selection system for Lactobacillus. Lactobacillus casei grew faster in inulin-containing medium than L. plantarum after transformation with pESIEW2, but did not grow when harbouring pLPEW1. Inulin-degrading activities of 90 mU/ml were found in culture medium of L. plantarum containing pLPEW1 or pESIEW2, and of 500 mU/ml in medium of L. casei (pESIEW2). Addition of 1 mMm isopropyl -d-thiogalactoside to the culture medium had no effect on growth and levanase expression in L. plantarum (pESIEW2) and L. casei (pESIEW2) strains. Levanase produced by L. casei (pESIEW2) has a size of 75 kDa and 72 kDa, corresponding to that of unprocessed and mature B. subtilis levanase, respectively, suggesting that the protein produced is recognized and processed by a signal peptidase.     

Different lactic acid bacteria and their combinations regulated the fermentation process of ensiled alfalfa: ensiling characteristics,dynamics of bacterial community and their functional shifts

The objectives of this study were to investigate the adaptation and competition of Lactobacillus plantarum, Pediococcus pentosaceus and Enterococcus faecalis inoculated in alfalfa silage alone or in combination on the fermentation quality, dynamics of bacterial community, and their functional shifts using single-molecule real-time (SMRT) sequencing technology. Before ensiling, alfalfa was inoculated with L. plantarum (Lp), P. pentosaceus (Pp), E. faecalis (Ef) or their combinations (LpPp, LpEf, LpPpEf) and sampled at 1, 3, 7, 14 and 60 days. After 60-days fermentation, the Lp-, Pp- and LpPp-inoculated silages had lower pH but greater concentrations of lactic acid were observed in Pp, LpEf and LpPpEf-inoculated silages. The inoculants altered the keystone taxa and the bacterial community dynamics in different manners, where L. plantarum, Weissella cibaria and L. pentosaceus dominated the bacterial communities after 14 days-fermentation in all treatments. The silages with better fermentation quality had simplified bacterial correlation structures. Moreover, different inoculants dramatically changed the carbohydrate, amino acid, energy, nucleotide and vitamin metabolism of bacterial communities during ensiling. Results of the current study indicate that effect of different inoculants on alfalfa silage fermentation was implemented by modulating the succession of bacterial community, their interactions and metabolic pathways as well during ensiling.     

Cloning of Lactobacillus plantarum IAM 12477 lysine biosynthetic genes encoding functional aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase, and dihydrodipicolinate reductase

Summary The lysine biosynthetic genes asd, dapA, and dapB, encoding aspartate semialdehyde dehydrogenase (ASADH), dihydrodipicolinate synthase (DHPS), and dihydrodipicolinate reductase (DHPR), respectively, have been cloned from Lactobacillus plantarum IAM 12477 by heterologous complementation to Escherichia coli mutants. The amino acid sequences of the cloned genes showed considerable similarities to the corresponding protein from other gram-positive bacteria. We identified the amino acids that correspond to key catalytic residues of ASADH, DHPS, and DHPR and found them to be conserved in the protein from L. plantarum. ASADH, DHPS, and DHPR activity was detected in the cell extracts of E. coli mutant harboring each gene, indicating that the cloned genes were functionally expressed in E. coli. The regulation of ASADH, DHPS, and DHPR were studied in the cell extracts of both the E.␣coli mutant harboring the gene and L. plantarum; however, those enzymes were found not to be regulated by the end products of the pathway. This paper represents a portion of the thesis submitted by M. N. Cahyanto to Osaka University as partial fulfillment of the requirements for the PhD degree.     

Phenotypic and genomic diversity of Lactobacillus plantarum strains isolated from various environmental niches

Lactobacillus plantarum is a ubiquitous microorganism that is able to colonize several ecological niches, including vegetables, meat, dairy substrates and the gastro‐intestinal tract. An extensive phenotypic and genomic diversity analysis was conducted to elucidate the molecular basis of the high flexibility and versatility of this species. First, 185 isolates from diverse environments were phenotypically characterized by evaluating their fermentation and growth characteristics. Strains clustered largely together within their particular food niche, but human fecal isolates were scattered throughout the food clusters, suggesting that they originate from the food eaten by the individuals. Based on distinct phenotypic profiles, 24 strains were selected and, together with a further 18 strains from an earlier low‐resolution study, their genomic diversity was evaluated by comparative genome hybridization against the reference genome of L. plantarum WCFS1. Over 2000 genes were identified that constitute the core genome of the L. plantarum species, including 121 unique L. plantarum‐marker genes that have not been found in other lactic acid bacteria. Over 50 genes unique for the reference strain WCFS1 were identified that were absent in the other L. plantarum strains. Strains of the L. plantarum subspecies argentoratensis were found to lack a common set of 24 genes, organized in seven gene clusters/operons, supporting their classification as a separate subspecies. The results provide a detailed view on phenotypic and genomic diversity of L. plantarum and lead to a better comprehension of niche adaptation and functionality of the organism.     

Cloning,Expression, Purification,and Characterization of a Novel Esterase from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>

Lactobacillus plantarum is an important lactic acid bacterium, usually found as natural inhabitant of food, such as fermented vegetables and meat products. However, little information about lactic acid bacteria, especially concerning L. plantarum, as a source of useful enzymes has been reported. The aim of this study was to clone, express in Escherichia coli, purify, and characterize an esterase from L. plantarum ATCC 8014. The esterase gene (1014 bp) was amplified and cloned in pET14b expression vector to express a His₆-tagged protein in E. coli. Recombinant L. plantarum esterase was purified by Ni-NTA resin, presenting an apparent molecular mass of about 38 kDa. It presented highest activity at pH 6.0 and 40°C. Also, it presented preference for p-nitrophenyl butyrate, but hydrolyzed more efficiently p-nitrophenyl acetate. Besides, this study shows, for the first time, CD data about secondary structure of an esterase from L. plantarum.     

Rapid Identification of <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">acidophilus</Emphasis> by Restriction Analysis of the 16S–23S rRNA Intergenic Spacer Region and Flanking 23S rRNA Gene

A rapid molecular approach was developed for the initial identification of Lactobacillus acidophilus strains which are difficult to identify using a single biochemical test. The 16S–23S rRNA intergenic spacer regions and flanking 23S rRNA genes of 19 strains of lactobacilli were amplified and the nucleotide sequences and restriction site polymorphisms were analyzed. AluI was the most useful of the restriction enzymes analyzed and produced reproducible digestion profiles in the L. helveticus, L. plantarum, and L. casei groups, as well as in L. acidophilus. This restriction fragment length polymorphism method may be useful for the identification of L. acidophilus strains in dairy products.     

The effects of using lactic acid bacteria inoculants in maize silage on the formation of biogenic amines

Silages from five ripened varieties of silage maize with dry matter contents ranging between 275 and 410 g/kg were prepared in five laboratory experiments. Whole-plant maize was fermented at 22°C and silages were then stored at the same temperature for 4 months. Spontaneously fermented silages were prepared as control variants and compared with silages inoculated with commercial strains of Lactobacillus plantarum, Lactobacillus buchneri and a mixed preparation Microsil containing L. plantarum, Lactobacillus casei, Enterococcus faecium and Pediococcus pentosaceus. The starter cultures were applied at doses 5·10⁵ and 5·10⁶ CFU/g of chopped maize. Seven biogenic amines and polyamines were extracted from silages with perchloric acid and determined as N-benzamides by micellar electrokinetic capillary chromatography. Common chemical criteria of silage quality were also determined. All three inoculants, mainly at the higher dose, decreased significantly contents of tyramine, putrescine and cadaverine, three undesirable amines occurring at the highest levels. L. plantarum was the most effective. Contents of histamine and tryptamine were low in all experimental silages. Also relatively low were levels of polyamines spermidine and mainly of spermine.     

Growth and Survival of Genetically Manipulated Lactobacillus plantarum in Silage

The growth and persistence of two genetically manipulated forms of Lactobacillus plantarum NCDO (National Collection of Dairy Organisms) 1193 have been monitored in grass silage. Both recombinants contained pSA3, a shuttle vector for gram-positive organisms that encodes erythromycin resistance. In one of the recombinants, pSA3 was integrated onto the chromosome, whereas in the other, a pSA3 derivative designated pM25, which contains a Clostridium thermocellum cellulase gene cloned into pSA3, was maintained as an extrachromosomal element. This extrachromosomal element is a plasmid. Rifampin-resistant mutants were selected for the recombinants and the parent strain. When applied to minisilos at a rate of 10⁶ CFU/g of grass, both the recombinants and the parent strain proliferated to dominate the epiphytic microflora and induced an increase in the decline in pH compared with that of the noninoculated silos. The presence of extra genetic material did not appear to disadvantage the bacterium in comparison with the parent strain. The selective recovery of both strains by using rifampin and erythromycin was confirmed by Southern hybridization. Interestingly, the free plasmid (pM25) appeared more stable in silage than was expected from studies in MRS broth. The plasmid was retained by 85% of the rifampin-resistant L. plantarum colonies isolated from a day 30 silo. These data answer an important question by showing that genetically manipulated recombinants of L. plantarum can proliferate and compete with epiphytic lactic acid bacteria in silage.     

Characterization of wine Lactobacillus plantarum by PCR-DGGE and RAPD-PCR analysis and identification of Lactobacillus plantarum strains able to degrade arginine

Summary Screening of strains isolated from red wine undergoing malolactic fermentation allowed the identification of lactic acid bacteria able to degrade arginine. A denaturing gradient gel electrophoresis approach, using the rpoB gene as the molecular target, was developed in order to characterize the isolated strains. Several strains were identified as Lactobacillus plantarum and were typed by RAPD-PCR with several randomly designed primers. Almost all of the␣L. plantarum strains identified were able to produce citrulline and ammonia, suggesting that the ability of␣L.␣plantarum to degrade arginine is a common feature in wine. During the characterization of the newly identified L.␣plantarum strains, the presence of genes coding for the arginine deiminase (ADI) pathway was observed in the strains able to produce citrulline, while the lack of this genes was observed in strain unable to produce citrulline. These results suggest that the degradation of arginine in L. plantarum is probably strain-dependent.