Screening and selection of lactic acid bacteria strains suitable for ensiling grass

AIM: Lactic acid bacteria (LAB) strains shown to have broad-spectrum antimicrobial activity were screened for potential as grass silage inoculants. The strains capable of rapidly lowering the pH of the grass matrix and with low proteolytic activity were assessed in laboratory-scale silos in a grass matrix containing natural microbial flora. METHODS AND RESULTS: Screening of nine candidate strains was performed first in a grass extract medium. The four most promising strains were selected on the basis of growth rate in the medium, capacity to reduce pH and ability to limit the formation of ammonia-N. The efficiency of the selected strains was further assessed in a laboratory-scale ensiling experiment. Untreated (no additive) and formic acid served as controls. All tested inoculants improved silage quality compared with untreated. With one exception (Pediococcus parvulus E315) the fermentation losses in the inoculated silages were even lower than in the acid-treated control silage. Pure lactic acid fermentation was obtained in the timothy-meadow fescue silage with all inoculants. The results obtained in the ensiling experiments were consistent with those of the screening procedure, which appeared to predict correctly the potential of LAB as silage inoculants. The strains with a low ammonia production rate in the grass extract medium behaved similarly in the silage. Especially in this respect the strain Lactobacillus plantarum E76 was superior to the other candidates. CONCLUSIONS: The screening method using grass extract proved to be useful in strain selection. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid screening method developed for the LAB strains provides a useful tool for more systematic product development of commercial inoculant preparations. Time consuming and laborious ensiling experiments can be limited only to the most promising strains.     

Comparative evaluation of plasmid profiling and ribotyping in the analysis of Lactobacillus plantarum strain heterogeneity in silage

F. DUFFNER AND M. O'CONNELL. 1995. Seventy-two Lactobacillus plantarum isolates were recovered from six uninoculated grass silages for the purposes of firstly evaluating the usefulness of (1) restriction endonuclease digestion of total genomic DNA, (2) plasmid profiling and (3) ribotyping in Lact. plantarum strain differentiation and secondly, examining the strain heterogeneity in well preserved silage.
The three methods for differentiation were applied to 72 of the isolates and allowed at least 32 different strains to be identified. Twenty-five different plasmid profiles were detected (26 if the absence of plasmids is included as a profile). Ribotyping with Eco RI identified only 11 patterns among the silage isolates. A variety of restriction enzymes was screened to increase the sensitivity of ribotyping to detect strain differences and Bam HI was used successfully for this purpose, differentiating all of the strains tested.
Two dominant strains (I and II) were identified in one particular silage, comprising 47% and 17% respectively of the isolates, while strains III and V comprised 37% and 25% of the Lact. plantarum population isolated from another of the silages.     

Investigations on the Glycerol Metabolism of Lactobacilli

S ummary . The glycerol metabolism of homofermentative and heterofermentative strains of Lactobacillus obtained either from Culture Collections or isolated from sausages has been investigated.
The results show that the homofermentative lactobacilli ( Thermobacterium and Streptobacterium ) produce mannose phosphate which is then metabolized to lactic acid and other byproducts.     

THE ENUMERATION OF LACTOBACILLI ON GRASS AND IN SILAGE

SUMMARY: For the enumeration of lactobacilli on grass and in silage the following medium has shown promise: peptone, meat extract and glucose, 10 g. each; tomato extract, 200 ml.; yeast autolysate, 50 ml.; Tween 80, 0.5 ml.; agar, 15 g., in a final volume of 1 1. and containing acetic acid/sodium acetate buffer in 0.2M concentration; pH 5–4. The medium was adjusted to pH 5–4 before sterilization and the requisite amount of concentrated pH 5.4 acetate buffer added just before plating. Double laver plates were used.
The only other silage organisms which in this medium formed colonies comparable in size with those of lactobacilli were heterofermentative streptococci and a micrococcus.     

Dominance of Lactobacillus plantarum strains in grass silage as demonstrated by a novel competition assay

An assay was developed for assessing the competitive ability of potential Lactobacillus plantarum silage inoculants. This assay was based on the ability of the test inoculant to outcompete a standard strain ( Lact. plantarum DCU101) co-inoculated at the same rate of 5 × 10⁵ colony forming units g^-1 of grass. Total populations of Lact. plantarum were enumerated with a selective medium and Lact. plantarum DCU101 was identified with a strain-specific DNA probe. The DNA probe was based on a small (2.2 kb), cryptic, indigenous plasmid which was cloned into pAT153, a multicopy cloning vector. Seven Lact. plantarum strains, six of which were isolated from well-preserved grass silages, were used to inoculate laboratory scale silos, and variation in strain dominance was monitored over the 14 d ensilage period.     

饲料乳酸菌的分离鉴定及优良菌株筛选

对从饲料玉米、高粱、麦秆及棉花中筛选出的乳酸菌进行分类鉴定和综合性分析。用MRS+CaCO3固体培养基从棉花中分离出乳酸菌18株、高粱中30株、饲料玉米中18株、麦秆中18株。经形态学、生理生化试验进行初步鉴定并按产酸试验,耐盐及耐酸试验挑选出32株产酸率强的乳酸菌对其进行16S rDNA分子鉴定。结果显示,32株菌都具有良好的耐盐、耐酸能力;经生理生化和16S rDNA基因序列鉴定可知32株乳酸菌分属于两个属,即乳杆菌属、肠球菌属,4个种,即干酪乳杆菌(Lactobacilluscasei)、肠道球菌(Entercoccus faecium)、植物乳杆菌(Lactobacillus plantarum)、海氏肠球菌(Entercoccus hirae)。4种饲料原料中肠道球菌普遍存在。除了这种乳酸菌以外,棉花有干酪乳杆菌、植物乳杆菌、海氏肠球菌,玉米和麦秆内有植物乳杆菌。从饲料中筛选出4株具有较强产酸能力的乳酸菌,可进一步研发成青贮饲料添加剂。     

A numerical taxonomic study of lactic acid bacteria from tropical silages

Lactic acid bacteria were isolated from various tropical silages made from either forage sorghum ( Sorghum bicolor cv. Sugardrip): three tropical grasses: hamil grass ( Panicum maximum cv. Hamil), pangola grass ( Digitaria decumbens ) and setaria ( Setaria anceps cv. Kazungula) either with or without addition of molasses and from mixtures of pangola or setaria grasses with leucaena ( Leucaena leucocephala ) or gliricidia ( Gliricidia sepium ). The 311 wild strains isolated and 13 reference strains were examined by 80 biochemical, physiological and morphological tests. A computer-assisted complete linkage analysis produced 16 clusters and five unclustered strains at 82% S. The largest cluster included 134 wild strains and the reference strain Lactobacillus plantarum . The results showed that Lactobacillus plantarum was the predominant lactic acid bacterium in all of the tropical silages.     

The fermentation of cell wall substances in grass silage and in potato pulp

Summary The amount of acid formed in grass silage was greater than could have been formed from the soluble sugars present, even when only a lactic fermentation took place. This seemed to point to fermentation of cell wall substances by lactic acid bacteria. Lactic acid fermentation in potato pulp always takes place with cell wall substances as substrates, as sugars are absent. It was found that galactose, probably occurring as galactan, and also some pectic acid were fermented in potato pulp. Some lactobacilli were isolated from potato pulp; streptobacteria which could ferment galactan but no pectic or galacturonic acid, and betabacteria which could ferment galacturonic acid but no galactan or pectic acid. A number of homofermentative lactobacilli were all found to belong to the speciesStreptobacterium casei. It was shown that a strain of this species could ferment galactan in potato pulp sterilised previously with ethylene oxide. Part of this work was carried out at the Netherlands Institute for Dairy Research, Ede, Netherlands.     

苜蓿青贮用乳酸菌复合系Al2的组成多样性

苜蓿是高蛋白的饲料作物,青贮是保存青草过冬的主要手段,但苜蓿是难青贮的作物,添加乳酸菌制剂是解决苜蓿青贮难的有效措施。本研究室通过连续的限制性培养筛选到苜蓿青贮用乳酸菌复合系Al2,用变性梯度凝胶电泳(DGGE)、平板分离及建立16S rDNA克隆文库3种手段相结合分析Al2的组成多样性,确认Al2复合菌系由7株菌组成,全部属于Lactobacillus。其中3株菌Al2-1i,Al2-2i,Al2-3i通过平板分离得到,分别属于L.plantarum(99.9%)、L.kimchii(99.4%)、L.farciminis(100%)。在Al2复合菌系的16S rDNA克隆文库中,上述7种菌的组成比例分别为55.21%、19.79%、14.58%、3.13%、3.13%、3.13%、1.03%。     

Gnotobiotic Silage

Selected strains of lactic acid bacteria isolated from grass silage were found to flourish when inoculated into irradiation-sterilized forage under gnotobiotic conditions. The acid content and pH of these silages resembled naturally fermented silage. Inoculation of gnotobiotic silage with Clostridium sporogenes and C. tyrobutyricum failed to cause any noticeable deterioration of silage quality.     

A Preliminary Investigation into the Role of Yeasts in the Ensiling Process

The activities of six unidentified yeasts isolated from silage were monitored in a defined environment prepared to simulate a grass silage. Lactic acid and acetic acid contents of the silages were higher when yeasts were included in the inoculum used and the yeasts appeared to be beneficial to preservation. The yeasts also contributed to the small quantities of ethanol which were detected. Where a yeast was used as the sole inoculum the pH of the silage remained high, but lactic acid and acetic acid as well as ethanol were present in the resulting silages.     

Deoxyribonucleic acid base composition of some members of the Micrococcaceae

Auletta, Angela E. (Catholic University, Washington, D.C.), and E. R. Kennedy. Deoxyribonucleic acid base composition of some members of the Micrococcaceae. J. Bacteriol. 92:28-34. 1966.-Thirty-seven strains from the genera Micrococcus, Staphylococcus, Gaffkya, and Sarcina were examined for deoxyribonucleic acid base composition and biochemical activity. Organisms were tested for production of catalase, coagulase, deoxyribonuclease, oxidase, phosphatase, hydrogen sulfide, indole, and acetoin; nitrate reduction; gelatin, starch, and urea hydrolysis; citrate and ammonium phosphate utilization; NaCl tolerance; growth at 10 and 45 C, and growth in litmus milk. They were tested for production of acid from dextrose and mannitol under anaerobic conditions, and for aerobic production of acid from dextrose, mannitol, lactose, sucrose, raffinose, maltose, xylose, and glycerol. Organisms could be divided into two groups on the basis of guanine-cytosine (GC) content. Group I had an average GC content of 32%, and included all organisms which produced acid from dextrose. Group II had an average GC content of 62%, and included those organisms incapable of producing acid from dextrose under anaerobic conditions. Sarcina ureae had a GC content of 43%.     

Characterization of a gram-positive broad-host-range plasmid isolated from Lactobacillus hilgardii

Two plasmids, pLAB1000 and pLAB2000 (3.3 and 9.1 kb, respectively), have been isolated from a grass silage strain of Lactobacillus hilgardii. Both plasmids were cloned in Escherichia coli and characterized through restriction mapping. A 1.6-kb XbaI-SacI fragment of pLAB1000 appeared to be sufficient for autonomous replication in Lactobacillus plantarum and in Bacillus subtilis. Different shuttle vectors for E. coli and gram-positive bacteria were developed using the pLAB1000 plasmid. These could stably be maintained in Lactobacillus, Enterococcus, and Bacillus under selective conditions. Plasmids sharing DNA homologies with pLAB1000 have been observed in different strains of the related species L. plantarum.     

Gnotobiotic Silage

Selected strains of lactic acid bacteria isolated from grass silage were found to flourish when inoculated into irradiation-sterilized forage under gnotobiotic conditions. The acid content and pH of these silages resembled naturally fermented silage. Inoculation of gnotobiotic silage with Clostridium sporogenes and C. tyrobutyricum failed to cause any noticeable deterioration of silage quality.     

Sources of Clostridia in Raw Milk on Farms

A PCR-denaturing gradient gel electrophoresis (DGGE) method was used to examine on-farm sources of Clostridium cluster I strains in four dairy farms over 2 years. Conventional microbiological analysis was used in parallel to monitor size of clostridial populations present in various components of the milk production chain (soil, forage, grass silage, maize silage, dry hay, and raw milk). PCR amplification with Clostridium cluster I-specific 16S rRNA gene primers followed by DGGE separation yielded a total of 47 operational taxonomic units (OTUs), which varied greatly with respect to frequency of occurrence. Some OTUs were found only in forage, and forage profiles differed according to farm location (southern or northern Québec). More clostridial contamination was found in maize silage than in grass silage. Milk represented a potential environment for certain OTUs. No OTU was milk specific, indicating that OTUs originated from other environments. Most (83%) of the OTUs detected in raw milk were also found in grass or maize silage. Milk DGGE profiles differed according to farm and sampling year and fit into two distinct categories. One milk profile category was characterized by the presence of a few dominant OTUs, the presence of which appeared to be more related to farm management than to feed contamination. OTUs were more varied in the second profile category. The identities of certain OTUs frequently found in milk were resolved by cloning and sequencing. Clostridium disporicum was identified as an important member of clostridial populations transmitted to milk. Clostridium tyrobutyricum was consistently found in milk and was widespread in the other farm environments examined.     

A survey of lactic acid bacteria in Italian silage

G razia , L. & S uzzi , G. 1984. A survey of lactic acid bacteria in Italian silage. Journal of Applied Bacteriology 56 , 373–379.
Lactic acid bacteria, isolated from Italian ensiled products, were represented by strains of the genera Lactobacillus and Leuconostoc . The predominant strains were heterofermentative lactobacilli, with Lactobacillus buchneri being the most frequent. Among homofermentative lactic acid bacteria, strains of Lact. plantarum and Lact. casei were recovered. Almost all strains utilized malic acid and showed good acid-tolerance, but only some of them were able to metabolize malic acid at extremely low pH; these were five homofermentative lactobacilli (4 Lact. plantarum and 1 Lacr. casei var. casei ) and two heterofermentative lactobacilli ( Lact. cellobiosus and Lactobacillus sp.).     

Arabinose fermentation by Lactobacillus plantarum in sourdough with added pentosans and alphaalpha-L-arabinofuranosidase: a tool to increase the production of acetic acid

Sixty-five strains of obligately and facultatively heterofermentative sourdough lactic acid bacteria were screened for their capacity to grow optimally in the presence of arabinose, ribose and xylose as carbon sources. Lactobacillus alimentarius 15F, Lact. brevis 10A, Lact. fermentum 1F and Lact. plantarum 20B showed higher growth rate, cell yield, acidification rate and production of acetic acid when some pentoses instead of maltose were added to the SDB medium. Lactobacillus plantarum 20B used arabinose also in a synthetic medium where complex growth factors such as yeast extract were omitted. Other Lact. plantarum strains did not show the same property. Pentosan extract was treated with alpha-L-arabinofuranosidase from Aspergillus niger or endo-xylanase from Bacillus subtilis to produce hydrolysates containing mainly arabinose and xylose, respectively. In particular, the hydrolysate containing arabinose substantiated the growth and the production of lactic acid and, especially, of acetic acid by Lact. plantarum 20B. Sourdough fermentation by Lact. plantarum 20B with addition of pentosan extract and alpha-L-arabinofuranosidase increased the acidification rate, titratable acidity and acetic acid content compared with traditional sourdough. A facultatively heterofermentative strain, Lact. plantarum 20B, also produced a sourdough with an optimal fermentation quotient.     

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.     

Lactic acid bacteria diversity in corn silage produced in Minas Gerais (Brazil)

The diversity of lactic acid bacteria (LAB) in silages produced in warm climate countries is not well known. This study aimed to identify and characterise the metabolic and genotypic aspects of autochthonous LAB isolated from corn silage produced in the state of Minas Gerais, Brazil. Eighty-eight LAB were isolated. To evaluate their performance at the strain level, all isolates were distinguished among strains using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) and repetitive extragenic palindromic PCR (REP-PCR) techniques. The organic acid and ethanol production were determined by high-performance liquid chromatography (HPLC). The fingerprints obtained by RAPD-PCR with a M13 primer were more discriminatory than those obtained with the REP-PCR technique using a (GACA)4 primer. Moreover, 28 representative isolates were identified as Lactobacillus acidophilus, L. buchneri, L. casei, L. diolivorans, L. hilgardii, L. paracasei, L. parafarraginis, L. plantarum, L. rhamnosus, L. zeae and Pediococcus acidilactici. Different fingerprinting profiles between isolates within the same species were observed. However, some strains isolated from different silages showed the same band profile, thus suggesting the presence of clusters with high similar fingerprints in silages from various regions. A variation in LAB diversity was observed in the silages of the evaluated regions, with L. rhamnosus and L. buchneri showing the highest distribution. Differences in organic acid production were observed among the strains belonging to the same species. This research contributes to a better understanding of the LAB community present in corn silage produced in warm climates. These strains will be studied as potential silage starters.