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.     

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.     

Studies on Streptococci

The population levels of bacteria in the contents and the walls of the gastrointestinal tract of gnotobiotic rats inoculated with lactic acid bacteria (streptococci, lactobacilli, and bifidobacteria) from humans and rats were determined. Lactobacilli as well as streptococci isolated from rats colonized in the digestive tracts of the gnotobiotic rats at a high population level, characteristically highest in the stomach. On the other hand, in the rats inoculated with human lactic acid bacteria, streptococci were dominant in the lower tract. The human lactobacilli or bifidobacteria did not colonize when the organisms in each genus were inoculated together with streptococci. However, when all three genera were inoculated together, lactobacilli and bifidobacteria colonized. Observations on the species of streptococci showed that the intestinal type of streptococci was found to colonize at a high population level, but the oral type was not. Strains of the same genus of lactic acid bacteria from humans and from rats showed different colonization patterns.     

Bacterial community associated with ensilage process of wilted guinea grass

Aims: To determine the effects of wilting, storage period and bacterial inoculant on the bacterial community and ensiling fermentation of guinea grass silage. Methods and Results: Fermentation products, colony counts and denaturing gradient gel electrophoresis (DGGE) profiles were determined. There was more lactic acid than acetic acid in all silages, but the lactic acid to acetic acid ratio decreased with storage time. This shift from lactic to acetic acid was not prevented even with a combination of wilting and bacterial inoculant. The DGGE analyses suggest that facultatively heterofermentative lactic acid bacteria (Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus pentosus) were involved in the shift to acetic acid fermentation. Conclusions: Lactic acid can dominate the fermentation in tropical grass silage with sufficient wilting prior to ensiling. Prolonged storage may lead to high levels of acetic acid without distinctive changes in the bacterial community. Significance and Impact of the Study: The bacterial community looks stable compared to fermentation products over the course of long storage periods in tropical grass silage. Acetic acid fermentation in tropical grass silage can be a result of the changes in bacterial metabolism rather than community structure.     

The Significance of Propionibacterium Species and Micrococcus lactilyticus to the Ensiling Process

The fermentation of lactic acid by two Propionibacterium spp. and by Micrococcus lactilyticus was examined in simulated silage. Fermentation occurred in silages which achieved a low pH (<4·0) but this resulted only in a small rise in pH and a secondary clostridial fermentation did not occur. Lactic acid-fermenting organisms resembling Propionibacterium spp. were isolated from authentic silages but not from fresh grass.     

Metabolite profiles of lactic acid bacteria in grass silage

The metabolite production of lactic acid bacteria (LAB) on silage was investigated. The aim was to compare the production of antifungal metabolites in silage with the production in liquid cultures previously studied in our laboratory. The following metabolites were found to be present at elevated concentrations in silos inoculated with LAB strains: 3-hydroxydecanoic acid, 2-hydroxy-4-methylpentanoic acid, benzoic acid, catechol, hydrocinnamic acid, salicylic acid, 3-phenyllactic acid, 4-hydroxybenzoic acid, (trans, trans)-3,4-dihydroxycyclohexane-1-carboxylic acid, p-hydrocoumaric acid, vanillic acid, azelaic acid, hydroferulic acid, p-coumaric acid, hydrocaffeic acid, ferulic acid, and caffeic acid. Among these metabolites, the antifungal compounds 3-phenyllactic acid and 3-hydroxydecanoic acid were previously isolated in our laboratory from liquid cultures of the same LAB strains by bioassay-guided fractionation. It was concluded that other metabolites, e.g., p-hydrocoumaric acid, hydroferulic acid, and p-coumaric acid, were released from the grass by the added LAB strains. The antifungal activities of the identified metabolites in 100 mM lactic acid were investigated. The MICs against Pichia anomala, Penicillium roqueforti, and Aspergillus fumigatus were determined, and 3-hydroxydecanoic acid showed the lowest MIC (0.1 mg ml(-1) for two of the three test organisms).     

New trends and opportunities in the development and use of inoculants for silage

Abstract: Inoculants are used as silage additives to improve preservation efficiency and to enhance animal performance. In most commercially available inoculants, homofermentative lactic acid bacteria (LAB) have been used because they are fast and efficient producers of lactic acid, improving natural silage fermentation. Specific LAB inuculants may also have beneficial effects on animal performance even if there is no effect on fermentation. However, these types of inoculants are not always advantageous. They do not necessarily prevent sermentation by clostridia in moist silages, and they sometimes impair the aerobic stability of grass and small grain silages. Therefore, new criteria for silage inoculants should be established which consider the specific needs of the crop being ensiled. New approaches which are being taken to develop improved inoculants for silage include the following: (1) using LAB isolates which are more specific to the target crops; (2) inclusion of heterofermentative LAB to produce volatile fatty acids to inhibit yeasts and moulds upon aerobic exposure; (3) inclusion of organisms other than LAB in inoculants to inhibit detrimental microorganisms; (4) selection or engineering of LAB strains to inhibit specific microorganisms; and (5) cloning and expression of genes which would enable selected LAB strains to utilize polysaccharides in crops which are low in soluble carbohydrates. Many of these new strategies for formulating inoculants are being tested, but further research is needed to determine the most successful approaches.     

Bacterial community dynamics during the ensilage of wilted grass

Aims:  Grass silage is the product formed by a natural lactic acid bacterial fermentation when grass is stored under anaerobic conditions, and represents an important ruminant feedstuff on farms during winter. Of the two commonly employed methods of ensiling forage, baled silage composition frequently differs from that of comparable precision-chop silage reflecting a different ensiling environment. The aim of this study was to investigate the dynamics of the silage fermentation in wilted grass and between ensiling systems.
Methods and Results:  Fermentation dynamics were examined using traditional methods of silage analyses, including microbial enumeration and analysis of fermentation products, and culture-independent terminal restriction fragment length polymorphism (T-RFLP). A successful fermentation was achieved in both systems, with the fermentation (increase in lactic acid bacteria and lactic acid concentration, decrease in pH) proceeding rapidly once the herbage was ensiled.
Conclusions:  Under controlled conditions, little difference in silage quality and microbial composition were observed between ensiling systems and this was further reflected in the T-RFLP community analysis.
Significance and Impact of the Study:  T-RFLP proved a potentially useful tool to study the ensilage process and could provide valid support to traditional methods, or a viable alternative to these methods, for investigating the dynamics of the bacterial community over the course of the fermentation.     

Degradation of fructans by epiphytic and inoculated lactic acid bacteria and by plant enzymes during ensilage of normal and sterile hybrid ryegrass

Degradation of grass fructans by epiphytic or inoculated lactic acid bacteria during ensilage was examined using both normal and sterile hybrid ryegrass. It was clear that even in the absence of bacteria fructan degradation occurred, but at a significantly slower rate than in normal grass which had not been inoculated with lactic acid bacteria. Fructan degradation in sterile herbage suggests that plant fructan hydrolases were partially responsible for this process in all herbages, irrespective of treatment. Inoculation of sterile herbage with a strain of Lactobacillus plantarum known to lack the ability to degrade grass fructans resulted in a slower rate of fructan breakdown than when inoculated with Lactobacillus casei subsp. paracasei , a confirmed fructan degrader. In the later stages of the fermentation of uninoculated normal herbage when water-soluble carbohydrate appeared to be limiting, lactic acid was fermented to acetic acid. However, this fermentation pathway was not observed in either of the inoculated silages. The results suggest that silage inoculant bacteria possessing fructan hydrolase activity may have potential for improving silage fermentation, particularly when late cut, low sugar grass containing a high proportion of fructans is ensiled.     

Dynamics in the microbiology of maize silage during whole‐season storage

Aims: To monitor seasonal variations in the microbiology of maize silage and to determine whether the risk of fungal spoilage varies during whole‐year storage. Methods and Results: A continuous survey of 20 maize silage stacks was conducted over a period from three to 11 months after ensiling. Filamentous fungi, yeasts and lactic acid bacteria (LAB) were enumerated at five time‐points, and cultivable species of filamentous fungi were identified. Significant differences in the numbers of filamentous fungi, yeast and LAB were detected. The highest numbers of fungi were five to seven and the lowest 11 months after ensiling, while the LAB decreased in numbers during the study. Filamentous fungi were isolated from all stacks at all time‐points. The most abundant toxigenic mould species were Penicillium roqueforti, Penicillium paneum and Aspergillus fumigatus. Conclusions: There are significant variations in the microbiology of maize silage over a whole storage season. The risk of fungal spoilage was highest 5–7 months after ensiling and lowest after 11 months. Significance and Impact of the Study: This information is valuable in the assessment of health risks connected with spoiled maize silage and may be useful in the management of maize silage stacks, when whole‐season storage is applied.     

Cellulolytic and non-cellulolytic bacteria in rat gastrointestinal tracts.

Lactobacillus and Bifidobacterium species were the predominant organisms isolated from small intestinal (jejunal) contents of rats, and lactic acid was the only organic acid detected. The numbers of cellulolytic bacteria in small intestines were low (approximately 10(3)/g). The fermentation in ceca was different from that in intestines, as, in addition to small amounts of lactic acid, high concentrations of volatile fatty acids were detected. The mixed cecal microflora was able to digest cellulose (pebble-milled Whatman no. 1) and cabbage. High numbers of cellulolytic bacteria were found (0.5 X 10(8) to 12.2 X 10(8)/g; 6% of total viable bacteria). The predominant celluloytic organism isolated was Bacteroides succinogenes. Ruminococcus flavifaciens was isolated from a few animals. The kinds and numbers of the predominant non-cellulolytic organisms isolated from rat ceca were similar to those described by previous workers.     

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.     

Ensilage of tropical grasses mixed with legumes and molasses

The effects of adding two legumes, Gliricidia sepium and Leucaena leucocephala, cv. Cunningham, and molasses on the fermentation characteristics of silages made from two tropical grasses (Pangola grass, Digitaria decumbens, and Setaria sphacelata cv. Kazungula) were investigated. Pangola grass silages contained significantly higher contents of water-soluble carbohydrates and lactic acid than did setaria silages after 100 days fermentation, but there were no significant differences between the two silages in populations of lactic acid bacteria and contents of total N and NH₃–N. Addition of either species of legume had no significant effect on fermentation acids and NH₃–N contents, and numbers of lactic acid bacteria. Addition of both legumes reduced NH₃–N production in the silages by 59% after 5 days' fermentation. Numbers of lactic acid bacteria were not significantly affected by the different treatments. Enterococcus faecalis represented 60% of the lactic acid bacteria isolated from the treated herbages prior to ensiling. By 100 days of fermentation, only lactobacilli were isolated: 82% homo-fermenters and 18% hetero-fermenters. Lactobacillus mesenteroides subsp. dextranicum was found only in the silage supplemented with 33% (w/w) legume. It was concluded that the low quality of tropical grasses used as feeds for ruminants may be significantly improved by ensiling these grasses with small amounts of molasses and with high-protein tree leaves.M. Tjandraatmadja and B.W. Norton are with the Department of Agriculture. The University of Queensland, Queensland, 4072, Australia; I.C. Mac Rae is with the Department of Microbiology, The University of Queensland, Queensland, 4072, Australia.     

A COMPARATIVE STUDY OF LACTOBACILLI FROM GRASS SILAGE AND OTHER SOURCES

Samples of 152 strains of lacto bacilli isolated from six different types of grass silage, which had been subjected to various treatments at the time of ensiling, and five strains isolated from other sources for comparative purposes, were tested by their reactions in litmus milk, yeast extract litmus milk, dextrose litmus milk, yeast extract dextrose litmus milk and in various carbohydrate broths. The metabolic products formed from dextrose by nineteen selected strains were identified and estimated.
The evidence obtained in this way pointed to the conclusion that the majority of lactobacilli in grass silage are strains of Streptobacterium plantarum, characterized by comparative inactivity in ntmns milk, bat markedly stimulated by addition of yeast extract. They produce from dextrose mainly lactic acid (usually the inactive form) with a small quantity of acetic acid. A strain was encountered which also produced alcohol and butyric acid, but the numbers of this type in silage was negligible. Considerable differences were found in the ability of the various strains to ferment carbohydrates.
Of the five strains isolated from dairy products three woe of the Streptobacterium plantarum type and two were of a kind which rapidly clotted litmus milk and exhibited reactions corresponding to the true milk strains.
The authors wish to record their thanks to the Research Council of Imperial Chemical Industries, Ltd., for a grant which rendered this work possible, to Dr S. J. Watson for supplying samples of grass and silage, and to Dr J. 6. Davis for valuable suggestions.     

Ensilage characteristics of three tropical grasses as influenced by stage of growth and addition of molasses

When molasses was added during ensilage of three tropical grasses [hamil grass (Panicum maximum cv. Hamil), pangola grass (Digitaria decumbens) and setaria (Setaria sphacelata cv. Kazungula)] the final pH, concentration of fermentation acids (except lactic acid) and NH₃–N content were all similar after 100 days of incubation. Pangola grass silage had significantly higher lactic acid content (66 g/kg dry matter) than the other two. Adding either 4 or 8% (w/w) molasses reduced NH₃–N, volatile fatty acid content and pH but increased lactic acid content in the final silages. Numbers of lactic acid bacteria remained approximately constant during the course of the fermentation, although large differences were noted in the species composition of the populations. At the time of ensiling, only Pediococcus spp. and Leuconostoc spp. were detected. By 5 days, the homo-fermentative population, notably Lactobacillus plantarum, dominated (43%) and remained dominant. Hetero-fermentative rods were only detected in the 100-day silage, where they represented 29% of the strains isolated. Homo-fermenters were more abundant in pangola (60%) and setaria (47%) silages than hamil (27%) silages. Homo-fermenter populations were lowest in the 12-week forage. Molasses additions increased homo-fermenter populations. Pangola grass gave the best quality silage but, since the water-soluble carbohydrate content in the grasses was insufficient to promote a strong lactic fermentation, the addition of 20 to 30 kg molasses/tonne should achieve satisfactory preservation.M. Tjandraatmadja and B.W. Norton are with the Department of Agriculture, The University of Queensland, Queensland, 4072, Australia; I.C. Mac Rae is with the Department of Microbiology, The University of Queensland, Queensland, 4072, Australia.     

Ecological studies on the occurrence of bacteria utilizing lactic acid at pH values below 4.5

Sites within two cheese factories, a silage tower and silage pit and two pet food factories were investigated for lactic acid utilizing bacteria. The bacteria isolated, together with known lactic acid utilizing and aciduric bacteria, were tested for their ability to 'alkalinize' a fermented meat product containing more than 20 g/1 lactic acid at pH > 4.3 a ten-fold dilution of this product and synthetic media, both at pH 4.5. Strains able to alkalinize the diluted product were 13 aerobic isolates (all Acetobacter spp.), nine anaerobic cultures, one strain of Megasphaera elsdenii and two strains of Clostridium tyrobutyricum. Eight of the aerobes could alkalinize undiluted product containing < 1 g/1 residual potassium sorbate, but had no effect on product containing > 3 g/1 sorbate; none of the anaerobic cultures was able to alkalinize undiluted product. Acetobacter spp., therefore, threatened the microbial stability of a fermented product containing < 3 g/1 potassium sorbate. The cultures able to alkalinize diluted product were all from sites contaminated with whey, silage or fermented product for at least 2 d, suggesting substantial accumulation of lactic acid utilizing bacteria at these sites.     

Metabolite Profiles of Lactic Acid Bacteria in Grass Silage

The metabolite production of lactic acid bacteria (LAB) on silage was investigated. The aim was to compare the production of antifungal metabolites in silage with the production in liquid cultures previously studied in our laboratory. The following metabolites were found to be present at elevated concentrations in silos inoculated with LAB strains: 3-hydroxydecanoic acid, 2-hydroxy-4-methylpentanoic acid, benzoic acid, catechol, hydrocinnamic acid, salicylic acid, 3-phenyllactic acid, 4-hydroxybenzoic acid, (trans, trans)-3,4-dihydroxycyclohexane-1-carboxylic acid, p-hydrocoumaric acid, vanillic acid, azelaic acid, hydroferulic acid, p-coumaric acid, hydrocaffeic acid, ferulic acid, and caffeic acid. Among these metabolites, the antifungal compounds 3-phenyllactic acid and 3-hydroxydecanoic acid were previously isolated in our laboratory from liquid cultures of the same LAB strains by bioassay-guided fractionation. It was concluded that other metabolites, e.g., p-hydrocoumaric acid, hydroferulic acid, and p-coumaric acid, were released from the grass by the added LAB strains. The antifungal activities of the identified metabolites in 100 mM lactic acid were investigated. The MICs against Pichia anomala, Penicillium roqueforti, and Aspergillus fumigatus were determined, and 3-hydroxydecanoic acid showed the lowest MIC (0.1 mg ml⁻¹ for two of the three test organisms).     

Bacterial spores in silage and raw milk

Spore-forming bacteria can survive food-processing treatments. In the dairy industry, Bacillus and Clostridium species determine the shelf-life of a variety of heat-treated milk products, mainly if the level of post-process contamination is low. In order to minimize problems caused by bacterial spores in foods and food production processes a chain management approach, from raw materials, ingredients and environmental sources to final product storage conditions, is most effective. Silage is considered to be a significant source of contamination of raw milk with spores. PCR-RAPD fingerprinting and heat resistance studies of populations of aerobic spore-formers isolated from grass and maize silage and from raw milk confirmed this assumption. Prevention of outgrowth of aerobic spores in silage will contribute to reduction of the total spore load of raw milk. Therefore, it is important that the silage fermentation process is controlled. Application of cultures of lactic acid bacteria or chemical additives can aid silage fermentation and improve aerobic stability. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Development of lactic acid bacteria during early stages of fermentation in fish silage

Summary The development of various lactic acid bacteria during the early stages of fermentation (1–6 days after ensiling) in fish silage was studied. The first type of organisms that grew fastest was the oval cocci (most of them resembledLeuconostoc mesenteroides andStreptococcus lactis) followed by round cocci (mostlyS. faecalis). The number of oval cocci increased rapidly one day after ensiling and then decreased after 2–3 days. The round cocci increased first after 2–3 days and then decreased slowly after 4–5 days. Lactobacilli began to increase in number (more than 10¹⁰ per g silage) first after 6 days. Thus the pH in the silage was mainly lowered by the action of streptococci. Also in MRS medium the pH was more rapidly lowered byS. faecalis than byLactobacillus plantarum and other rods.