Fatty Acid and Isoprenoid Quinone Composition in the classification of Bacteroides melaninogenicus and Related Taxa

Aerobic deterioration of lucerne, maize and wheat silages was characterized by rapid increases in yeast and mould flora which oxidized lactic and volatile acids resulting in increased temperature and pH. While populations of yeasts and moulds were similar, temperature increases were slightly greater for silages inoculated with Lactobacillus acidophilus and Candida spp. After 48 h the pH of the inoculated silages was higher in general and concentrations of acids were lower than controls. Bacterial growth was slight although continued lactic acid production was probable. In contrast to lucerne and maize silages, the pH of wheat silage remained stable during this period because of high butyric levels, but temperature and yeast populations increased. After 48 h the pH rose above 5 in maize and lucerne, and bacterial growth and metabolic activity resumed resulting in volatile and non-volatile acid production from carbohydrate fermentation and deamination of amino acids. During this phase of aerobic deterioration yeast growth slowed or stopped, but temperatures remained high and pH continued to climb probably because of production of ammonia. The changes in gross composition of the silages did not follow any particular pattern. Losses in dry matter were small (2.5–4.0%) and changes in individual components probably reflect this loss rather than substantial changes. Protein availability in the lucerne silages undoubtedly decreased, as protein losses were high. It is concluded that the aerobic deterioration of silage is enhanced by the addition of L. acidophilus and Candida spp. at ensiling.     

The effect of a propionic acid bacterial inoculant applied at ensiling on the aerobic stability of wheat and sorghum silages

The effect of a new strain ofPropionibacterium shermanii (PAB), applied at ensiling, on the aerobic stability of wheat and sorghum silages was studied in several experiments under laboratory conditions. In the one experiment with wheat and in those with sorghum a lactic acid bacteria (LAB) inoculant (Lactobacillus plantarum andPediococcus cerevisiae) was also included. After treatment, the chopped forages were ensiled in 1.5-L anaerobic jars which were sampled in triplicate on predetermined dates to follow fermentation dynamics. At the end of the experiments, the silages were subjected to an aerobic stability test. The PAB inoculant improved the aerobic stability only in one experiment with wheat, in which the decrease in pH was very slow; the final pH remained relatively high (4.5). The PAB-treated silages contained 19.5±2.0 g of propionic acid per kg of dry matter. In the experiments with sorghum, the control and PAB-inoculated silages were stable, whereas LAB-inoculated silages deteriorated. The results suggest that PAB can survive in and improve the aerobic stability of only slow-fermenting silages which are prone to aerobic deterioration.     

Studies on the Significance of Three Bacillus species to the Ensiling Process

The activities of Bacillus coagulans, Bacillus licheniformis and Bacillus polymyxa were examined in a defined environment prepared to simulate a grass with low contents of dry matter and water-soluble carbohydrates. All of these organisms produced lactic and acetic acids, and their growth was not suppressed by these fermentation products nor by low pH. The acid content of silages made by inoculation with lactic acid bacteria alone and in combination with each of the bacilli was greater than in the silages containing each of the bacilli alone. The acid content of the silages with lactic acid bacteria was not increased by the presence of bacilli. It is concluded that bacilli can compete with lactic acid bacteria for substrate but contribute little to the fermentation and in this context should be discouraged.     

Effect of NaCl-tolerant lactic acid bacteria and NaCl on the fermentation characteristics and aerobic stability of silage

NaCl-tolerant lactic acid bacteria (LAB) strains LC-10 ( Lactobacillus casei ) and LP-15 ( Lact. plantarum ) and NaCl were used as additives to sorghun ( Sorghum bicolor ). Numbers of LAB were significantly ( P < 0·05) higher in all the additive-treated silages than in the control silage at an early stage of ensiling. During the fermentation process, addition of NaCl or LAB effectively inhibited the growth of aerobic bacteria and clostridia, but not yeasts. All the additive-treated silages had significantly ( P < 0·05) lower pH, ammonia nitrogen content, dry matter loss and gas production but significantly ( P < 0·05) higher lactic acid content and residual water soluble carbohydrates compared with the control silage. The improvement in silage quality was in the order : LAB > NaCl > control. Yeast counts were high in all additive-based silages and they increased during the exposure of the silages to air. As a result, these silages suffered aerobic deterioration, whereas the control silage was stable. The results confirmed that the NaCl or LAB improved fermentation quality but did not prevent aerobic deterioration of the silage.     

The effect of applying lactic bacteria at ensilage on the chemical and microbiological composition of vetch, wheat and alfalfa silages

The effect of applying a commercial lactic acid bacterial inoculant, at 5.6 × 10⁴ cfu/g fresh material, to vetch, wheat, direct-cut and wilted alfalfa silages has been studied under laboratory conditions, and on wheat also under farm conditions. Dry matter losses in the inoculated vetch and alfalfa silages were smaller than in the control silages, due to improved fermentation in the former as indicated by a faster and larger pH decrease and by a faster and larger lactic acid build-up. Volatile fatty acid analysis also indicated more efficient fermentation patterns in the inoculated vetch and alfalfa silages with less ethanol, acetic and butyric acids compared with the respective control silages. The inoculant suppressed enterobacteria and clostridia in the inoculated direct-cut alfalfa silage. The inoculant did not have a great effect on the wheat silages.     

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.     

Fermentation patterns of forage sorghum ensiled under different environmental conditions

The effects of temperature, aerobic and anaerobic conditions in the silo and plant characteristics [water-soluble carbohydrate (WSC) contents, growing season] on the fermentation characteristics of a tropical forage species, Sorghum bicolor cv. sugar-drip, were investigated. Silages fermented in oxygen-impermeable bags were well preserved and had low pH (3.7), high lactic acid [72 g kg^–1 dry matter (DM) 80% of total acids], and low butyric acid (0.12 g kg^–1 DM) and ammonia nitrogen (NH₃–N) (57 g kg^–1 total nitrogen contents. Conversely, the use of oxygen-permeable bags as silos allowed aerobic decomposition of the ensiled forages. Increasing the incubation temperature lowered the population of lactic acid bacteria, reduced lactic acid production and caused the pH to rise. The heterofermentative Leuconostoc spp. predominated on fresh forages but homofermentative Lactobacillus plantarum began to dominate after 5 and 8 days of fermentation. Heterofermentative lactobacilli, notably Lactobacillus brevis, were dominant among the isolates obtained from 100-day silages. Varying the WSC contents, by crushing and/or chopping the forage, and growing season did not significantly affect the fermentation quality of the resulting silages. It was concluded that the maintenance of anaerobic conditions is essential if good quality silage is to be produced from tropical forage species.     

The effect of Propionibacterium acidipropionici, with or without Lactobacillus plantarum, on the fermentation and aerobic stability of wheat, sorghum and maize silages

AIMS: To determine the effect of Propionibacterium acidipropionici, alone or in combination with Lactobacillus plantarum, on the fermentation and aerobic stability of wheat, sorghum and maize silages. METHODS AND RESULTS: The inoculants were applied at 1.0 x 10(6) CFU g(-1). Silages with no additives served as control. Fresh forages were sampled prior to ensiling. Three jars per treatment were sampled on days 2, 4, 8, 16 and 60 after ensiling, for chemical and microbiological analysis. At the end of the ensiling period, the silages were subjected to an aerobic stability test. The P. acidipropionici-inoculated silages had significantly higher levels of acetic and propionic acid than the L. plantarum or P. acidipropionici + L. plantarum-inoculated silages (P < 0.05). Therefore, yeast activity was impaired in the P. acidipropionici-inoculated silages. As a result, P. acidipropionici decreased CO(2) production and improved aerobic stability of wheat, sorghum and maize silages. However, the combination of P. acidipropionici + L. plantarum did not improve aerobic stability of the silages. CONCLUSIONS: The P. acidipropionici was very effective in protecting the wheat, sorghum and maize silages exposed to air under laboratory conditions, probably because the acidic environment under ensiling conditions is favourable for this micro-organism. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of P. acidipropionici, as a silage inoculant can improve the aerobic stability of silages by inhibition of yeast activity.     

Inhibition of Listeria monocytogenes by defined lactic microflora in gnotobiotic silages of lucerne,fescue, ryegrass and maize — Influence of dry matter and temperature

The development of L. monocytogenes was studied in gnotobiotic silages of four plant species (ryegrass, fescue, lucerne, maize) with various dry matter concentrations and incubation temperatures. The microflora of these silages was perfectly defined and included Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus casei, Pediococcus spp., Enterobacter liquefaciens and Clostridium tyrobutyricum. The results show that in silages of good quality not only is there no development of L. monocytogenes, but the cells initially present are eliminated. It is therefore most unlikely that a silage of good quality with a pH lower than 5 plays a contaminating role. The hypothesis according to which silages, especially those of bad quality, contain factors favouring contamination seems to be more plausible.     

Fermentative profile and lactic acid bacterial dynamics in non-wilted and wilted alfalfa silage in tropical conditions

This study was conducted to evaluate the fermentative profile and microbial populations of wilted and non-wilted alfalfa silages ensiled with or without inoculant and the population dynamics of lactic acid bacteria (LAB) of wilted alfalfa plant and theirs silage. A 2?×?2?×?6 factorial arrangement was used, with the absence or presence of wilting (W), with and without bacterial inoculant (I) and six fermentation periods (P) (1, 3, 7, 14, 28 and 56 days), in a completely randomized design, with three replicates. The alfalfa was slightly wilted for 6 h and increased the dry matter content from 133.9 to 233.4 g/kg. It was performed the cultivation, followed by the isolation of LAB from samples of alfalfa forage before ensiling and its silage only in non-inoculated silages, after different fermentation periods. DNA was extracted from the isolated strains of LAB; the 16S rRNA gene sequences were amplified by PCR and the sequences were compared to those available from the GenBank database. Wilting provided silages with lower pH, ammonia nitrogen and acetic acid concentrations. The wilting process did not alter the amount of LAB; however, it affected the LAB diversity of the silages. The Lactobacillus plantarum was the predominant species in non-wilted and wilted silages.

     

Acid-bile, antibiotic resistance and inhibitory properties of propionibacteria isolated from Turkish traditional home-made cheeses

In this study, a total of 29 Propionibacterium spp. were isolated from traditional home-made Turkish cheese samples. As a result of the identification, isolates were identified as Propionibacterium freudenreichii subsp. freudenreichii (15 strains), Propionibacterium jensenii (12), and Propionibacterium thoenii (2). All isolates and 5 reference strains were examined for their abilities to survive at pH 2.0, 3.0, 4.0, 5.0 and in the presence of 0.06, 0.15 and 0.30% bile salts, their influence on the growth of food-borne and spoilage bacteria, as well as their sensitivity against 11 selected antibiotics. Only seven propionibacteria strains survived in both the acidic and bile salt environments. Propionibacterium spp. strains strongly inhibited growth of the Escherichia coli ATCC 11229 and Shigella sonnei Mu:57 strains (91%). All propionibacteria strains were sensitive to a majority of the antibiotics used in the investigations. Overall, dairy propionibacteria showed high antibacterial activity, resistance to pH 4.0, 5.0, high resistance to bile salts and will provide an alternative source to Lactobacillus and Bifidobacterium as probiotic culture.     

Growth of Escherichia coli O157 in poorly fermented laboratory silage: a possible environmental dimension in the epidemiology of E. coli O157

Laboratory silages, inoculated with either c. 1000 cfu g-1, an atoxigenic Escherichia coli O157 or a toxigenic E. coli O157 isolate, were made in plastic bags which permitted limited aerobic spoilage. Replicate bags of each treatment were opened at weekly intervals after incubation at 20 degrees C. In all silages the fermentation was slow and aerobic spoilage with visible moulding ocurred at the tie ends after 7 d. In all the aerobically spoiled silages Enterobacteriaceae reached over 107 cfu g-1 within 1 week. The E. coli in control silages increased from barely detectable levels to 104 cfu g-1 within 13 d; over the same period both strains of E. coli O157 increased from 103 to 107 cfu g-1. The increases in the poorly fermented interior of the silage bags were initially similar but declined slightly as the pH fell. It is suggested that faecal contamination of grass followed by poor silage management may be a factor in the persistence of E. coli O157 carriage in ruminants.     

青藏高原垂穗披碱草青贮饲料中乳酸菌的多样性及低温发酵菌株的筛选

【目的】探究青藏高原垂穗披碱草青贮饲料中乳酸菌的多样性,筛选在低温条件下(10、15和25°C)生长性能较好的优良菌株。【方法】将垂穗披碱草青贮饲料中分离纯化的乳酸菌进行形态特征观察及16S r RNA基因测序鉴定;用MRS液体培养基在10、15和25°C条件下分离、初筛乳酸菌,选取高吸光度值的菌株作为优势菌株。用绿汁发酵液在10、15和25°C条件下培养测定其p H值,选取低p H值菌株作为优势菌株,并综合MRS培养基筛选结果确定优良菌株。【结果】从不同温度和发酵阶段的垂穗披碱草青贮饲料中共分离得到108个乳酸菌菌株,它们分属于6个属、18个种。其中,清酒乳杆菌LS-24在15°C条件下发酵液p H值显著降低(P0.05),戊糖片球菌PP-63在发酵初期生长速度较快,植物乳杆菌LP-21在15°C条件下发酵液p H值降至3.9且有最大活菌数。【结论】在青藏高原垂穗披碱草青贮饲料中发现的乳酸菌属基本涵盖了前人在常温青贮饲料中发现的所有属,但种数略少;在108株菌中,清酒乳杆菌LS-24、戊糖片球菌PP-63和植物乳杆菌LP-21在低温条件下均表现出较好的繁殖和发酵特性,可作为青贮饲料低温发酵的备选菌株。     

Characterization and enhanced production of enterocin HJ35 byEnterococcus faecium HJ35 isolated from human skin

A strain named as HJ35 was isolated from the skin of sixty-five men and fourteen women for acne therapy, in order to find an effective antimicrobial agent againstPropionibacterium acnes. Isolate HJ35 was identified asEnterococcus faecium based on 16 rDNA sequence and produced enterocin HJ35 having antimicrobial activities against most lactic acid bacteria,Enterococcus spp.,Staphylococcus aureus, S. epidermidis, Clostridium perfringens, some bacilli,Micrococcus flavus, Listeria monocytogenes, L. ivanovii, Escherichia coli, Pseudomonas fluorescens andPropionibacterium acnes, in the modified well diffusion method. Especially, enterocin HJ35 showed a bactericidal activity againstPropionibacterium acnes P1. The antimicrobial activity of enterocin HJ35 was disappeared completely with the use of protease XIV. But enterocin HJ35 activity is very stable at high temperature (up to 100°C for 30 min), in wide range of pH 3.0∼9.0), and by treatment with organic solvents. The apparent molecular mass of enterocin HJ35 was estimated to be approximately 4∼4.5 kDa on detection of its bactericidal activity after SDS-PAGE. In batch fermentation ofE. faecium HJ35, enterocin HJ35 was produced at the midlog growth phase, and its maximum production was obtained up to 2,300 AU/mL at the late stationary phase. By employing fed-batch fermentation, the enhanced production of enterocin HJ35 was achieved up to 12,800 AU/mL by feeding with 10 g/L glucose or 6 g/L lactate.     

Enzyme assisted ensiling of alfalfa with enzymes by solid substrate fermentation

A crude enzyme preparation, obtained by solid substrate fermentation (SSF) with a Gliocladium spp. and added at the 5% level to wilted or non-wilted alfalfa, improved the fermentation characteristics and stability of alfalfa silages as effectively as commercial preparations, Novo-Nordisk Celluclast 1.5 L and Viscozyme 120 L, applied at the 0.025% level. The effective dose of the crude enzyme costs about one-fourth of the cost of the commercial enzymes.     

Characterization and identification of Pediococcus species isolated from forage crops and their application for silage preparation.

Pediococcus species isolated from forage crops were characterized, and their application to silage preparation was studied. Most isolates were distributed on forage crops at low frequency. These isolates could be divided into three (A, B, and C) groups by their sugar fermentation patterns. Strains LA 3, LA 35, and LS 5 are representative isolates from groups A, B, and C, respectively. Strains LA 3 and LA 35 had intragroup DNA homology values above 93.6%, showing that they belong to the species Pediococcus acidilactici. Strain LS 5 belonged to Pediococcus pentosaceus on the basis of DNA-DNA relatedness. All three of these strains and strain SL 1 (Lactobacillus casei, isolated from a commercial inoculant) were used as additives to alfalfa and Italian ryegrass silage preparation at two temperatures (25 and 48 degrees C). When stored at 25 degrees C, all of the inoculated silages were well preserved and exhibited significantly (P < 0.05) reduced fermentation losses compared to that of their control in alfalfa and Italian ryegrass silages. When stored at 48 degrees C, silages inoculated with strains LA 3 and LA 35 were also well preserved, with a significantly (P < 0.05) lower pH, butyric acid and ammonia-nitrogen content, gas production, and dry matter loss and significantly (P < 0.05) higher lactate content than the control, but silages inoculated with LS 5 and SL 1 were of poor quality. P. acidilactici LA 3 and LA 35 are considered suitable as potential silage inoculants.     

The effects of concentrate energy source on feed intake and rumen fermentation parameters of dairy cows offered a range of grass silages

The effects of concentrate energy source on feed intake and rumen fermentation parameters of lactating dairy cattle, offered one of three grass silages differing in fermentation and intake characteristics, were evaluated in a partially balanced changeover design experiment involving four rumen fistulated dairy cows. Three silages were harvested using different management practices prior to and at ensiling. Silages A and C and silage B were harvested from primary or secondary regrowths either untreated or treated with a bacterial inoculant. For silages A, B and C, dry matter (DM) concentrations were 334, 197 and 183 g/kg (S.E. 3.1), pH values 4.00, 4.79 and 4.80 (S.E. 0.042) and ammonia nitrogen (N) concentrations were 123, 319 and 295 g/kg total N (S.E. 20.0), respectively. Two concentrates were formulated to contain similar crude protein, effective rumen degradable protein, digestible undegradable protein and metabolisable energy concentrations but using different carbohydrate sources to achieve a wide range of starch concentrations. For the low and high starch concentrates starch concentrations were 17 and 304 g/kg DM and acid detergent fibre concentrations were 170 and 80 g/kg DM, respectively. The silages were offered ad libitum, supplemented with 10 kg fresh concentrate daily. For silages A, B and C, DM intakes were 10.9, 7.2 and 8.6 kg/day and concentrate energy sources did not alter (P>0.05) intake. Increasing the level of starch in the concentrate decreased the molar concentration of acetate (P<0.05) and tended to increase the molar concentration of propionate (P<0.1). Silage type altered the molar concentration of acetate (P<0.01) and the acetate:propionate ratio (P<0.05). There were no silagetype×concentrate interactions (P>0.05) on silage intake or rumen fermentation parameters. It is concluded that when concentrate and silage form equal proportions of the diet, the composition of the silage has an over-riding influence on rumen fermentation parameters. Furthermore, the changes in milk fat concentration, observed in a concurrent production study, due to changes in silage and concentrate types can be accounted for by changes in the ratio of lipogenic to glucogenic precursors in the rumen fluid.     

Temperate forages ensiled with molasses or fresh cheese whey: Effects on conservation quality, effluent losses and ruminal degradation

Effects of ensiling and adding molasses or increasing levels of fresh cheese whey on the conservation and rumen degradability of temperate pasture silages were evaluated. Forage from three paddocks of mixed grass and legume pastures was used to make 45 silages (15 silages per paddock) with 5 treatments, including silage without additives (control), silage with 15 g/kg dehydrated molasses and silage with 20, 50 and 100 g/kg fresh cheese whey. The chemical composition and fermentation quality (i.e., pH, ammonia N, loss of dry matter (DM) and neutral detergent fibre (NDF), effluent production) of the silages were determined. Fresh and ensiled materials were evaluated for in situ rumen degradability. Ensiling reduced DM and NDF rumen degradability (P<0.01). When additives were employed, the reduction of DM degradability of the silages decreased (P≤0.03). Addition of molasses led to the lowest pH (P<0.01) and DM losses (P<0.01), and highest DM degradability (P<0.01). The conservation and DM degradation results of dried molasses silage was superior to those of fresh whey silages. In general, an increase in the level of whey increased DM degradability (P≤0.03), but linearly increased effluent production (P<0.01) and losses (P<0.01).     

Propionic acid fermentation of lactose by Propionibacterium acidipropionici: effects of pH

Batch propionic acid fermentation of lactose by Propionibacterium acidipropionici were studied at various pH values ranging from 4.5 to 7.12. The optimum pH range for cell growth was between 6.0 and 7.1, where the specific growth rate was approximately 0.23 h(-1). The specific growth rate decreased with the pH in the acids have been identified as the two major fermentation products from lactose. The production of propionic acid was both growth and nongrowth associated, while acetic acid formation was closely associated with cell growth. The propionic acid yield increased with decreasing pH; It changed from approximately 33% (w/w) at pH 6.1-7.1 to approximately 63% at pH 4.5-5.0. In contrast, the acetic acid yield was not significantly affected by the pH; it remained within the range of 9%-12% at all pH values. Significant amounts of succinic and pyruvic acids were also formed during propionic acid fermentation of lactose. However, pyruvic acid was reconsumed and disappeared toward the end of the fermentation. The succinic acid yield generally decreased with the pH, from a high value of 17% at pH 7.0 to a low 8% at pH 5.0 Effects of growth nutrients present in yeast ex-tract on the fermentation were also studied. In general, the same trend of pH effects was found for fermentations with media containing 5 to 10 g/L yeast extract. However, More growth nutrients would be required for fermentations to be carried out efficienytly at acidic pH levels.     

Survival of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> in Wilted and Additive-Treated Grass Silage

Grass was field-dried to 3 different dry matter (DM) levels (200, 430 and 540 g/kg) and inoculated with 10⁶–10⁷ cfu/g of a Listeria monocytogenes strain sharing a phagovar occasionally involved in food-borne outbreaks of listeriosis. Formic acid (3 ml/kg) or lactic acid bacteria (8·10⁵/g) with cellulolytic enzymes were applied only to forages with low and intermediate DM levels. Forages were ensiled in laboratory silos (1700 ml) and were stored at 25°C for 30 or 90 days. After 90 days of storage, L. monocytogenes could not be detected in any silo, except one with the high dry matter grass without additive. After 30 days of storage, between 10² and 10⁶ cfu L. monocytogenes/g silage were isolated from the untreated silages. Increasing the DM content from 200 to 540 g/kg did not reduce listeria counts possibly because of the lower production of fermentation acids (higher pH). In silages treated with additives, counts of L. monocytogenes were always lower than in silages without additive. In wet silages (DM 200 g/kg) both additives were effective, but in the wilted silages (DM 430 g/kg) only the bacterial additive reduced listeria counts below detection level. Listeria counts were highly correlated to silage pH (r = 0.92), the concentration of lactic acid (r = -0.80) and the pooled amount of undissociated acids (r = -0.83).