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.     

A PHYSICAL-CHEMICAL DIFFERENCE IN ANTIBODIES AGAINST THE S AND R VARIANTS OF A SINGLE BACTERIAL STRAIN

1. Rabbits were immunized with Bact. typhosum 0 901 S and 0 901 R, over a long period. Homologous and heterologous strains were sensitized with sera obtained from weekly bleedings. Agglutination titer was recorded, and the isoelectric points of the bacteria maximally sensitized were determined. 2. 0 901 S maximally sensitized with homologous immune serum had isoelectric points which became more alkaline as immunization progressed, covering a range of pH 4.8 to 5.5. 3. Strain 0 901 R maximally sensitized with homologous immune serum had isoelectric points which became more alkaline as immunization progressed, covering the range of pH 5.0 to 5.9. 4. Both 0 901 S and 0 901 R maximally sensitized with heterologous serum had isoelectric points lower than when sensitized with homologous serum. 5. The isoelectric points of both forms sensitized with increasing concentrations of homologous immune serum were determined. Increasing concentrations of homologous immune serum shifted the isoelectric point of 0 901 R from less than 2.2 for the unsensitized bacteria progressively to the alkaline side until the maximum values previously mentioned were reached. Increasing concentrations of homologous immune serum conferred upon 0 901 S isoelectric points which became only slightly more alkaline in maximal sensitization. 6. The electrophoretic mobilities of 0 901 S and 0 901 R, in each case maximally sensitized with homologous hyperimmune serum, were found to differ significantly over the whole range of pH studied.     

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.     

Growth and survival of lactic acid bacteria in lucerne silage

A rifampicin-resistant variant of two strains of Lactobacillus plantarum, one strain of Pediococcus acidilactici, and one strain of Enterococcus faecium were used for the experimental production of lucerne silage. Laboratory silage without inoculants served as a control. Counts of total anaerobes, total lactic acid bacteria (LAB), lactobacilli, pediococci, and enterococci were determined on days 14, 21, 30, 49, and 60 of lucerne fermentation. LAB dominated in silage microflora, reaching a percentage between 59 and 95 % of total anaerobes. Lactobacilli were found as a predominant group of LAB during the whole study. Lactobacilli reached numbers 8.74 log CFU/g in treated silage and 8.89 log CFU/g in the control at the first observation. Their counts decreased to 4.23 and 4.92 log CFU/g in treated silage and the control, respectively, on day 63 of fermentation. Similar decreases were observed in all bacterial groups. The treated silage samples possessed lower pH (4.2 vs. 4.5 in control samples) and contained more lactic acid compared to control silage. The identity of re-isolated rifampicin-resistant bacteria with those inoculated to the lucerne was evaluated by fingerprinting techniques. The fingerprint profiles of re-isolated bacteria corresponded to the profiles of strains used for the treatment. It could be concluded that supplemented LAB dominated in laboratory silage and overgrew naturally occurring LAB.     

Bacteriological and chemical changes occurring in Bunker-stored silage covered with biodegradable coating

AIMS: To evaluate the efficacy of a biodegradable silage coating for the ability to protect timothy (Phleum pratensa) type silage against spoilage and its quality under natural conditions. METHODS AND RESULTS: Triplicate mini-silos of silage were prepared for three treatments (1: uncoated; 2: coated with biodegradable coating and 3: sealed with plastic), two types of storage (unprotected or protected from rain) and 10 sampling times (0, 7, 14, 21, 28, 35, 42, 56, 63 and 70 days postensiling). Triplicate mini-silos were opened at each sampling time for microbiological (total aerobic bacteria, lactic acid bacteria, moulds and yeasts) and biochemical analyses [pH, dry matter (DM), water-soluble sugars (WSC), lactic (LA), acetic, propionic and butyric acids content]. The study showed that at day 70, counts of moulds and yeasts in silages protected against rain and coated with biodegradable coating were 5.98 log CFU g(-1) when compared with 5.92 and 3.62 log CFU g(-1) in samples from plastic-sealed silage and uncoated silage, respectively. The pH was low and stable pH (4.34) when compared with uncoated (7.17) and plastic sealed (8.34) silages (P < or = 0.05). A DM, WSC and LA content of 421.7, 13.4 and 20.9 g kg(-1) was, respectively, observed. For silage stored outdoors, a level of moulds and yeasts of 3.77 log CFU g(-1) of silage was also observed in silages coated with biodegradable coating after 28 days of storage. A stable pH showing a mean value of 4 was also observed. The pH, DM, WSC and LA content were, respectively, 4.18, 341.1, 13.34 and 31.8 g kg(-1) in these samples. After 70 days of storage, the level of moulds and yeasts on silage sealed with biodegradable coating was 7.73 log CFU g(-1). A DM, WSC and LA content of 291.9, 5.56 and 10.0 g kg(-1) was, respectively, observed. CONCLUSIONS: When compared with uncoated silage, the application of biodegradable coating can preserve the quality of silage for up to a month when exposed to rain and up to 70 days when protected from rain. SIGNIFICANCE AND IMPACT OF THE STUDY: Results emphasize the possibility of the use of a biodegradable coating as an alternative to plastic film for sealing horizontal bunker silos.     

沙颍河下游城市黑臭内河沉积物微生物群落季节变化特征

为了研究沙颍河下游城市黑臭内河不同季节沉积物微生物群落特征,对安徽省阜阳市黑臭内河中清河、七渔河表层沉积物进行16S rDNA高通量测序。结果发现:黑臭河流中沉积物的微生物多样性指数均不高,但是表现出一定的变化规律,即春季>冬季≥夏季>秋季;通过冗余性分析发现微生物多样性受季节与沉积物pH影响较显著。分析沉积物门水平上的微生物群落结构发现,季节、温度、TN及SOM对微生物影响较大。变形杆菌、厚壁菌门、绿弯菌门、疣微菌门、拟杆菌门和放线菌门等优势菌门的相对丰度在季节水平上存在差异,春季厚壁菌门、绿弯菌门、放线菌门和酸杆菌门相对丰度较高,其中绿弯菌门和酸杆菌门是已知指示污染的微生物,变形杆菌门相对较少。秋季疣微菌门与拟杆菌门相对丰度显著减小,变形杆菌门相对其他季节显著增加。样品中共发现16个硫酸盐还原菌(SRB)菌属,其中Desulfoprunum是丰度最高的菌属。春季沉积物中SRB的类群最多,相对丰度最大;硫酸盐还原菌群与SO42-、TN、SOM、Cl–等呈显著正相关。上述结果为营养盐控制时机的选择从而有效避免河流中黑臭物质的产生提供了一定参考。     

An investigation into the sources of lactic acid bacteria in grass silage

During the 1983 silage making season an investigation was carried out to determine the contribution made by farm machinery to the numbers of lactic acid bacteria present on grass. Although no lactic acid bacteria were isolated from the standing crop, up to 10³/g were found in the grass as it entered the clamp. Examination of machinery in close contact with the grass revealed lactic acid bacteria on the mower blades and on the forks of the rough-terrain vehicle used to pack the grass into the clamp. During the 1984 season the occurrence of lactic acid bacteria in particular batches of grass from the standing crop through to silage after 2 d in the clamp was studied. There was a gradual increase in numbers of lactic acid bacteria in the grass during the process of mowing and collection. Wilting resulted in a greater initial increase in numbers but non-wilted silage had the highest count after 48 h. The majority of organisms isolated from machinery before silage making were streptococci. After silage making the number and proportions of organisms changed, with numbers increasing in all cases, particularly from the mower with lactobacilli and streptococci predominating which indicates that passage through the machinery served to inoculate the grass.     

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.     

BACTERIAL CHANGES IN EXPERIMENTAL LABORATORY SILAGE

SUMMARY: In preliminary experiments with grass silage made in the laboratory under controlled conditions bacterial numbers reached a peak earlier in silage held at 30° than at 22°, although maximum numbers tended to be greater at the lower temperature. Wilting the grass before it was ensiled delayed bacterial action, while chopping the grass hastened the action and helped to ensure a low pH in the silage.     

Biological ensiling of sugarcane tops

The following lactic acid bacteria were isolated from sugarcane tops silage:Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus brevis, Lactobacillus delbrueckii, Pediococcus cerevisiae, Leuconostoc mesenteroides andStreptococcus lactis. The isolates were grown in a synthetic medium and the final pH, sugar uptake and the effect of adding CaCO₃ and L1, L2 and L3 factors was observed. The importance of the buffering capacity of the ensiled material for the silage process with a view to the occurrence of lactic acid bacteria is discussed.     

低温乳酸菌复合系的构建、培养条件优化及在玉米干秸杆微贮中的应用

[目的]研究筛选得到的低温乳酸菌复合菌系(LAC-1)的最佳培养条件及应用于玉米干秸杆微贮的效果.[方法]通过连续限制性培养的方法,以pH为指标进行低温乳酸菌复合系的筛选;利用单因素试验、Box-Benhnken试验设计和响应面的分析方法对低温乳酸菌复合系的培养条件进行优化.[结果]筛选获得培养液pH下降迅速、变化稳定及接种干玉米秸杆后可降低发酵体系pH、提高发酵饲料感官品质的低温乳酸菌复合菌系LAC-1.培养时间对LAC-1的pH降低速度影响最大,温度次之,接种量影响最小;响应面分析LAC-1最佳的培养条件为培养温度9.5℃、接种量3.3％和培养时间139 h.经过优化,LAC-1培养过程中pH下降到4.0的时间比优化前缩短48 h,乳酸菌数提高了20.5％.LAC-1接种于干玉米秸秆发酵5d时,秸秆发酵体系pH降至4.1;与对照相比,秸秆发酵饲料可散发出甜酸香味,外观质地松散、色泽鲜亮,可溶性碳水化合物含量降低了46.3％,霉菌、恶臭醋酸杆菌和酵母菌数量显著降低,乳酸菌数量提高了33.3％.[结论]接种LAC-1可促进干玉米秸秆低温微贮进程,提高饲料品质.     

Amylolytic lactic acid bacteria in fish silage

An ∝aL-amylase activity has been observed in lactic acid bacteria occurring initially in fermented fish silage. The organisms belong to the genus Leuconostoc . The main fraction of the amylolytic enzyme produced by one of the isolated bacteria is cell-bound and is released into the medium at a late stage of growth. Treating cells with ultrasound or Triton X-100 increases enzyme activity in the culture filtrate. The pH range for enzyme activity is 5.0–7.0, with an optimum at pH 6.0. The enzyme is extremely labile at pH 8.0 and is inactivated at temperatures above 50°C at pH 5.8. Two enzyme fractions were found by isoelectric focusing, the main one at pH 5.00 and another at pH 4.5. Chromatography on DEAE cellulose gave two active peaks.     

Diversity of a stable enrichment culture which is useful for silage inoculant and its succession in alfalfa silage

Alfalfa is a kind of forage that is difficult to ensile for good quality. Therefore, inoculants are always used to enhance the preservation of alfalfa silage. Through continuous restricted subcultivation, a lactic acid bacteria community (Al2) was selected from well-fermented alfalfa silage, which sharply decreased the pH level and produced a large amount of lactic acid. The adding of Al2 to alfalfa at ensiling resulted in a more rapid drop in pH and higher levels of lactic acid, and it also reduced the ammonia-nitrogen content significantly (P < 0.01). Plate isolation, denaturing gradient gel electrophoresis (DGGE) and the construction of a 16S rRNA gene clone library were used to identify the composition diversity of the Al2 community; seven strains were detected in the community, the predominant strain belonging to Lactobacillus plantarum. Samples of alfalfa silages of duration 0, 2, 5, 10, 20 and 30 days were studied with DGGE analysis. The DGGE band patterns of Al2-treated and non inoculated were rather different, and the components of Al2 were the dominant bacteria in Al2-treated silages, especially L. plantarum, while Pediococcus pentosaceaus was predominant in naturally fermented alfalfa silage.     

玉米秸秆青贮过程中优势细菌多样性分析

通过从发酵0、1、3、5、15、30和60d自然发酵的青贮玉米(CK)和采用菌剂处理的青贮玉米(处理1)上取样,分别进行pH值测定和菌群总DNA的提取。总DNA经纯化后采用引物341F-GC和518R进行细菌16S rDNA V3区PCR扩增,扩增产物进行变性梯度凝胶电泳(DGGE)分离并回收优势条带和变化明显的条带进行测序分析,了解秸秆饲料发酵过程中的优势细菌多样性及动态变化规律,从而为现有青贮接种菌的改良奠定理论基础。研究结果表明:采用菌剂处理的青贮玉米pH值下降更快,其优势细菌种类更丰富,且各优势菌的丰度比对照组中相应菌的丰度更强。     

The survival of silage inoculant lactic acid bacteria in rumen fluid

AIMS: To determine whether lactic acid bacteria (LAB) used in inoculants for silage can survive in rumen fluid (RF), and to identify those that survive best. METHODS AND RESULTS: Twelve commercial silage inoculants were added at 107 CFU ml-1 to strained RF (SRF) taken from dairy cows, with and without 5 g l-1 glucose and incubated in vitro at 39 degrees C. Changes in pH, LAB numbers and fermentation products were monitored for 72 h. In the inoculated RF with glucose, the pH decreased and numbers of LAB increased. The inoculants varied with regard to their effect on pH change and growth. In the SRF, both with and without glucose, the pH values of the inoculated samples were generally higher than those of the uninoculated controls throughout most of the incubation period. This may suggest a positive effect on the rumen environment. CONCLUSIONS: LAB used in silage inoculants can survive in RF in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first step in studying the probiotic potential of silage LAB inoculants for dairy cattle. The survival of these LAB in RF may enable them to interact with rumen microorganisms and to affect rumen functionality.     

乳酸杆菌体内外对阴道菌群及pH值生物学影响的研究

本研究利用从正常妇女阴道内分离出来的乳酸杆菌,从微生态学角度进行体内外生物拮抗试验,以探索其对阴道菌群及生境ｐＨ的生物学影响。结果表明：乳酸杆菌于体内外对阴道的葡萄球菌、肠杆菌、酵母菌有生物抑制作用;对生境的ｐＨ有生物学影响,主要表现为使生境ｐＨ下降。通过上述生物学影响调节生境菌群平衡及内环境的稳定。     

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.     

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.).     

QUANTIFICATION OF THE SINUSOIDAL TRAJECTORY IN TISSUE MASS AND CONDITION INDICES OF A BIVALVE (EGERIA RADIATA) IN THE CROSS RIVER, NIGERIA

Variation in monthly values of dry tissue mass (DTM) and twodifferent types of condition indices of the bivalve Egeria radiata(Lamarck, 1804) was studied for 32 consecutive months. To quantifythe seasonal trajectory of variation these data were fittedto one- and two-compartment sinusoidal models; and also to aquadratic, cubic and quartic models. The cyclic events in thevariation of the DTM and both indices of condition were adequatelydescribed by the one-compartment sinusoidal model whose parametersare biologically interpretable. The fit of the data is progressivelyimproved as one moves from the quadratic through the cubic tothe quartic model whose fit was best. Although biological meaningcould be found for the one-compartment sinusoidal model, thebiological meaning of the coefficients of the parabolic-basedmodels are obscure. Multiple regression analysis shows thatof all the four environmental parameters (temperature, salinity,pH and phytoplankton) tested, temperature has the greatest effecton DTM and one of the condition indices, while pH has the greatestimpact on the other condition index. This implies that the twoindices are not only intrinsically different, but that theyrespond to different sets of factors. Inter- and intra-specificcomparison of the many recommended ‘standard’ indicesof condition found in the literature, especially with regardto their innate sensitivity to seasonal variation in internal(e.g. state of gravidity) and external (physicochemical parametersof water) factors is necessary. (Received 1 May 1996; accepted 30 August 1996)