Selection and application of Streptococcus bovis as a silage inoculant.

Three strains of Streptococcus bovis, a homolactic bacterium capable of utilizing starch, were evaluated for growth kinetics and ability to decrease the pH of alfalfa silage. A selected strain was evaluated for its competitiveness as an inoculant with Enterococcus faecium, an organism used in inoculants, and for its ability to enhance the effect of a commercial inoculant. Testing was completed over three studies using wilted alfalfa (28 to 34% dry matter) ensiled into laboratory silos. Treatments were control, E. faecium, E. faecium and commercial inoculant, S. bovis, and S. bovis and commercial inoculant. Replicate silos were emptied and analyzed at 0.5, 1, 2, 4, 8, and 40 days for pH, fermentation products, and nitrogen fractions. S. bovis alone lowered the pH quicker and improved silage parameters early in the fermentation compared with E. faecium, the commercial inoculant, and control treatments. When combined with a commercial inoculant, S. bovis lowered pH more quickly than the commercial inoculant alone and E. faecium plus commercial inoculant. At 40 days, S. bovis combination had lower pH and ammonia nitrogen and acetate contents than the E. faecium combination. Starch in the silage was not utilized by S. bovis as had been anticipated. Results indicate that S. bovis was more effective than E. faecium as a silage inoculant and could enhance a commercial inoculant on low-dry-matter alfalfa.     

Characterization of Streptococcus bovis bacteriophages.

About 25 Streptococcus bovis bacteriophages were isolated from abattoir wastes, bovine rumen fluid, and lysogenic strains of S. bovis. Eight phages were selected and characterized by morphology, stability, rate of adsorption, single-step growth curve, serum neutralization, and antigenic relationship. Two distinct morphological phage types were found, one of which has not been previously reported for group D streptococci.     

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.     

Streptococcus bovis as a host for the expression of cloned polysaccharidase genes

The shuttle vector, pVA838, was used to introduce the xynD gene from the cellulolytic rumen anaerobe, Ruminococcus flavefaciens 17, into Streptococcus bovis JB1. Expression of xylanase and -(1,3-1,4)-glucanase activity due to xynD was demonstrated in culture supernatants. SDS-PAGE zymograms revealed the full-length xynD 90 kDa product, together with some proteolytic products. Activities due to the cloned xynD gene, and to the R. flavefaciens 17 endA endoglucanase gene present in the construct pVACMC1, decreased after early growth stages in batch cultures of S. bovis JB1, reflecting the sensitivity of the cloned R. flavefaciens 17 enzymes to inactivation in the presence of accumulated lactic acid. Of xynD activity, 40–80% was detected in the culture supernatant, indicating recognition of the xynD signal peptide by S. bovis JB1.     

Lasalocid-catalyzed proton conductance in Streptococcus bovis as affected by extracellular potassium.

The effect of extracellular potassium on lasalocid-catalyzed proton conductance in Streptococcus bovis 24 was measured by using the fluorescent probe 9-aminoacridine. Increasing external potassium concentration resulted in decreased proton flux into S. bovis cells exposed to the ionophore. These results suggest that lasalocid catalyzes K+/H+ exchange diffusion in S. bovis cells.     

Nisin resistance of Streptococcus bovis

The growth of Streptococcus bovis JB1 was initially inhibited by nisin (1 microM), and nisin caused a more than 3-log decrease in viability. However, some of the cells survived, and these nisin-resistant cells grew as rapidly as untreated ones. To see if the nisin resistance was merely a selection, nisin-sensitive cells were obtained from agar plates lacking nisin. Results indicated that virtually any nisin-sensitive cell could become nisin-resistant if the ratio of nisin to cells was not too high and the incubation period was long enough. Isolates obtained from the rumen were initially nisin sensitive, but they also developed nisin resistance. Nisin-resistant cultures remained nisin resistant even if nisin was not present, but competition studies indicated that nisin-sensitive cells could eventually displace the resistant ones if nisin was not present. Nisin-sensitive, glucose-energized cells lost virtually all of their intracellular potassium if 1 microM nisin was added, but resistant cells retained potassium even after addition of 10 microM nisin. Nisin-resistant cells were less hydrophobic and more lysozyme-resistant than nisin-sensitive cells. Because the nisin-resistant cells bound less cytochrome c, it appeared that nisin was being excluded by a net positive (i.e., less negative) charge. Nisin-resistant cells had more lipoteichoic acid than nisin-sensitive cells, and deesterified lipoteichoic acids from nisin-resistant cells migrated more slowly through a polyacrylamide gel than those from nisin-sensitive cells. These results indicated that lipoteichoic acids could be modified to increase the resistance of S. bovis to nisin. S. bovis JB1 cultures were still sensitive to monensin, tetracycline, vancomycin, and bacitracin, but ampicillin resistance was 1,000-fold greater.     

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.     

Nisin Resistance of Streptococcus bovis

The growth of Streptococcus bovis JB1 was initially inhibited by nisin (1 μM), and nisin caused a more than 3-log decrease in viability. However, some of the cells survived, and these nisin-resistant cells grew as rapidly as untreated ones. To see if the nisin resistance was merely a selection, nisin-sensitive cells were obtained from agar plates lacking nisin. Results indicated that virtually any nisin-sensitive cell could become nisin-resistant if the ratio of nisin to cells was not too high and the incubation period was long enough. Isolates obtained from the rumen were initially nisin sensitive, but they also developed nisin resistance. Nisin-resistant cultures remained nisin resistant even if nisin was not present, but competition studies indicated that nisin-sensitive cells could eventually displace the resistant ones if nisin was not present. Nisin-sensitive, glucose-energized cells lost virtually all of their intracellular potassium if 1 μM nisin was added, but resistant cells retained potassium even after addition of 10 μM nisin. Nisin-resistant cells were less hydrophobic and more lysozyme-resistant than nisin-sensitive cells. Because the nisin-resistant cells bound less cytochrome c, it appeared that nisin was being excluded by a net positive (i.e., less negative) charge. Nisin-resistant cells had more lipoteichoic acid than nisin-sensitive cells, and deesterified lipoteichoic acids from nisin-resistant cells migrated more slowly through a polyacrylamide gel than those from nisin-sensitive cells. These results indicated that lipoteichoic acids could be modified to increase the resistance of S. bovis to nisin. S. bovis JB1 cultures were still sensitive to monensin, tetracycline, vancomycin, and bacitracin, but ampicillin resistance was 1,000-fold greater.     

A transglucosylase of Streptococcus bovis

1. A transglucosylase has been separated from the α-amylase of Streptococcus bovis by chromatography of the cell extract on DEAE-cellulose. 2. The transglucosylase can synthesize higher maltodextrins from maltotriose, but maltose, isomaltose and panose do not function as donors. 3. Iodine-staining polysaccharide may be synthesized from maltotriose provided that glucose is removed. Synthesis from maltohexaose results in dextrins of sufficient chain length to stain with iodine, but again maltodextrins of longer chain length are formed when glucose is removed from the system. 4. The transglucosylase degrades amylose in the presence of a suitable acceptor, transferring one or more glucosyl residues from the non-reducing end of the donor to the non-reducing end of the acceptor. With [¹⁴C]glucose as acceptor the maltodextrins produced were labelled in the reducing glucose unit only. 5. The acceptor activities of 25 sugars have been compared with that of glucose. Maltose has 50%, methyl α-glucoside has 15%, isomaltose and panose each has 8% and sucrose has 6% of the accepting efficiency of glucose. Mannose and sorbose also had detectable activity. With the exception of maltose all these sugars produced a different series of dextrins from that obtained with glucose. 6. It was concluded that S. bovis transglucosylase transfers α-(1→4)-glucosidic linkages in the same manner as D-enzyme, but some differences in specificity distinguish the two enzymes. Unlike D-enzyme, S. bovis transglucosylase can transfer glucosyl units, producing appreciable amounts of maltose both during synthesis from maltotriose and during transfer from amylose to glucose. 7. No evidence was found that the transglucosylase was extracellular. The enzyme is cell-bound, and is released by treatment of the cells with lysozyme and by suspension of the spheroplasts in dilute buffer. 8. The transglucosylase may be responsible for the storage of intracellular iodophilic polysaccharide that occurs when the cells are grown in the presence of suitable carbohydrate sources.     

Azospirillum as a potential inoculant for agriculture

Azospirillum sp. contribute to increased yields of cereal and forage grasses by improving root development in properly colonized roots, increasing the rate of water and mineral uptake from the soil, and by biological nitrogen fixation. A better understanding of the basic biology of the Azospirillum—root interaction, aided by the application of genetic engineering techniques, may lead to greater efficiency in its use as a biofertilizer.     

Identification and characterization of the proBA operon of Streptococcus bovis.

A genomic DNA library of the rumen bacterium Streptococcus bovis was constructed in Escherichia coli, and recombinant plasmids able to complement proA and proB mutations of the host were found. Southern hybridization and restriction analysis showed that a 3.5-kb fragment of S. bovis DNA contained two genes, organized in an operon and coding for enzymes functionally similar to the glutamyl phosphate reductase-glutamyl kinase enzyme complex that in E. coli catalyzes the first step of proline biosynthesis. Complementation of the E. coli mutations was observed with the fragment inserted in both orientations, which suggested that the S. bovis proBA operon was transcribed from its own promoter. Genetic and biochemical data suggested that the proline biosynthetic pathway of S. bovis is similar to the one previously characterized for E. coli.     

Lasalocid-catalyzed proton conductance in Streptococcus bovis as affected by extracellular potassium

The effect of extracellular potassium on lasalocid-catalyzed proton conductance in Streptococcus bovis 24 was measured by using the fluorescent probe 9-aminoacridine. Increasing external potassium concentration resulted in decreased proton flux into S. bovis cells exposed to the ionophore. These results suggest that lasalocid catalyzes K+/H+ exchange diffusion in S. bovis cells.     

Identification of intracellular amylase activity in Streptococcus bovis and Streptococcus salivarius

The ruminal bacterium Streptococcus bovis has been demonstrated to produce an extracellular amylase activity. We previously reported on the cloning of a gene from S. bovis encoding for what was initially believed to be the extracellular amylase. DNA sequence analyses indicated that the amylase produced by the cloned gene did not match the N-terminus amino acid sequence of the purified extracellular amylase and contained no apparent leader sequence for secretion. Analyses of crude extracts demonstrated the presence of an intracellular amylase in S. bovis JB1 that differed in molecular weight (56,000) from that of the extracellular amylase (70,000). The 56,000 molecular weight amylase was identical to the amylase produced by Escherichia coli containing the cloned amylase gene. Low levels of intracellular amylase activity were also detected in other strains of S. bovis and also Streptococcus salivarius. Introduction of the plasmid pVA838 containing the cloned amylase gene into S. bovis and S. sanguis resulted in enhanced intracellular amylase production by both organisms. The amylase gene has been sequenced, and analysis of the deduced amino acid sequence for the amylase indicates a high degree of similarity with secreted amylases from Bacillus species.Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Effects of polyphenol oxidase on lipolysis and proteolysis of red clover silage with and without a silage inoculant (Lactobacillus plantarum L54)

Polyphenol oxidase (PPO) has been shown to reduce proteolysis and lipolysis in red clover through deactivation of proteolytic and lipolytic enzymes and/or through formation of protein–phenol–lipid complexes. This experiment investigated the time course of both lipolysis and proteolysis in two red clover lines with different PPO activities with and without addition of a silage inoculant to help understand the action of PPO in the silo, and its potential effects on protein and glycerol-based lipid conservation, and to determine effects of a more rapid pH reduction with inoculation on PPO activity. Four silages were prepared from high or low PPO precision chopped red clover in 60 test-tube-silos, each containing 120 g fresh weight: (a) high PPO red clover without inoculation (H−), (b) low PPO red clover without inoculation (L−), (c) high PPO red clover with inoculation (H+), and (d) low PPO red clover with inoculation (L+). Each treatment had three replicates for each time point of 1, 2, 4, 8 and 90 days. The inoculant used was Lactobacillus plantarum strain L54 applied at a rate of 10⁶ CFU/g fresh weight. Silage pH was reduced (P < 0.001) by inoculation with no effect of PPO. Inoculation had no effect on either lipolysis or free amino acid release, although more (P < 0.01) soluble protein and less (P < 0.01) ammonia-N was in inoculated silages. H silages had a lower level of both proteolysis (release of free amino acids, P < 0.05) and lipolysis (loss of membrane lipid, P < 0.01) than L red clover silages. Results indicate that PPO reduced proteolysis and lipolysis in the silo and that inoculation had no adverse effects on PPO activity.     

Protoplast Formation and Regeneration of Streptococcus bovis Cells

The simultaneous use of hen egg lysozyme and mutanolysin, N-acetylmuramidase SG, showed synergistic effects on the lytic action and more effectiveness for protoplast formation of Streptococcus bovis IFO 12058. The frequency of regeneration reached 100% after 7 days of incubation under the improved conditions and by a method in which protoplasts were overlaid with the agar medium layer.