Antibacterial activity of lactic acid bacteria included in inoculants for silage and in silages treated with these inoculants

AIMS: To determine antibacterial activity in lactic acid bacteria (LAB) silage inoculants and in wheat and corn silages which were treated with these inoculants. METHODS AND RESULTS: Wheat and two corn silages were prepared in 0.25 l sealed glass jars. Inoculant treatments were prepared for each type of silage with each of 10 LAB silage inoculants at inoculation rate of 10(6) CFU g(-1). Untreated silages served as controls. Antibacterial activity was determined in the inoculants and in their respective silages with Micrococcus luteus and Pseudomonas aeruginosa. Antibacterial activity was detected in nine of the 10 inoculants whereas such activity in the silages varied. Control silages did not have antibacterial activity. CONCLUSIONS: Many LAB silage inoculants have antibacterial activity and in some cases this activity is imparted on inoculated silages. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was conducted as part of a broader research objective, which is to find out how LAB silage inoculants enhance ruminant performance. The results of this study indicate that LAB silage inoculants produce antibacterial activity, and therefore, have a potential to inhibit detrimental micro-organisms in the silage or in the rumen.     

Biogenic amine production in grass, maize and total mixed ration silages inoculated with Lactobacillus casei or Lactobacillus buchneri

AIMS: To investigate the effects of inoculating Lactobacillus casei or Lacobacillus buchneri on the production of biogenic amines (BA) in silage. METHODS AND RESULTS: Wilted festulolium (Lolium perenne x Festuca pratensis), whole crop maize or a total mixed ration, consisting of wet brewer grains, lucerne hay, cracked maize, sugarbeet pulp, soyabean meal and molasses, was ensiled with or without the inoculation of either L. casei (>10(6) CFU g(-1)) or L. buchneri (>10(6) CFU g(-1)). Silages were opened after 60 days of storage, and the concentrations of histamine, tyramine, putrescine and cadaverine were determined. The inoculation of L. casei decreased all the BA regardless of the silage type. The effects of L. buchneri varied between the three silages; the tyramine and putrescine were increased in maize but were lowered in festulolium. Histamine was reduced in festulolium and the by-products, whereas no change was found in the maize silage. None of the inoculant strains produced the four BA in a synthetic medium, accounting for the actual ensiling except for tyramine and putrescine in maize. CONCLUSIONS: Wide variation would be found in the production of BA owing to the ensiling materials. The inoculation of L. casei can lower the BA concentration, while the effects of L. buchneri may vary considerably. The screening of BA-producing activity may help to reduce the risk of BA contamination in inoculated silage. SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of decarboxylase-negative L. buchneri can enhance the aerobic stability of silage without a concern regarding the large production of putrefactive BA.     

Presence of sourdough lactic acid bacteria in commercial total mixed ration silage as revealed by denaturing gradient gel electrophoresis analysis

Aims: To characterize the bacterial communities in commercial total mixed ration (TMR) silage, which is known to have a long bunk life after silo opening. Methods and Results: Samples were collected from four factories that produce TMR silage according to their own recipes. Three factories were sampled three times at 1‐month intervals during the summer to characterize the differences between factories; one factory was sampled 12 times, three samples each during the summer, autumn, winter and spring, to determine seasonal changes. Bacterial communities were determined by culture‐independent denaturing gradient gel electrophoresis. All silages contained lactic acid as the predominant acid, and the contents appeared stable regardless of factories and product seasons. Acetic acid and 1‐propanol contents were different between factories and indicated seasonal changes, with increases in warm seasons compared to cool seasons. Both differences and similarities existed among the bacterial communities from each factory and product season. Lactobacillus parabuchneri was found in the products from three of four factories. Various sourdough lactic acid bacteria (LAB) were identified in commercial TMR silage; Lactobacillus panis, Lactobacillus hammesii, Lactobacillus mindensis, Lactobacillus pontis, Lactobacillus frumenti and Lactobacillus farciminis were detected in many products. Moreover, changes owing to product season were distinctive, and Lact. pontis and Lact. frumenti became detectable in summer products. Conclusion: Sourdough LAB are involved in the ensiling of commercial TMR silage. Silage bacterial communities vary more by season than by factory. The LAB species Lact. parabuchneri was detected in the TMR silage but may not be essential to the product’s long bunk life after silo opening. Significance and Impact of the Study: Commercial TMR silage resembles sourdough with respect to bacterial communities and long shelf life. The roles of sourdough LAB in the ensiling process and aerobic stability are worth examining.     

Survival of silage lactic acid bacteria in the goat gastrointestinal tract as determined by denaturing gradient gel electrophoresis

Aims: To determine the survival rate of silage lactic acid bacteria (LAB) in the ruminant gastrointestinal tract. Methods and Results: Wilted Italian ryegrass (Lolium multiflorum Lam.) silage (containing 1·9 × 10⁶ CFU LAB g^?1) was fed ad libitum to three goats equipped with rumen cannulae. Silage was given alone or with concentrates at a 1 : 1 ratio on a dry matter basis. Rumen fluid was then obtained 2, 4 and 8 h after the morning feeding. Denaturing gradient gel electrophoresis was performed to compare LAB communities in silage, rumen fluid and faeces. The LAB detected in the wilted silage included Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus murinus and Lactobacillus sakei. Bands indicative of Lact. murinus were detected in either the rumen fluid or faeces, whereas the bands indicative of Lact. plantarum, Lact. brevis and Lact. sakei were not. Although the rumen fluid LAB counts and volatile fatty acid concentrations were higher in goats fed silage plus concentrates compared with those fed silage alone, the LAB communities themselves remained unaffected. Sampling times and goat‐to‐goat variations did not affect the LAB communities found in the rumen fluid. Conclusion: LAB communities found in the gut are not remarkably affected by the consumption of silage LAB, even when the silage is accompanied by concentrates that facilitate gut fermentation. Significance and Impact of the Study: Although silage can improve probiotic function, it may be difficult for silage LAB to survive the digestive process in the ruminant gastrointestinal tract.     

Microbiological quality of raw milk produced in Estonia

Aims: The microbial quality of farm bulk‐tank raw milk produced in Estonia during years 2004–2007 was investigated. Methods and Results: Bulk‐tank milk samples were analysed for lactic acid bacteria count (LABC), psychrotrophic bacteria count (PBC), aerobic spore‐forming bacteria count (ASFBC), total bacterial counts using BactoScan and somatic cell count (SCC) using Fossomatic. Randomly selected psychrotrophic isolates were subjected to 16S–23S PCR‐ribotyping. LABC remained below 10⁴ CFU ml^?1 in most samples, while psychrotrophic micro‐organisms dominated in 60% of farms. PBC ranged from 4·2 × 10² to 6·4 × 10⁴ CFU ml^?1, and ASFBC varied from 5 to 836 CFU ml^?1. Conclusions: In general, the microbiological quality of the farm bulk‐tank milk was good – more than 91% of samples contained <50 000 CFU ml^?1, and SCC in the majority of samples did not exceed the internationally recommended limits. Genus Pseudomonas spp. was the dominating spoilage flora with Pseudomonas fluorescens as the prevailing species. Significance and Impact of the Study: Specific bacterial groups (LABC, PBC and ASFBC), not analysed routinely by dairies, were determined in bulk‐tank raw milk of numerous dairy farms during 4‐year period. Based on the survey, dairy plants can better control their supply chains and select farms (milk) for the production of specific products, i.e. milk with low PBC and high LABC for cheesemaking.     

Growth of yeasts, lactic and acetic acid bacteria in palm wine during tapping and fermentation from felled oil palm (Elaeis guineensis) in Ghana

AIM: To investigate the microbiological and biochemical changes which occur in palm wine during the tapping of felled oil palm trees. METHODS AND RESUlts: Microbiological and biochemical contents of palm wine were determined during the tapping of felled oil palm trees for 5 weeks and also during the storage. Saccharomyces cerevisiae dominated the yeast biota and was the only species isolated in the mature samples. Lactobacillus plantarum and Leuconostoc mesenteroides were the dominated lactic acid bacteria, whilst acetic acid bacteria were isolated only after the third day when levels of alcohol had become substantial. The pH, lactic and acetic acid concentrations during the tapping were among 3.5-4.0%, 0.1-0.3% and 0.2-0.4% respectively, whilst the alcohol contents of samples collected within the day were between 1.4% and 2.82%; palm wine which had accumulated over night, 3.24% to 4.75%; and palm wine held for 24 h, over 7.0%. CONCLUSION: Accumulation of alcohol in palm wine occurs in three stages during the tapping and marketing with the concurrent lactic and acetic acid fermentation taking place as well. SIGNIFICANCE AND IMPACT OF THE STUDY: Yeasts, lactic and acetic acid bacteria are all important in the fermentation of palm wine and influence the composition of the product.     

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.     

The sourdough microflora. Interactions between lactic acid bacteria and yeasts: metabolism of amino acids

Co-culture of Lactobacillus brevis subsp. lindneri or L. plantarum with Saccharomyces cerevisiae or S. exiguus from sourdough did not modify the yield of the yeasts but gave higher growth rates and final yields of both lactic acid bacteria (LAB) than in their respective mono-cultures. Co-cultures of L. brevis subsp. lindneri with S. cerevisiae or S. exiguus in a medium without valine or leucine, which are essential for growth of the LAB, led to growth of the LAB due to excretion of these amino acids by the yeasts.The authors are with the Institute of Dairy Microbiology, Faculty of Agriculture, University of Perugia, Via S. Costanzo, 06100 Perugia, Italy     

PCR-based dENATURING GRADIENT GEL ELECTROPHORETICtypification and identification of the microbial consortium present in Kefir grains

Kefir grains have a complex microbiological composition that makes it difficult to obtain an optimal and constant starter culture necessary for the production of a quality Kefir beverage. The microbes present in the grains have in the past been identified using traditional methods such as growth on selective media and morphological and physiological characteristics. The aim of this study was to typify and identify the complex microbial community present in mass cultured, traditionally cultured and Irish Kefir grains by PCR amplification of a variable part of the ribosomal RNA (rRNA) genes in Eubacteria and yeasts and resolving these PCR fragments by denaturing gradient gel electrophoresis (DGGE). Unique PCR-based DGGE fingerprints were obtained for the Eubacterial and yeast species present in the three different grain types. A part of the Eubacterial and yeast rRNA genes were sequenced and compared to sequences available on NCBI. The phylogenetic relatedness of the amplified and sequenced lactobacilli was determined. Different bands in the Eubacterial and the yeast DGGE profiles of the mass cultured grains were identified to species level. A DGGE marker was constructed providing a quick reference for the identification of the members of the Eubacterial microbial population in mass cultured Kefir grains.     

Alcoholic fermentation and its prevention by Lactobacillus buchneri in whole crop rice silage

AIMS: To evaluate the fermentation characteristics and the effects of Lactobacillus buchneri inoculation in ensiling whole crop rice. METHODS AND RESULTS: Laboratory-scale silages were prepared from whole crop rice harvested at yellow-ripe stage. The crop was ensiled for 2 months with and without inoculation of L. buchneri at 10(4), 10(5) and 10(6) CFU g(-1). The effect of prolonged ensiling was also studied by using the same crop; the silos were opened at 1, 3, 6 and 12 months, while the inoculation was made at 10(5) CFU g(-1). Enhanced alcoholic fermentation was found in untreated silage; the sum of ethanol and 2,3-butanediol were seven times higher at 2 months than those of lactic and volatile fatty acids, while the differences were diminished at 12 months owing to the reduction of ethanol in the late ensiling period. Inoculation of L. buchneri inhibited the alcohols; however, ethanol yet prevailed over the fermentation until 6 months, after which acetic acid became the main product in the inoculated silage. Regardless of inoculation and ensiling period, yeasts were not found in whole crop rice silage. CONCLUSIONS: Substantial amounts of ethanol and 2,3-butanediol would be produced in silage prepared from whole crop rice. The alcoholic fermentation can be suppressed when inoculated with L. buchneri. SIGNIFICANCE AND IMPACT OF THE STUDY: Inoculation of L. buchneri could be an option to prevent ethanol fermentation in silage.     

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.     

Effects of copper supplement on growth and viability of strains used as starters and adjunct cultures for Emmental cheese manufacture

Aims: To determine the effects of supplemented copper (Cu²⁺) on growth and viability of strains used as starters and adjunct cultures for Emmental cheese manufacture. Methods and Results: Thirteen strains belonging to Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus rhamnosus, Streptococcus thermophilus or Propionibacterium freudenreichii species were exposed to various copper concentrations in the proper growth medium at relevant growth temperatures, and the effects of supplemented copper on bacterial growth and cell viability were determined by optical density and pH measurements, also by platings. Among the species considered, L. delbrueckii was the most copper resistant and S. thermophilus the most sensitive to copper. Anaerobic conditions increased this sensitivity significantly. There was also a considerable amount of variation in copper resistance at strain level. Conclusions: Copper resistance is both a species- and strain-dependent property and may reflect variability in copper-binding capacities by cell wall components among species and strains. In addition, the chemical state of copper may be involved. Significance and Impact of the Study: This study revealed that copper resistance is a highly variable property among starter and adjunct strains, and this variability should be considered when strains are selected for Emmental cheese manufacture.     

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.