Diversity and probiotic characterisation of yeast isolates in the bovine gastrointestinal tract.

The use of yeasts as a feed supplement for cattle can promote animal development and performance. However, for the positive results to be consistent, strains with probiotic properties must be selected. The objective of this study was to isolate and identify yeasts present in the bovine feces and evaluate their probiotic potential together with strains previously isolated from the rumen (preliminary study). A total of 193 isolates were studied, including 139 isolates (19 species) from fecal samples from 11 different animals (Bos taurus and Bos indicus) and 54 strains previously isolated from rumen fluid (Bos taurus). The yeast population in the feces ranged from 3.51 to 4.99 log CFU/g, with Candida pararugosa being the most abundant (isolated from the feces of six samples analysed). Isolates were selected that had negative results in the safety tests (hemolytic activity, DNAse, and gelatinase) and had percentages greater than 35 and 70% for hydrophobicity and auto-aggregation, respectively. In addition, selected isolates had percentages greater than 77.7 and 74.7% for coaggregation with pathogenic strains of Escherichia coli and Clostridium perfringens, respectively. The isolates with percentage growth at 39 °C greater than 64.6% and viability greater than 96.7% were selected for survival testing under bovine gastrointestinal conditions. After the tests, the seven best isolates were selected, belonging to the species Candida pararugosa (L60, CCMA 928 and CCMA 930) and Pichia kudriavzevii (L97, L100, CCMA904, CCMA 907). The selected isolates were exopolysaccharide producers. Based on the results of the evaluated properties, the seven selected isolates were classified as potential probiotics for cattle.

Criteria for lactic acid bacteria screening to enhance silage quality.

The main challenge of ensiling is conserving the feed through a fermentative process that results in high nutritional and microbiological quality while minimizing fermentative losses. This challenge is of growing interest to farmers, industry and research and involves the use of additives to improve the fermentation process and preserve the ensiled material. Most studies involved microbial additives; lactic acid bacteria (LAB) have been the focus of much research and have been widely used. Currently, LABs are used in modern and sustainable agriculture because of their considerable potential for enhancing human and animal health. Although the number of studies evaluating LABs in silages has increased, the potential use of these micro-organisms in association with silage has not been adequately studied. Fermentation processes using the same strain produce very different results depending on the unique characteristics of the substrate, so the choice of silage inoculant for different starting substrates is of extreme importance to maximize the nutritional quality of the final product. This review describes the current scenario of the bioprospecting and selection process for choosing the best LAB strain as an inoculant for ensiling. In addition, we analyse developments in the fermentation process and strategies and methods that will assist future studies on the selection of new strains of LAB as a starter culture or inoculant.

Silage fermentation-updates focusing on the performance of micro-organisms.

Advances in micro-organism identification techniques have resulted in increased knowledge of the diversity of prokaryotes and eukaryotes in silage. Such knowledge has enhanced the understanding of how fermentation occurs in forage crops with different characteristics and how the process can be improved to enhance silage quality. Undesirable micro-organisms can grow in silage when fermentation does not occur properly. Such micro-organisms may be pathogenic and/or produce toxic metabolic compounds; however, information on the consequences of these metabolites on the health of animals that consume silage is still lacking. The major challenge of ensilage is to produce high-quality feed that is nutritional, sanitary and stable, with a high dry matter recovery rate, in a process involving no interventions during fermentation and considerable variation in the characteristics of the substrates. It is important to note that each substrate has particularities and that we can only improve fermentation if we fully understand microbial diversity. This review is intended to update information related to the fermentation profile of silage, focusing on microbial diversity.

Identification and characterization of yeasts from bovine rumen for potential use as probiotics.

AIMS: The aim was to isolate, identify and characterize yeasts present in rumen fluid and to select strains showing potential as probiotics. METHODS AND RESULTS: Rumen fluid was sampled from 4 herds of dairy and beef cattle and 77 yeast isolates were identified. Initial screening was based on the capacity to maintain viability in a medium with different ruminal conditions. A second screening in fresh rumen fluid to assess the growth of inoculated yeasts and evaluate in vitro neutral detergent fibre digestibility (NDF-D), pH and acid accumulation was conducted. The yeast population ranged from 3·84 to 6·76 log10 CFU per ml. The main species of yeast found were Pichia kudriavzevii, Candida rugosa, C. pararugosa, C. ethanolica and Magnusiomyces capitatus. Strains CCMA 933 (C. rugosa) and CCMA 970 (C. pararugosa) showed greater ability to survive in ruminal fluid and stimulated the production of acids. Isolate CCMA 967 (C. ethanolica) survived and improved the NDF-D. CONCLUSION: Pichia kudriavzevii was the dominant yeast found in the cattle herds. Strains CCMA 933, CCMA 970 and CCMA 967 showed properties that could be useful as potential probiotics for cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was the first to select yeasts from the rumen fluid, with the potential to be used as probiotic, based on the ruminal conditions.

Short communication: Effect of washing method, grinding size, and the determination of an indigestible fraction on in situ degradation of starch in mature corn grain.

The objectives of this study were to determine (1) the effect of grinding size (1, 2, 4, and 6 mm) to determine effective ruminal disappearance (ERD); (2) the most adequate method to estimate the rapidly degradable fraction (A); (3) a time point to measure the indigestible fraction (C); and (4) the viability of using fewer time points to estimate starch fractional disappearance rate (kd) of mature corn grain. Fraction A was determined by rinsing in a bucket or washing machine, rumen immersion followed by bucket or washing machine, and water immersion for 30 min followed by bucket or washing machine. Ruminal in situ incubations were performed at 48, 72, 96, and 120 h to determine fraction C, and at 0 (washing machine), 3, 6, 12, 18, 24, and 48 h to determine the kinetics of starch disappearance. Models were used with either 2 or 3 pools and kd was determined by the linear slope of the log-transformed bag residues as a proportion of incubated samples over time. The ERD was calculated as A + B [kd/(kd + kp)], where kp is the ruminal fractional passage rate = 16.0% h-1. Data were analyzed using PROC MIXED of SAS (SAS Institute Inc., Cary, NC) with the fixed effects of run (for fraction A analysis only) method (either washing or model), grinding size, and method by grinding size interaction, with cow as a random effect. Correlation between estimates calculated using all time points or combinations of 2 and 3 time points were determined using PROC CORR. Fraction A was reduced as grinding size increased, but was not altered by washing method. Samples ground at 6 mm had greater fraction C than other grinding sizes at 48, 72, or 96 h, but not at 120 h. Model affected the slowly degradable fraction (B) values solely, but the difference was minor (0.5 percentage units). Greater fractions B and C but reduced kd and ERD were observed as grinding size increased. Based on correlation analysis the 2-pool model, incubation times of 0, 3, and 48 h were suitable to evaluate ruminal starch degradation kinetics in mature corn. Ruminal in situ incubation at 120 h highlighted the lack of a fraction C of starch (0.13% of starch). Washing method did not affect determination of fraction A of starch. Ruminal in situ incubations of 0, 3, and 48 h for starch degradation kinetics using a 2-pool model were adequate for mature ground corn, but 120 h of incubation is suggested to confirm the existence or absence of a fraction C. Grinding size affected starch degradation kinetics and fraction A determination.

Effect of the inoculation of sugarcane silage with Lactobacillus hilgardii and Lactobacillus buchneri on feeding behavior and milk yield of dairy cows.

Despite its low NDF digestibility, sugarcane is an option for feeding dairy cattle in tropical regions. We evaluated the effect of sugarcane silages inoculated with CCMA 0170 (LH; an epiphytic bacteria isolated from sugarcane) or with NCIMB 40788 (LB; a commercial strain isolated from temperate grasses) on dairy cow performance and feeding behavior. The microbial inoculums were previously grown in the laboratory to obtain 5 log cfu/g of fresh forage. Nine tons of each inoculated silage and a noninoculated control silage (CON) were harvested from the same field and stored for at least 35 d in experimental 20 × 2.1 × 0.4 m bunker silos. Fifteen Holstein cows in late lactation (336 ± 175 days in milk at the start of the experiment) received the treatments in five 3 × 3 Latin squares with 21-d periods. The diets contained 20% of DM of sugarcane silage and 41% of DM of corn silage. Milk yield was increased from 18.0 kg/d for CON to18.8 kg/d for LH, but LB did not elicit a detectable increase in milk yield (18.1 kg/d). The daily yields of fat, protein, lactose, and total solids were increased by LH. Daily DMI and total tract apparent digestibility of nutrients did not differ among treatments. Both inoculated silages reduced acetate and increased butyrate proportions in ruminal VFA, but only LH silage reduced the acetate to propionate ratio (3.0 vs 3.3). First meal duration was shorter for CON compared to LH and LB. The proportion of daily intake between 0700 and 1300 h tended to be increased, and the proportion between 1900 and 0700 h was reduced by LH. The inoculation of sugarcane silage with affected rumen fermentation profile and feeding behavior of late lactation dairy cows, increasing the yield of milk solids.

Lining bunker walls with oxygen barrier film reduces nutrient losses in corn silages.

The objective of this study was to evaluate 2 systems for covering corn silage in bunker silos. The first system consisted of a sheet of 45-µm-thick oxygen barrier film (OB, polyethylene + ethylene-vinyl alcohol) placed along the length of the sidewall before filling. After filling, the excess film was pulled over the wall on top of the silage, and a sheet of polyethylene was placed on top. The second system involved using a standard sheet (ST) of 180-µm-thick polyethylene film. Eight commercial bunker silos were divided into 2 parts lengthwise so that one-half of the silo was covered with OB and the other half with a ST system. During the filling, 3 net bags with chopped corn were buried in the central part (halfway between the top and bottom of the silo) of the bunkers (CCOR) in 3 sections 10 m apart. After filling, 18 net bags (9 per covering system) were buried 40 cm below the top surface of the 3 sections. These bags were placed at 3 distances from the bunker walls (0 to 50 cm, 51 to 100 cm, and 101 to 150 cm). During unloading, the bags were removed from the silos to determine the dry matter (DM) losses, fermentation end products, and nutritive value. The Milk2006 spreadsheet was used to estimate milk per tonne of DM. The model included the fixed effect of treatment (7 different locations in the bunker) and the random effect of the silo. Two contrasts were tested to compare silages in the top laterals (shoulders) with that in the CCOR (CCOR vs. OB and CCOR vs. ST). Three contrasts compared the corresponding distances of the silage covered by the 2 systems (OB50 vs. ST50, OB100 vs. ST100 and OB150 vs. ST150). Variables were analyzed with the PROC MIXED procedure of the SAS at the 5% level. The OB method produced well-fermented silages, which were similar to CCOR, whereas the OB system showed less lactic acid and greater pH and mold counts compared with CCOR. The ST method had 116.2 kg of milk/t less than the CCOR, as the OB system and the CCOR were similar (1,258.3 and 1,294.0 kg/t, respectively). Regarding the distances from the walls, the effects were more pronounced from 0 to 101 cm. The OB50 and OB100 silages had better quality and lower mold counts and DM losses than ST50 and ST100. The OB system reduced DM and nutrient losses at the shoulders in farm bunker corn silages compared with no sidewall plastic. The OB film should lap onto the crop for at least 200 cm so that 150 cm are covered outward from the wall.

Fermentation profile and identification of lactic acid bacteria and yeasts of rehydrated corn kernel silage.

AIMS: The aim of this study was to evaluate the chemical and microbiological characteristics and to identify the lactic acid bacteria (LAB) and yeasts involved in rehydrated corn kernel silage. METHODS AND RESULTS: Four replicates for each fermentation time: 5, 15, 30, 60, 90, 150, 210 and 280 days were prepared. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and PCR-based identification were utilized to identify LAB and yeasts. Eighteen bacteria and four yeast species were identified. The bacteria population reached maximum growth after 15 days and moulds were detected up to this time. The highest dry matter (DM) loss was 7·6% after 280 days. The low concentration of water-soluble carbohydrates (20 g kg-1 of DM) was not limiting for fermentation, although the reduction in pH and acid production occurred slowly. Storage of the rehydrated corn kernel silage increased digestibility up to day 280. CONCLUSIONS: This silage was dominated by LAB but showed a slow decrease in pH values. This technique of corn storage on farms increased the DM digestibility. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was the first to evaluate the rehydrated corn kernel silage fermentation dynamics and our findings are relevant to optimization of this silage fermentation.

Occurrence of mycotoxins and yeasts and moulds identification in corn silages in tropical climate.

AIMS: This study was aimed to identify yeasts and moulds as well as to detect mycotoxin in corn silages in southern Minas Gerais, Brazil. METHODS AND RESULTS: Corn silages from 36 farms were sampled to analyse dry matter, crude protein, ether extract, ash, neutral detergent fibre, nonfibre carbohydrates and mycotoxins contents, yeasts and moulds population, pH and temperature values. The mycotoxins found in high frequency were aflatoxin in 77·7% of analysed samples, ochratoxin (33·3%) and zearalenone (22·2%). There was no significant correlation between the mycotoxin concentration and the presence of moulds. The pH was negatively correlated with ochratoxin concentration. Aspergillus fumigatus was identified in all silages that presented growth of moulds. Ten different yeast species were identified using the culture-dependent method: Candida diversa, Candida ethanolica, Candida rugosa, Issatchenkia orientalis, Kluyveromyces marxianus, Pichia manshurica, Pichia membranifaciens, Saccharomyces cerevisiae, Trichosporon asahii and Trichosporon japonicum. Another six different yeast species were identified using the culture-independent method. CONCLUSIONS: A high mycotoxin contamination rate (91·6% of the analysed silages) was observed. The results indicated that conventional culturing and PCR-DGGE should be combined to optimally describe the microbiota associated with corn silage. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information about the corn silage fermentation dynamics and our findings are relevant to optimization of this silage fermentation.

Fermentative profile and bacterial diversity of corn silages inoculated with new tropical lactic acid bacteria.

AIMS: This study aimed to evaluate the effects of inoculation of strains of lactic acid bacteria (LAB) isolated from sugarcane grown in a Brazil on the quality of corn silage. METHODS AND RESULTS: Three strains of Lactobacillus buchneri (UFLA SLM11, UFLA SLM103 and UFLA SLM108), five strains of Lactobacillus plantarum (UFLA SLM08, UFLA SLM41, UFLA SLM45, UFLA SLM46 and UFLA SLM105), and one strain of Leuconostoc mesenteroides (UFLA SLM06) were evaluated at 0, 10, 30, 60 and 90 day after inoculating corn forage. The inoculation of the LAB strains did not influence the chemical composition of the silage, but pH, acetic acid and 1,2-propanediol were affected by treatment. The silages inoculated with UFLA SLM11 and SLM108 contained the lowest yeast and filamentous fungi counts during fermentation. Bacteria belonging to the Enterobacteriaceae family, Clostridium genus were detected in the silages inoculated with Lact. buchneri UFLA SLM 11, 103 and 108, as shown by DGGE analysis. Silages inoculated with Lact. buchneri UFLA SLM 11 showed higher aerobic stability. CONCLUSIONS: The Lact. buchneri UFLA SLM11 strain was considered promising as a starter culture or inoculant for corn silages. SIGNIFICANCE AND IMPACT OF THE STUDY: The selection of microbial inoculants for each crop promotes improvement of silage quality. Studies on the chemical and microbiological characteristics of silage provide useful information for improving ensiling techniques.

The use of Lactobacillus species as starter cultures for enhancing the quality of sugar cane silage.

Sugar cane (Saccharum spp.) is a forage crop widely used in animal feed because of its high dry matter (DM) production (25 to 40 t/ha) and high energy concentration. The ensiling of sugar cane often incurs problems with the growth of yeasts, which leads to high losses of DM throughout the fermentative process. The selection of specific inoculants for sugar cane silage can improve the quality of the silage. The present study aimed to select strains of lactic acid bacteria (LAB) isolated from sugar cane silage and to assess their effects when used as additives on the same type of silage. The LAB strains were inoculated into sugar cane broth to evaluate their production of metabolites. The selected strains produced higher concentrations of acetic and propionic acids and resulted in better silage characteristics, such as low yeast population, lower ethanol content, and lesser DM loss. These data confirmed that facultative heterofermentative strains are not good candidates for sugar cane silage inoculation and may even worsen the quality of the silage fermentation by increasing DM losses throughout the process. Lactobacillus hilgardii strains UFLA SIL51 and UFLA SIL52 resulted in silage with the best characteristics in relation to DM loss, low ethanol content, higher LAB population, and low butyric acid content. Strains UFLA SIL51 and SIL52 are recommended as starter cultures for sugar cane silage.

Selection of tropical lactic acid bacteria for enhancing the quality of maize silage.

The objective of this study was to select lactic acid bacteria (LAB) strains isolated from silage and assess their effect on the quality of maize silage. The LAB strains were inoculated into aqueous extract obtained from maize to evaluate their production of metabolites and pH reduction. The ability to inhibit the pathogenic and silage-spoilage microorganisms' growth was evaluated. Nine LAB strains that showed the best results were assessed in polyvinyl chloride experimental silos. The inoculation of the LAB strains influenced the concentration of lactic and acetic acids and the diversity of Listeria. The inoculation of silages with Lactobacillus buchneri (UFLA SLM11 and UFLA SLM103 strains) resulted in silages with greater LAB populations and improvements after aerobic exposure. The UFLA SLM11 and SLM103 strains identified as L. buchneri showed to be promising in the treatment of maize silage.

Identification and characterization of yeasts in sugarcane silages.

AIMS: To enumerate the micro-organisms and to identify the yeast species present during the ensilage of different sugarcane (Saccharum spp.) cultivars. METHOD: Samples of sugarcane silage were collected at 10, 20, 30 and 40 days from the start of fermentation. Population levels of lactic acid bacteria (LAB), mesophilic facultative anaerobic (MFA) bacteria, filamentous fungi and yeasts were determined. Nine species of yeasts were classified according to traditional methods and confirmed using molecular techniques. CONCLUSIONS: LAB dominated the ensiling process of sugarcane, although yeasts were present at relatively high population levels throughout the whole fermentation period. The detected species of yeasts varied according to sugarcane cultivar and time of fermentation. Torulaspora delbrueckii was the predominant yeast, followed by Pichia anomala and Saccharomyces cerevisiae. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of the population of micro-organisms in general, and of yeasts in particular, present during the fermentation of sugarcane is of fundamental importance in the development of more effective ensiling processes.