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.

     

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.     

Broiler litter supplementation improves storage and feed-nutritional value of sawdust-based spent mushroom substrate

A study was conducted to determine the effect of broiler poultry litter (BL) supplementation to spent mushroom substrate (SMS) on its storage and feed-nutritional value improvement. In Exp. 1, the sawdust-based SMS from a king oyster mushroom (Pleurotus eryngii) farm was mixed with BL at 0%, 25%, 50%, 75% and 100% levels on a wet basis and deepstack stored for short-term (1-wk) and long-term (3-wk). At 1-wk of short-term deepstack storage, SMS with more than 50% BL levels showed favorable conservation. At 3-wk of long-term storage, all treatments except for BL 100% had a serious fungal problem. Based on chemical parameters, BL-blending to SMS practically improved the feed-nutritional value of the mixtures. Since the deepstacking method was not effective for long term storage, in Exp. 2 SMS ensiled with or without BL was attempted to improve long-term (3-wk) storage. All the ensiled treatments (SMS 100%, SMS 75%+BL 25% and SMS 50%+BL 50%) had desirable fermentation. As in deepstacking, BL-blending to SMS improved the nutritive value of the ensiled mixtures. The populations of total bacteria, lactic acid bacteria and yeast were highest (P<0.05) when 75% SMS was blended with 25% BL. In conclusion, blending 50% or more BL with SMS was effective for the short-term (1-wk) deepstack storage. For long-term (3-wk) storage of SMS, an ensiling method was effective with or without the addition of BL.     

The effect of an inoculant and enzymes on fermentation and nutritive value of sorghum straw silages

The objectives of this study were to determine the effect of inoculant, enzymes and inoculant-enzymes mixture on fermentation quality, nutritive value, and microbial changes of sorghum straw silage. Sorghum straws were collected and treated with distilled water (control), inoculant, enzymes and inoculant+enzymes prior to ensiling. Three bag silos for each silage (denoted C, I, E and I+E, respectively) were opened after 3, 7, 11, 15, 30 and 60 days for chemical and microbial analyses. For all the silages, there was a rapid decline in pH during the first 3 days of ensiling. Relative to silage C, all the treatment (I, E and I+E) had higher (P<0.05) lactic acid concentration at all ensiling periods. Population of LAB during all ensiling time was numerically greater for treated than control silages. Separate addition of two additives, especially for enzymes, can effectively (P<0.05) decrease aNDF and ADF concentration. Treatments with enzymes (E, I+E) can also improve significantly silage IVDMD and IVNDFD concentration. These results indicated that the addition of additives can improve the sorghum straw silage fermentation quality at different extent.     

Preservation of crab or shrimp waste as silage for cattle

Five experiments were conducted to either ferment fresh shrimp or crab waste with molasses, molasses and bacterial inoculant, or to preserve it with salt. Experiment 1 was a 4 × 2 factorial arrangement. Crab waste was combined with 0, 5, 10, or 15% liquid molasses, and stored in mini-silos (15 l) with or without lids for 14 days. The addition of molasses slightly decreased pH and offensive odors; mini-silo temperatures without lids were higher than those with lids. Experiment 2 was a 5 × 2 factorial arrangement designed to enhance fermentation. Fresh shrimp waste was combined with 0, 10, 15, 20, or 25% dry molasses and 0 or 1.0 × 10⁸ colony forming bacteria/g inoculant and ensiled for six days. As the level of molasses increased, dry matter and lactic acid increased but, the pH, crude protein, ammonia acetic, butyric, and propionic acid concentrations decreased. Significant molasses by inoculant interactions occurred which were highly variable for each acid. Evidence of fermentation was supported by production of lactic acid at all levels of molasses. The pH decreased from 7.7 in the untreated waste to an average of 7.4 for the 10, 15 and 20% molasses treated wastes to 6.8 in the 25% molasses treated waste. The high pH was an indication that the waste may be unstable with longer storage (> 6 days). Therefore, in Experiment 3, designed as a 2 × 2 factorial arrangement, shrimp waste treated with 15 and 20% molasses, with or without inoculant was ensiled for 21 days to test stability. By day 21, shrimp waste had deteriorated as indicated by a mean pH of 7.5, low lactic acid, and high butyric acid concentration, an unacceptable odor, and the presence of mold on the surface of the samples.In Experiments 4 and 5, shrimp or crab waste was combined with salt at 0, 2.5, 5.0, 7.5, 10.0, and 12.5%. Increasing levels of salt decreased crude protein percent, ammonia concentration, and lactic and volatile fatty acids while increasing the pH and improving the acceptability of the odors in both the shrimp and crab wastes. Treatment of crustacean waste with 7.5% or greater salt was more effective at preserving crude protein and minimizing odor than either dry or liquid molasses.     

Effect of ensiling sudax fodder with broiler litter and Candida yeast on the changes in different fiber fractions

Sudax fodder was ensiled in laboratory silos without or with the addition of 20, 30 or 40% broiler litter and 6% molasses, each without or with yeast inoculant (Candida utilus). Different fiber fractions were analyzed at the start of the ensiling and at 10-day intervals thereafter for 40 days. NDF, ADF, cellulose and lignin increased and cell contents decreased as a result of fermentation during ensiling. The addition of increasing levels of broiler litter not only decreased NDF, hemicellulose and cellulose, with a simultaneous increase in ADF content, but also extended the time course of fermentation during ensiling. The yeast inoculant failed to produce any significant effect on the fermentation characteristics or on any of the fiber fractions of the silage.     

Bioconversion of corn straw by coupling ensiling and solid-state fermentation

A two-stage process that combined solid-state fermentation (SSF) and ensiling was used for bioconversion of corn straw, in order to increase nutritional value and palatability for animal feed. SSF of corn straw increased the level of protein from 6.7% to 14.7% and decreased the cellulose by 38.0% and hemicellulose by 21.2%. Cellulase and xylanase were produced during SSF. After SSF, the fermented substrate was directly ensiled by inoculating with lactic acid bacteria (LAB). In situ produced enzymes and bacterial inoculation resulted in a rapid drop in pH, a high level of lactic acid production, partial degradation of cell wall components and generation of reducing sugars (RSs). Efficiency of ensiling at 25 degrees C, 30 degrees C, 35 degrees C, 40 degrees C was evaluated. Temperature influenced the effect of ensiling; the higher the temperature, the shorter the ensiling period. The combined fermentation upgraded the nutritional value, enhanced the efficiency of ensiling and reduced bioprocessing costs.     

Effect of deep stacking and ensiling broiler litter on chemical composition and pathogenic organisms

The feasibility of processing broiler litter by deep stacking and ensiling was evaluated prior to its use as feed ingredient. Fresh broiler litter was collected from the peri-urban area of Islamabad, Pakistan. Broiler litter was deep stacked at 15%, 25% and 35% moisture; ensiled at 40% moisture, alone or with 5% added sugarcane molasses; and ensiled with rumen contents at 60:40 and 50:50, wet basis. Deep stacking was done in 1.2×1.2×1.2 m bins and ensiling was done in 210 l metal drums double lined with polyethylene. Broiler litter deep stacked at 15% moisture showed a lower rise in temperature than litter deep stacked at 25% and 35% moisture. Maximum temperature was recorded at 40 cm depth for litter stacked with 25% moisture. Overall, deep stacking had no effect on the chemical composition of broiler litter. Deep stacked litter was devoid of lactic acid, but the processing was effective in the destruction of pathogens. Desirable fermentation was achieved in all the silages, with significant reductions (P<0.05) in pH and water-soluble carbohydrates, and increase (P<0.05) in lactic acid. The highest pH and lowest lactic acid concentration was recorded for silages containing broiler litter and rumen contents, 60:40, wet basis. No pathogenic microbes were observed in the ensiled mixtures.     

Effects of formic acid,molasses and inoculant additives on corn silage composition,organic matter digestibility and microbial protein synthesis in sheep

This study examined the production of com silages with low or high lactic acid concentrations, provided by the addition of formic acid (0.5%), molasses (5%) or microbial inoculant (homofermentative lactic acid bacteria, 10 g/tonne). After the fermentation period, sheep were fed the silages to determine true and apparent digestibility of the organic matter and microbial protein synthesis. The experiment were carried out with four KıvırcıkxMorkaraman sheep, 1.5 years old, fixed with cannula in their rumen and duodenum.Lactic acid concentrations were significantly higher in silages treated with enzyme or molasses compared to other specific treatments. Acetic acid concentration was highest in silage treated with formic acid, and lowest in silage treated with molasses (P < 0.05). The by-pass of crude protein was highest in silage treated with formic acid.     

Effects of chemical additives and ensiling time on whole plant wheat silage microbial profiles inferred by phenotypic and 16S ribosomal DNA analyses

The objectives of this study were to test the effects of various chemical additives, and ensiling time periods on microbial profiles of whole plant wheat silage (WPWS). WPWS was harvested at dough maturation stage, and ensiled with sodium chloride (T), sodium bicarbonate (N), sucrose (S), or urea (U) at 1% dry matter (DM) (w/w). Each WPWS treatment (i.e. T, N, S, and U) was ensiled in 1 dm³ duplicate anaerobic jars for 7, 14, 28, 35, 42, 49, and 56 days. The highest lactic acid concentration was detected for U (5.9% DM) compared to the control (0.5% DM) at day 56. Similarly, the highest ammonia concentration was observed for U (9.3% DM) compared to the control; T, N, and S treatments averaged 2.4% DM. A higher lactic acid bacteria (LAB) count was observed between days 14 and 56 for all treatments relative to control WPWS, which averaged 6.7 and 5.6 c.f.u./g of fresh material, respectively. The highest quality silage was observed for N and U treated WPWS, with an absence of yeasts and molds from days 7 to 49. Identification of bacterial isolates at days 7 and 56 from each individual silage treatment was performed based on phenotypic (biochemical tests), and molecular (16S rDNA partial gene sequence) methods. 16S rDNA sequence results identified U7 and N56 isolates as Enterococcus faecium, and demonstrated positive galactose, ribose, lactose, maltose, mannitol, mannose, sucrose, and trehalose fermenting characteristics. In addition, most isolates exhibited growth potential in 6.5% NaCl. Overall sequence alignment results supported phenotypic identification, and indicated that U and N treated WPWS were LAB (Enterococcus) dominant compared to the control (non-LAB).     

Lactic acid fermentation of food waste for swine feed

This study was conducted to determine the effects of lactic acid bacteria (LAB, Lactobacillus salivarius) inoculation on the microbial, physical and chemical properties of food waste mixture (FWM) stored at ambient temperature (25 degrees C) for 10 and 30 days. A complete pig diet including restaurant food waste, bakery by-product, barley and wheat bran, and broiler poultry litter was amended with LAB at the levels of 0.1%, 0.2%, 0.5% and 1.0% and fermented anaerobically. These treatments were compared with intact FWM before storage and non-anaerobically stored FWM. Non-anaerobic storage of FWM showed microbial putrefaction with the loss (P < 0.05) of water and water soluble carbohydrate (WSC) and increases (P < 0.005) in protein and fiber. Anaerobic fermentation of FWM with or without LAB seemed effective in both 10- and 30-day-storage. The addition of LAB inoculants to FWM showed a linear trend (P < 0.05) toward an increase in the number of total and lactic acid bacteria and toward the nutritional improvement with WSC increased and fiber decreased. Long-term (30 days) storage resulted in consistent reduction (P < 0.05) in numbers of total and lactic acid bacteria and pH and showed little change in chemical components, compared with short-term (10 days) storage. On the basis of these results, LAB inoculation improved fermentative characteristics of FWM. Among anaerobic treatments, further WSC increase and NDF reduction did not occur (P > 0.05) when LAB-added levels were over 0.2%. Based on these observations the optimum level of LAB addition to FWM was 0.2%.     

Ensiling olive cake with and without molasses for ruminant feeding

The purpose of the current experiment was to study the ensiling properties of olive cake with and without added molasses at 2-6% (w/w). The results indicate that molasses enhanced the ensiling fermentation of olive cake, as evidenced from higher lactic acid content and higher lactic acid bacteria numbers at 4% and 6% added molasses. However, when applied at 4% and 6% molasses increased fermentation losses up to 9.4%, probably due to larger yeast population. Polyphenols which could interfere with protein utilization by ruminants, decreased during ensiling by about 40%. It is concluded that molasses added at 3% could improve the ensiling fermentation of olive cake without substantial losses.     

Persistence of Escherichia coli O157:H7 in barley silage: effect of a bacterial inoculant

AIMS: The effect of a lactic acid producing bacterial (LAB) inoculant on the elimination of Escherichia coli O157:H7 from barley forage was assessed. METHODS AND RESULTS: Triplicate mini-silos were prepared for four treatments and six sampling times (1, 3, 7, 15, 30 and 42 d post-ensiling). The treatments were (i) 10(5) cfu g(-1) Pediococcus pentosaceus and Propionibacterium jenzenii (P2); (ii) 10(5) cfu g(-1) E. coli O157:H7 strain 3081 and 10(5) cfu g(-1) E. coli Biotype 1 strains 719IE10, 719IE14 and 614ME49 (EC); (iii) P2 + EC; and (iv) the control (sterile distilled water). Triplicate mini-silos were opened at each sampling time for pH, volatile fatty acid (VFA) and lactate determinations and E. coli, E. coli O157:H7 and LAB were enumerated. On d 3 and 7, numbers of E. coli O157:H7 in P2 + EC were significantly lower than in EC (P < 0;05). Escherichia coli O157:H7 was not detected in P2 + EC and EC at 7 and 15 d post-ensiling, respectively. On d 15 through 42, E. coli Biotype 1 was not detected in P2 + EC or EC. Populations of LAB were higher in P2 and P2 + EC than in the control and EC on d 3 and 7 (P < 0.05). After 3 d of ensiling, lactate levels were higher (P < 0.05) and pH was lower (P < 0.05) in P2 and P2 + EC as compared to the control and EC. Bacteriocins of P2 were not found to be inhibitory to E. coli O157:H7 using the agar-spot procedure. Escherichia coli O157:H7 inoculated into the control silage at a level of 10(3) cfu g(-1) and exposed to aerobic conditions at 22 degrees C was not detected after 1 d and remained undetectable for the 28 d exposure period. CONCLUSIONS: Silage inoculant P2 increased lactate levels and decreased pH more rapidly during ensiling, which appeared to hasten the elimination of E. coli O157:H7 from the silage. SIGNIFICANCE AND IMPACT OF THE STUDY: Results emphasize the importance of adequate ensiling since E. coli O157:H7 may be maintained and spread among cattle through feed.     

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.     

Microbial and chemical changes during the spontaneous ensilage of grape pomace

Pilot scale fermentations with grape pomace from two different wineries were investigated during the 24 weeks of the ensiling period, along with laboratory scale experiments in which the environmental temperatures were held constant at 20, 25, 30 and 35 °C. During this period, yeast and lactic acid bacteria (LAB) counts were made, after which the identity of both groups of organisms was studied, as were the major microbial metabolites present. Major microbial and chemical alterations occurred during the first 3 weeks of ensilage, leaving a more stable product differing significantly from the initial substrate. The results obtained indicated that after initial growth, yeast and LAB populations undergo progressive inactivation at environmental temperatures above 20 °C, although LAB seem to adjust better to this specific, post-fermentation environment. Homofermentative species of Lactobacillus were the dominant LAB. The initial yeast flora of non- Saccharomyces species was replaced by a typical wine yeast flora, i.e. predominantly Saccharomyces cerevisiae. At the chemical level, major alterations were due to an alcoholic fermentation and a malolactic conversion within the first 3 weeks.     

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.     

Anaerobic lactic acid degradation during ensilage of whole crop maize inoculated with lactobacillus buchneri inhibits yeast growth and improves aerobic stability

Aerobic deterioration of silages is initiated by (facultative) aerobic micro-organisms, usually yeasts, that oxidize the preserving organic acids. In this study, a Lactobacillus buchneri strain isolated from maize silage was evaluated for its potential as a bacterial inoculant that enhances aerobic stability of silages. In four experiments, chopped whole crop maize (30-43% dry matter (DM)) was inoculated with Lact. buchneri and ensiled in laboratory silos. Uninoculated silages served as controls. Analysis of silages treated with Lact. buchneri at levels of 103-106 cfu g-1 after about 3 months of anaerobic storage showedthat acetic acid and 1-propanol contents increased with inoculum levels above 104 cfu g-1,whereas lactic acid decreased. Propionic acid, silage pH and DM loss increased withinoculum levels above 105 cfu g-1. Time course experiments with maize inoculated with Lact. buchneri at 4 x 104-2 x 105 cfu g-1 showed that up to 7-14 d after ensiling, Lact. buchneri had no effect on silage characteristics. Thereafter, the lactic acid content of the inoculated silages declined and, simultaneously, acetic acid and, to a lesser extent, propionic acid and 1-propanol, accumulated. Inoculation reduced survival of yeasts during the anaerobic storage phase and inhibited yeast growth when the silage was exposed to O2, resulting in a substantial improvement in aerobic stability. The results indicate that the use of Lact. buchneri as a silage inoculant can enhance aerobic stability by inhibition of yeasts. The ability of the organism to ferment lactic acid to acetic acid appears to be an important underlying principle of this effect.     

Effect of ensiling on the nutritive value of low quality roughage rice hulls. II. Chemical studies

The sequence of the effects of delayed and immediate sealed silos as well as the role of nutrient additives such as molasses and/or urea in the ensiling process of rice hulls and the subsequent nutritive value of the product has been shown. There were considerable variations in the chemical constituents during the ensiling of rice hulls among the different treatments under both delayed and immediate sealed silos. In all cases, when compared with the controls, the crude protein, soluble sugars, lactic acid, and fat contents were markedly increased, while the carbohydrate polymers and silica contents were significantly decreased. It was concluded that the treatment of rice hulls + 2% molasses for two months ensiling period under anaerobic conditions can be considered as the optimum conditions for the production of good quality silage from the low grade roughages rice hulls. Changes in pH values also confirmed the improving effect of the combination of molasses addition and the immediate sealing of the silos.     

The effect of treatment with urea, sorbic acid, or dehydration on orange peel silage

The purpose of this work was to study the effects of 1% urea, 0.05% sorbic acid (SA) and moderate dehydration (by oven at 35°C for 1 h) on the ensiling process of orange peels and on reducing fermentation losses.

Fresh peels of the various treatments, in 10-kg triplicates, were ensiled in specially designed anaerobic containers for 35 days. Chemical and microbial analyses were performed on the peels and on seepage samples taken during the ensiling period.

The dominant microbial populations of the peel silage were lactobacilli (10⁸ g per DM) and yeasts (10⁵ g per DM) and the major fermentation product was ethanol (16% in DM of the control silage). There were differences between the treatments, with SA being the only treatment effective in reducing DM losses to 15%, compared with > 30% for the other treatments. Chemical results indicate a more efficient fermentation process with SA.     

Effects of ensilage on storage and enzymatic degradability of sugar beet pulp

Ensiling was investigated for the long-term storage of Sugar Beet Pulp (SBP). Eight strains of lactic acid bacteria (LAB) and a non-inoculated control were screened based on their ability to rapidly reduce pH, produce a large amount of lactic acid and inhibit undesirable fermentations. Lactobacillus brevis B-1836 (LAB #120), Lactobacillus fermentum NRRL B-4524 (LAB #137) and a non-inoculated control were selected for further research to determine the effects of LAB inoculation level and packing density on SBP silage quality and sugar yield upon enzymatic hydrolysis. Both SBP preservation and prevention of cellulose and hemicellulose loss were better when SBP was treated with LAB #137 compared to LAB #120 and the non-inoculated control. Additionally, the ensiling process was found to significantly improve the enzymatic digestibility of SBP by as much as 35%. The results suggest that ensiling may be a promising technology for SBP stabilization and pretreatment for bioconversion to products.