Modern biotechnology in ensiling

The performance of lactic acid bacteria in silage production: A review of modern biotechnology for silage improvement

Ensiling, a microbial-driven process employed for preserving fresh forage in both bio-refineries and animal production, triggers significant biochemical transformations. These changes have spurred the exploration of novel silage additives, with a particular emphasis on the potential of microbial strains that exhibit superior biopreservation capabilities. Lactic acid bacteria (LAB) species have gained widespread recognition for their diverse applications as additives in the fermentation of crops and forage biomasses during ensiling. Nonetheless, recent variations in silage quality might be attributed to a lack of comprehensive information on the gene expression and molecular mechanisms of the microbiota involved in silage production. Contemporary research efforts have been directed toward uncovering nutrient-rich animal feed solutions through enhanced LAB inoculants. This review aims to shed light on the role of LAB inoculants in silage production and the modern biotechnological methods, including metabolomics, proteomics, metagenomics, genomics, transcriptomics, and genetic manipulation. These powerful tools are instrumental in the identification, enhancement, and development of high-performance LAB strains. Additionally, the review outlines emerging trends and prospective developments in LAB advancement for the enhancement of silage, which holds significant promise for breakthroughs in sustainable agriculture and improved animal feed production.

Received: 2021-03-23 | Accepted: 2021-04-12 | Available online: 2021-12-31 https://doi.org/10.15414/afz.2021.24.04.297-300 The aim of this research was to find out the changes in fermentation parameters of whole-crop rye silage after adding the biological additive. Two variants of rye silage were used in the experiment: variant C (silage without additive) and variant A (silage with the addition of additive). The wilted whole-crop rye was treated with strains of lactic acid bacteria ( Pediococcus acidilactici , Lactobacillus paracasei and Lactococcus lactis 1.25x10 11 CFU.g -1 ) in a dose of 2 g of additive + 25 ml of water per 1 ton of matter. Both silage variants were ensiled with a vacuum pack device and after 2 months of storage, average samples were taken to determine the dry matter content, fermentation products, acidity of water extract, pH and the degree of proteolysis by appropriate methods. Treatment of rye silage with a microbial silage additive affected the quality of rye silage by a statistically significant (P <0.05) higher content of lactic acid and acidity of water extract. The lower content of acetic acid, alcohols, pH value and the degree of proteolysis were also statistically significant (P <0.05). The results confirmed the positive effect of the addition of Pediococcus acidilactici , Lactobacillus paracasei and Lactococcus lactis on the quality of the fermentation process of rye silage. Keywords: rye, silage, biological silage additives, fermentation quality References Adesogan, A. T. (2014, April). Avoiding the two greatest silage problems. In Proceedings of the 50th Florida dairy production conference (pp. 9–17). Alba-Mejía, J. E. A., Skládanka, J., Delgado, A. H., Klíma, M., Knot, P., Doležal, P. and Horký, P. (2016). The effect of biological and chemical additives on the chemical composition and fermentation process of Dactylis glomerata silage. 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The aim of this study is to investigate the application of two lactic acid bacteria and dry condensed molasses fermentation solubles (CMS) in the making and preservation of corn silage. Forage corn materials are divided into eight treatment groups as follows: (i) control, (ii) B2 (Lactobacillus plantarum B2, 1 × 109 cfu kg-1 ), (iii) LAS (Lactobacillus buchneri 40788, 3 × 108 cfu kg-1 ), (iv) B2 + LAS, (v) CMS (35 g kg-1 , fresh weight), (vi) B2 + CMS, (vii) LAS + CMS and (viii) B2 + LAS + CMS. The silage composition and aerobic stability are determined after ensiling for 90 days. Furthermore, the digestibility of the silage product and gas production are evaluated using a trotro digestion procedure. The assay results indicate that the CMS supplementation and B2 inoculation significantly increased lactic acid concentration (P < 0.01). However, they also reduced the content of acetic acid and silage pH at the initial fermentation stage. The CMS supplemented with B2 (B2 + CMS) showed an improvement in the quality of silage, but a significant decrease in aerobic stability (P < 0.01). The B2 + LAS + CMS treatment yielded an increase in acetic acid production during the late fermentation period and is able to extend the aerobic stability of corn silage. Furthermore, this study shows that CMS supplementation alone can significantly improve the digestibility of the in vitro dry matter (P < 0.01) and the microbial protein synthesis efficiency (P = 0.01). In addition, the CMS supplementation is beneficial for enhancing the aerobic stability of corn silage. These results suggest that the combination of CMS supplementation and a suitable inoculation lactic acid bacterial strain can be highly promising for enhancing the higher quality and stability of corn silage. © 2020 Society of Chemical Industry.

Plant essential oils have played an important role in the field of antibiotic alternatives because of their efficient bacteriostatic and fungistatic activity. As plant essential oils are widely used, their activity to improve the quality of plant silage has also been explored. This review expounds on the active ingredients of essential oils, their bacteriostatic and fungistatic activity, and mechanisms, as well as discusses the application of plant essential oils in plant silage fermentation, to provide a reference for the development and application of plant essential oils as silage additives in plant silage fermentation feed.

Alfalfa poses challenges for ensiling because of its elevated protein levels, low amounts of water-soluble carbohydrates, low dry matter content, and high buffering capacity. As a result, there has been a recent push to improve silage production using additives. In recent years, silage additives have been employed to enhance the quality of alfalfa silage. Bacterial additives are employed to enhance the quality of crop silage, with a particular emphasis on hay silage. A primary objective of incorporating lactic acid bacteria into silage is to inhibit the proliferation of undesirable microorganisms, including Clostridium and Enterobacteriaceae. This is achieved by swiftly elevating the hydrogen ion concentration to a threshold that is inhospitable for the growth of these detrimental bacteria. Recent insights into the functions of bacterial additives in crop silage suggest significant potential for enhancing silage, not just as a fermented feed, but also to deliver probiotic substances that can benefit animal health. This article provides a comprehensive overview of the silage preparation process and critically assesses a range of studies concerning the quality of silage, as well as the impact of bacterial additives on alfalfa silage. The quality of silage can be enhanced by incorporating different bacterial inoculants, which help during fermentation, storage, and feeding by improving fermentation processes, encouraging beneficial microbial diversity, and inhibiting harmful microorganisms. Alfalfa is the most important forage, and microbial additives can enhance its silage preparation in a cost-effective and environmentally friendly way.

Probiotics may improve ruminal and/or intestinal conditions and according to FAO/WHO, 2002 which states “mono or mixed strains of living microorganisms which confer desirable health benefits on the host when used adequately”. It should be nonpathogenic, able to give a viable cell count, has a positive effect on the health of the host, and enhance the functions of the intestinal tract. The most commonly used probiotics are from Lentilactobacillus genus formerly quoted as Lactobacillus acidophilus, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus salivarius, Bifido bacterium spp., Enterococcus faecium, Enterococcus faecalis, Streptococcus thermophilus, Escherichia coli bacteria, and other probiotic fungi such as Saccharomyces cerevisiae and Saccharomyces boulardii. Probiotics may benefit farm animals by improving feed efficiency, body weight gain, milk yield and immune response. In silages, the major claim is regarding the microbiome, coming either from the wild original microbial population endophytic and epiphytic or mainly from silage microbial additives. After silage fermentation, microbiome is largely changed towards less diversity of viable species of bacteria, yeasts and fungi and many different compounds resulted from the substrate undergone through the metabolic pathways. The survival of the original/added microbiome throughout the silage fermentation still remains as a key question and results are largely influenced across trials. Only recently, the use of next-generation sequencing and PacBio single-molecule real-time sequencing technology offered high-throughput and enabled the discovery of a vast majority of microbiota and relative abundances of various microbes in the community to the genus and species precision. The addition of microbial additives during ensiling not only has shown positive effects on silage quality by altering the bacterial community but also changed microbiome and metabolites in the digestive tract of ruminants. Moreover, undisputably metabolites in the rumen were correlated with the bacterial communities. The lactic acid bacteria inoculants in silage production might be beneficial for animal performance and health by influencing the bacterial community and metabolites in the digestive tract of ruminants. Lactic acid bacteria (LAB) may increase intake and digestibility. The positive effects on digestibility are related to degrading feruloyl ester linkages and modifying the matrix structure of forage lignocellulose based on the feruloyl esterase-producing Lactobacillus (Lactiplantibacillus) plantarum. LAB can also act as antimicrobial agents by producing bacteriocins or organic compounds harmful to microbial membranes such as 3-phenyllactic acid. The enhancement of the antioxidant capacity to mitigate oxidative stress in ruminants fed silages was also linked to the free ferulic acid. Some biofunctional metabolites, such as bacteriostatic, antioxidant, anti-inflammatory compounds, and neurotransmitters have been detected in silage too. More precise research tools and targeting the right parameters related to health and metabolism are allowing us to assure the probiotic effects of silages but we still need more deep learning.

It is difficult to make good quality of silage from alpine gramineous from the Qinghai Tibetan plateau. The effects of lactic acid bacteria (LAB) on the fermentation quality and aerobic stability of Siberian wildrye silage were studied in southeast of the Qinghai Tibetan plateau. Siberian wildrye materials were freshly cut at the sprouting stage, flowering stage, and milky stage. Silage was prepared by using a small‐scale silage fermentation system (bag silos). Lactobacillus plantarum (LP, 5 × 108 cfu/kg FM), Lactobacillus buchneri (LB, 5 × 108 cfu/kg FM) and their mixture (LP+LB, 5 × 108 cfu/kg FM) as silage additives were separately added to ensiled forages, and no additive served as control (CK). These bag silos were kept at room temperature (<15°C), and the silage qualities were analyzed after 60 days of ensiling. The number of indigenous LAB on fresh materials was less than that of yeasts and molds, and LAB species showed specification adapted to low temperature. LAB inoculated silages had lower (P < 0.05) pH value, NH 3‐N/TN and butyric acid content compared with control silage. Silage treated with LB had higher contents of acetic acid, propionic acid, WSC and CP. However, the aerobic stability of silages inoculated with LAB did not differ significantly between stages (P > 0.05). When fermentation characteristics, chemical composition, and aerobic stability were considered, treatment with L. plantarum resulted in high quality of Siberian wildrye silage harvested at the flowering stage in the alpine region.

Lactic acid bacteria (LAB) inocula play a key role in the preservation and fermentation of forage crops within inoculated silages. LAB is a significant group of the bacterial community as they successfully reduce pH, inhibit the survival of undesirable microorganisms and control nutrient loss in fermented silage. Ensiled plants and metabolites such as simple plant carbohydrates have been utilized by LAB (homo-fermentative and hetero-fermentative LAB) to initiate the production of organic acids including lactic and acetic acids. LAB as a biological silage additive provides stable feed value and secondary metabolic products during rapid anaerobic primary silage fermentation. They are able to ferment a large number of forage crops and also to reduce pH levels in fermented forages, which helps to suppress the growth of spoilage microorganisms. Furthermore, silage inoculants can enhance silage quality, nutritional recovery and shelf life of the inoculated product. When ingested silage, Lactobacilli in the rumen may degrade secondary plant metabolites as part of the rumen microbiota, along with endogenous enzymes. Also, the forages harvesting time are key factors in the development of essential metabolites particularly carbohydrates and proteins which is essential nutrition for LAB survival and production of organic acids. The higher population of LAB could reduce the pH faster and control of deleterious microbial growth in silage. This review presents LAB function in silage production and the potential impacts of its fermentative activity. In addition, the advantage of LAB additives in silage production is discussed, with a focus on recent literature.

Silage is an essential part of the rations for ruminants. Preparation and production of high-quality silage is vital to improving the quality and efficiency of animal husbandry. The nutritive value and fermentation quality of silage is dependent on not only the type and quality of the forage crop, but also the ensiling technique and epiphytic microbiota. Lactic acid bacteria (LAB), as an important silage additive and feed probiotics, play an important role in improving the quality of silage forage and regulating the growth performance and health of ruminants. LAB offer various advantages for ruminants as potential probiotics and are able to improve nutrient acquisition, growth performance and immune function stimulation in ruminants. In this paper, we review the current knowledge on the effects of LAB on silage quality and ruminant health. The comprehensive and in-depth understanding of the biological functions of important microorganisms, like LAB, in the silage fermentation process, the mechanisms of action, and the physiological effects on ruminant health is useful for developing highly effective compound silage inoculants, and for providing a scientific basis for improving physiological health of ruminants.

The successful ensiling of lucerne (Medicago sativa L.) depends on a rapid acidification in the silo and consequently relies on a sufficient proliferation of, particularly homofermentative, lactic acid bacteria. Similarly, growth of spoilage bacteria, such as enterobacteria and clostridia, must be suppressed and silage additives are therefore frequently applied to promote favourable conditions during ensiling. Three silage additives or soil were applied during lucerne ensiling and investigated for their effects on silage quality characteristics and abundances of total bacteria as well as the bacterial key players Lactobacillus spp., homofermentative Lact. plantarum, heterofermentative Lact. buchneri, Clostridium spp. and Enterobacteriaceae after 30 days of storage. Inoculation with viable Lact. plantarum resulted in highest concentration of this species and excellent silage quality, i.e. high lactic acid concentration coupled with low acetic acid and ammonia-nitrogen concentrations. A sodium nitrite and hexamine-based additive did not support growth of lactic acid bacteria, which was also apparent by higher pH and low lactic acid concentration. No effect of treatments was found on spoilage-related enterobacteria and clostridia, even not when adding soil to lucerne to increase initial clostridial contamination. However, soil treatment resulted in increased ammonia-nitrogen and acetic acid concentrations. Consequently, among the bacterial key players, lactic acid bacteria concentrations were related to silage quality. Regarding spoilage bacteria, however, alterations in silage quality characteristics were not reflected in the abundances of enterobacteria and clostridia. Future investigations should underpin the present findings and help to understand how silage additives affect microbial key players and silage fermentation.

Broussonetia papyrifera L. (paper mulberry) is an alternative woody plant, which can used to replace part of the protein feed for ruminants. Ensiling is an effective way to preserve fresh pasture and to solve the problem of stable storage and feed conversion of paper mulberry in the rapid growth period. However, low dry matter (DM), water-soluble carbohydrate, and lactic acid bacteria (LAB) reduce the quality of paper mulberry silage. This study assesses the influence of wilting time (0 h and 3.5 h; lighting: 3.43 × 104 Lux) and three additives (Enterococcus durans, CL; cellulase, CE; and formic acid, FA) on the fermentation quality, aerobic stability, and bacterial community of whole plant B. papyrifera silage. The whole plant B. papyrifera sample was mowed and wilted for 0 h and 3.5 h, and then had CL, CE, or FA added, followed by 60 days of ensiling. The results show all silage samples had high fermentation quality with pH below 4.2, ammonia-nitrogen below 100 g/kg DM, and no detectable butyric acid. The additives protected the DM and the crude protein from protease activity (p &lt; 0.05), and CL was the most effective among them. Furthermore, wilting time influenced the silage’s bacterial communities, but overall, CL treatment had the greatest impact on bacterial communities. Wilting time and formic acid treatment significantly improved aerobic stability (p &lt; 0.05). Enterococcus was positively correlated with lactic acid (LA), while negatively correlated with LA and Weissella (p &lt; 0.001). Enterococcus was identified as the main driver of the whole plant paper mulberry ensiling process in the present study. In conclusion, compared to other additives, LAB is the most effective and economical to improve the fermentation quality and reduce the protein degradation of whole plant paper mulberry silage. Our findings provide a theoretical basis to improve the quality and production of paper mulberry silage.

Proteolysis During Ensilage of Forages Varying in Soluble Sugar Content

Salmon processing waste can create disposal issues unless a fish meal plant is located nearby. Other preservation methods, such as fermentation with lactic acid bacteria (LAB), are less energy intensive, but require an added carbohydrate. In this study, pink salmon heads (raw, smoked or cooked) were mixed with potatoes at different ratios and incubated with LAB. An initial pH drop was observed, with concurrent production of lactic acid, for all salmon–potato silages within 24 h; however, only silages composed of 100% potato or salmon with added sucrose became stable and remained at or below pH 4.8 for 60 days. Increasing the potato content of the potato–salmon silage increased the initial acidification, but did not prevent a rise in pH during storage. This study suggests that discarded agricultural products such as potatoes might be useful as a carbohydrate supplement for LAB preservation of fish by-products. PRACTICAL APPLICATIONS Pink salmon by-products, such as heads and viscera, create disposal issues for fish processors in Alaska. However, preserving these high-protein wastes through natural acidification (using lactic acid bacteria) requires an added carbohydrate to promote fermentation during silage production. Agricultural waste products such as discarded potatoes represent a convenient source of fermentable carbohydrate. Once preserved, fish processing waste offers an inexpensive feed source for agricultural animals, or as aquaculture feeds or local compost for vegetable farming.

The objective of this research was to investigate effects of different additives on the fermentation quality, aerobic stability and rumen degradation of mixed silage composed of amaranth and corn straw. The mixture ratio of amaranth to corn straw was 78%: 22%. Three additives were selected in this study and five groups were as follows: control group (CON, without additive), lactic acid bacteria group (LAB, 5 mg/kg, Lactobacillus plantarum ≥ 1.6×1010 CFU/g and L. buchneri ≥ 4.0×109 CFU/g), glucose group (GLU, 30 g/kg), cellulase group (CEL, 2 mg/kg) and lactic acid bacteria, glucose and cellulase group (LGC, added at the same levels as in individual group). The period of ensiling was 60 days. Fermentation quality, chemical composition and aerobic stability of mixed silage were analyzed. Four cows with permanent ruminal fistula were selected as experimental animals. Nylon bag technique was used to study rumen degradation characteristic of dry matter (DM), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF) of mixed silage. Compared with CON group, the addition of different silage additives could improve mixed silage quality of amaranth and corn straw to some extent. Combining three additives significantly increased (P < 0.05) the DM, CP and lactic acid contents, whereas decreased (P < 0.05) the ADF and NDF contents as well as pH and ammonia nitrogen/total nitrogen. Moreover, the aerobic stability and rumen degradation of DM, CP and NDF were significantly improved (P < 0.05) in LGC group when compared to other groups. In conclusion, the combined addition of lactic acid bacteria, glucose and cellulase increased DM, CP and lactic acid contents as well as lactic acid bacteria count, decreased NDF and ADF contents and aerobic bacteria and mold counts, improved aerobic stability and rumen degradation of amaranth and corn straw mixed silage.