Evaluation of Natural Chitosan as a Silage Additive: A Metabolomic-Metagenomic Approach

Abstract

Ruminant livestock methane emissions contribute significantly to agriculture's massive environmental burden. Current animal nutrition research has focused on its ability to modify rumen fermentation in order to reduce methane emissions. Chitosan is a linear polysaccharide made up of two repeating units, Dglucosamine and N-acetyl-D-glucosamine, joined together by β-(1→4)-linkages made up of N-acetylglucosamine (GlcNAc) units. On the other hands, silage is a type of fodder preservation technology. Non-optimal ensilage can lower DM content and cause mold to emerge on silage produced. Chitosan added to silage reduced methane generation, improved nutritional and fermentative quality, and reduced yeast and mold on silage product. Pre-research examined the influence of commercial chitosan and crab shells, a source of chitin, on in vitro rumen fermentation properties. RJ4 (treatment with 1% commercial chitosan) reduces gas production, while RJ2-RJ3 (20-30% swimming crabs shell of DM) also reduces gas production and in vitro organic matter digestibility. Despite the fact that crab shells can reduce gas production, they are less effective than commercial chitosan. This implies that the crab shells should be further processed into chitosan to increase their value and efficacy as rumen modifiers. The first stage focuses on how to obtain the best chitosan product from various methods and sources. Crab shells and wooden grasshoppers were used as chitosan source species, while conventional and green chemistry extraction methods were used as chitosan extraction variations. Green chemistry extraction using crab shells as a source of chitosan produces more chitosan with higher yield, DD, and solubility. The Gram-positive bacteria Clostridium acetobutylicum and the Gram-negative bacteria Escherichia coli were used to evaluate this. At a concentration of 2,000 ppm, the green chemistry extraction method (M2) using crab shell (P1) as the source considerably decreased the growth of Clostridium acetobutylicum (P<0.05). The second stage, chitosan which is gets from the first stage was applied on TMR silage and evaluated this physical, chemical and microbiological quality, in vitro rumen fermentation characteristics and in sacco degradability. The level of chitosan consists of SA (TMR silage with distilled water control, 0% chitosan), SB (TMR silage with 1% acetic acid control, 0% chitosan), SC (TMR silage with 0.5% chitosan in 1% acetic acid), SD (TMR silage with 1% chitosan in 1% acetic acid) and SE (TMR silage with 1.5% chitosan in 1% acetic acid). Chitosan at a dosage of 1–1.5% (SD and SE treatments) improves the nutritional (lowering ether extract, increasing protein content, tends reducing weight loss and maintain the optimal pH for ensilage) and microbiological properties (increase LAB count as well as tend to reduce Clostridium population) of TMR silage without impairing its physical state. The addition of 1-1.5% chitosan (SD and SE treatments) has been shown to decrease total gas production, total protozoa, average methane production, acetateproportion and the acetate/propionate ratio, increasing propionate proportion. The degradation of dry matter was reduced by the inclusion of chitosan (SE treatment) whereas the degradation of organic matter and crude protein was enhanced which is evaluated by in sacco analysis The third stage discusses the metabolome profile in silage and rumen fluid samples, as well as microbiome dynamics in rumen fluid and the correlation between the metabolome and microbiome in rumen fluid. This study found 308 metabolites, including 227 significant different compounds detected from silage samples. A total of 144 metabolites with variable important of projections (VIP) scores > 1 can be used as differentiating metabolites. L-Valine has the greatest significant difference VIP value of potential metabolite marker, and the area of this metabolite increases significantly as the level of chitosan supplementation increases. While, in the rumen fluid sample, 33 metabolites were discovered, with 20 significant different metabolites observed. A total of 13 metabolites with VIP scores > 1 can be used as metabolite markers. The potential metabolite marker 1- Methyl-1,2,3,4-tetrahydro-Î2-carboline-3-carboxylic acid (MTCA), a harmala alkaloid, has the highest significant difference VIP value. As the amount of chitosan added increased, this metabolite decreased (SD ad SE treatments). Proteobacteria, Bacteroidota, and Firmicutes were the most abundant of the 10 phyla found. Nevertheless, treatments with varying chitosan concentration had no effect on the relative abundances of these three phyla. To a greater extent than other factors, the TMR silage's composition appears to determine the relative abundance of these phyla. With the addition of chitosan, the abundance of nearly every phylum and some genera of bacteria decreased. In this study, the relationship between the metabolome (amine and indoles compounds) in rumen fluid and the microbiome (Bacteroidota and Firmicutes) appears to be mostly negative. The correlation between Ruminobacter and 2,4- xylidine has a moderately strong positive correlation (r = 0.74, P<0.05). Biogenic amines can be acquired by ruminants through both dietary sources and microbial metabolites in the rumen; furthermore, biogenic amines are often produced by the decarboxylation of certain amino acids. While, negative correlations between metabolites and the rumen microbiome include 2,4-xylidine with Succiniclasticum (r = -0.66, P<0.05) and Veillonellaceae UCG-001 (r = -0.68, P<0.05), followed by 2-Oxindole with Bacteroidales BS11 gut group (r = -0.74, P<0.05) and F082 (Bacterodoita) (r = -0.66, P<0.05). Bacteroidetes became the prevalent bacterium in animals fed a high starch diet, on the other hand glucose can block indole production, which could explain why the Bacteroidota group and the indoles compound have a negative connection.