Enhancing silage quality of Elephant grass and Indigofera legume using shrimp shell-derived chitin and chitosan additives

Rakhmad Perkasa Harahap, Nahrowi¹, Sri Suharti¹, Anuraga Jayanegara¹ and Saitul Fakhri²

Faculty of Agriculture, Tanjungpura University, 78124, Pontianak, Indonesia
rakhmad@faperta.untan.ac.id
¹ Faculty of Animal Science, IPB University, 16680, Bogor, Indonesia
² Faculty of Animal Husbandry, University of Jambi, 36361, Jambi, Indonesia

Abstract

The study aimed to analyse the potential of shrimp shell-derived chitin and chitosan as alternative additives in Elephant grass and Indigofera legume silages on their silage quality profile, in vitro rumen fermentative profile and methane emission. The Elephant grass and Indigofera legume were treated in control, adding shrimp shell 10 g/kg fresh forage, chitin 10 g/kg fresh forage and chitosan 10 g/kg fresh forage, each in four replicates. Samples were put into mini-silos and stored for 30 days. The resulting data were analyzed using a randomized block factor design where the first factor was forage species and the second was additive types. The results showed that chitosan significantly decreased dry matter losses and pH on silage quality profile compared to the other additive types (p<0.01). Chitosan increased the concentration of ammonia (NH₃) and acetic acid on silage quality profile (p<0.01). Indigofera legume silages produced higher NH₃ concentrations than Elephant grass silages on silage quality profile (p<0.01). Chitosan increased propionate (C₃), dry matter digestibility (DMD) and organic matter digestibility (OMD) on in vitro rumen fermentative profile (p<0.01). However, chitosan decreased the concentration of NH₃, total volatile fatty acid (TVFA), acetate (C₂), total gas and methane (CH₄) emission at an incubation time 24 hours compared to the other additive types on in vitro rumen fermentative profile (p<0.01). In conclusion, chitosan enhanced the silage quality profile, in vitro rumen fermentative profile and reduced methane emissions than shrimp shell and chitin.

Keywords: in vitro ruminal fermentation, shrimp shell-derived, silage additive

Introduction

Elephant grass (Pennisetum purpureum) is a widely cultivated grass in Indonesia and other tropical countries with a cut-and-carry system. It is known for its good nutritional value and high productivity. The provision of grass needed to be increased to meet the nutritional requirement of ruminant livestock, particularly the protein requirement. Legumes rich in crude protein (CP) were typically supplemented for a ruminant. In recent years, a legume species widely developed as a ruminant feed in tropical country especially in Indonesia was Indigofera zollingeriana. The legume has a high nutritional value, such as CP content of 27-31%, CP digestibility of 75-87%, neutral detergent fiber (NDF) of 49-57% and acid detergent fiber (ADF) of 32-38%, dry matter digestibility (DMDi) of 72-81% and total low tannin content 0.09-0.65% (Abdullah 2010). In Indonesia and many other tropical countries, the quality and quantity of feed decreases during the dry season. Forage also has a perishable nature due to its high-water content. Therefore, at the time of harvest with high productivity, typically during the rainy season, it is necessary to preserve the forage and ensiling is an appropriate preservation method. However, Indigofera zollingeriana is a legume that contains higher CP than Elephant grass, so legumes are difficult to lower the pH in the silage process than grass. Silage mixtures with an increased proportion of legumes exhibited elevated levels of crude protein, ash content, pH value and NH3-N (Goyal & Tiwana 2016). High protein legumes could also present proteolysis during ensilage, requiring additive silage. Legume silages exhibit a greater buffering capacity in comparison to grass silages, hence potentially leading to a less optimal fermentation process and heightened proteolysis (Jayanegara et al 2019).

Recent research has been focusing on the potential of additives to address the challenges, in improving silage fermentation and quality. Natural bioactive compounds have recently been explored regarding their potential use as silage additives, particularly those originating from plants (Niderkorn & Jayanegara 2021). More recently, is the use of chitin and chitosan which're polymers derived from crustaceans like shrimp. Chitosan has become recognized for its considerable biological functions, encompassing antibacterial, antioxidant and enzyme activity inhibitory effects (Romanazzi et al 2018). They possess properties that can help regulate the ecosystem during silage fermentation promoting beneficial lactic acid bacteria while suppressing undesirable microorganisms. Research studies have demonstrated that chitosan exhibits a beneficial impact on the process of silage fermentation (de Morais et al. 2021). This is evidenced by its ability to decrease losses associated with fermentation and enhance silage quality and nutrient preservation (Del Valle et al 2018; Harahap et al 2023).

The present study investigates the potential utilization of chitin and chitosan, obtained from shrimp shells, as silage additives to enhance the overall characteristics of Elephant Grass and Indigofera legume silage. This study promotes a sustainable technique to enhance silage quality by integrating waste recycling with forage conservation. Additionally, it presents a novel method for utilizing by-products derived from the shrimp industry. The study aimed to analyse the potential of shrimp shell-derived chitin and chitosan as alternative additives in Elephant grass and Indigofera legume silages on their silage quality profile, in vitro rumen fermentative profile and methane emission.

Materials and methods

Approved ethically

This experiment used ruminal contents (solid and liquid fractions) as microbial inoculum, maintained at pH 6.5 and temperature at 39°C. Two fistulated Ongole crossbreed bulls from the Biotechnology Research Center of the Indonesian Institute of Science, Cibinong, Bogor, Indonesia, were used to collect ruminal contents before feeding in the morning. The two fistulated Ongole is fed two times in the morning and evening with a mixture of several types of local grasses in Indonesia. The method of collecting the rumen fluid from fistula bulls, according to Duffield et al (2004). The bulls were kept up according to the Animal Welfare Rules of the Indonesian Institute of Sciences. The Faculty of Animal Science, IPB University, Bogor, Indonesia, affirmed all the experimental conventions.

Harvesting, treatments and ensiling

The feed additive used was shrimp shell (0% degree of deacetylation), chitin (from shrimp shell; 65.3% degree of deacetylation) and chitosan (from shrimp shell, degree of deacetylation 87.5%; viscosity 50 cps; pH 7.1; solubility 99%) from the same batch of processing produced at CV. Bio Chitosan Indonesia. Elephant grass (Pennisetum purpureum) and Indigofera legume (Indigofera zollingeriana) were the forages species employed in this study and both were acquired from the Agrostology Field Laboratory of the Faculty of Animal Science, IPB University, Bogor, Indonesia. The forage’s part was the edible part, with a harvest age of 40 days for Elephant grass and six months for Indigofera legume. The Elephant grass was cut into pieces of about 3 cm.

A randomized block factorial design contained two factorials with four replications. Elephant grass and Indigofera legume, two separate forage plants, were the first factor. The second factor was the type of additive, which included fresh forage without additives (control), the addition of shrimp shell at 10 g/kg fresh forage, chitin at 10 g/kg fresh forage and chitosan at 10 g/kg fresh forage.

Treatments were dispensed at random into HDPE bottles that were 1 L as mini-silos. For the purpose of avoiding the gas scape, mini-silos with valves were built. For 30 days, those silages used in the tests were kept at room temperature (20–25 °C). According to Gandra et al 2018, mini silos were weighed immediately upon the opening to record dry matter losses. Silage was split into two equal parts using a comparable ratio. To test the quality of the silage, the first portion was mixed with distilled water (1:7 w/v) and filtered (Kondo et al 2014). The second portion underwent in vitro rumen fermentation testing and a chemical composition analysis after being oven-dried at 60 °C for 24 hours. It was then finely crushed to pass a 1 mm sieve size.

Nutritional composition analysis

The nutritional composition of the silage samples, including dry matter (DM), crude protein (CP), ether extract (EE), neutral detergent fiber (NDF), acid detergent fiber (ADF), neutral detergent insoluble crude protein (NDICP) and acid detergent insoluble crude protein (ADICP) were all determined. DM was ascertained by placing the sample in an oven set at 105°C for 24 hours. In order to determine the N content, CP was examined using a Micro Kjeldahl apparatus. EE was analyzed using Soxhlet extraction and the ash content was analyzed using a furnace at 600°C (AOAC 2005). Because proteins typically include 16% N, the CP was calculated by multiplying the N content by 6.25. The contents of the NDF and ADF were established following Van Soest et al (1991). The CP content of the NDF and ADF residues was then further examined to calculate NDICP and ADICP, respectively (Licitra et al 1996). The identification of these chemical components was done twice.

Silage quality profile

After the ensiling process, the supernatant's pH, partial volatile fatty acid (VFA) concentration and ammonia (NH₃) concentration were measured. A Jenway Model 3505 pH meter that was calibrated to pH 7 and pH 4 was used to measure the pH level. While NH ₃ was determined using the Conway micro diffusion method as described by Jayanegara et al (2016), then partial VFA concentration was determined using General Laboratory Procedure (1966) method utilizing gas chromatography (GC 14A, Shimazu Corp, Kyoto, Japan). These evaluations were carried out twice.

In vitro rumen fermentation profile

Samples of ground silage were incubated in vitro with a rumen fluid:buffer mixture (Theodorou et al 1994). A total of 75 mg of treatment substrate was put into a 125 mL serum bottle, then 50 mL of liquid and 75 mL of rumen buffer fluid were added as incubation media which had been saturated using CO ₂ gas. After that, the mixture between the treatment and rumen buffer fluid in the bottle was then closed using a rubber cover and then incubated in a water bath at 39°C for 24 hours. Using a 50-cc plastic syringe with a needle, gas production was released and measured at 2, 4, 6, 8, 10, 12 and 24 hours after incubation. All bottles were manually shaken as soon as the gas output was measured. The gas is gathered in the gas reservoir so that the methane content may be examined. By injecting the gas into a gas chromatograph (Shimadzu 8A GC, Shimadzu Corp., Kyoto, Japan) fitted with a flame ionization detector, the concentration of methane was determined (Wang et al 2017).

To determine rumen fermentation and digestibility parameters, a different set of serum bottles that had received comparable samples was incubated for 24 hours. Each incubation bottle's solids and supernatant were divided using a centrifuge. In order to determine the silage quality, the resultant supernatant was examined for the partial VFA and NH ₃ concentrations mentioned above. In order to determine the samples' dry matter digestibility (DMD) and organic matter digestibility (OMD), samples were subsequently treated with pepsin-HCl at 39 °C for 24 hours. After filtering, the residue was dried in an oven for 24 hours at 105°C. The digestibility was estimated by deducting the residue from the initial sample volume and correcting for the blank sample.

Statistical analysis

The collected data were analysed using a variety of factorial statistical models where the first factor was different forage species (Elephant grass and Indigofera legume). The second factor was the type of additive (without additives, shrimp shell 10 g/kg fresh forage, chitin 10 g/kg fresh forage and chitosan 10 g/kg fresh forage). The LSMEANS statement was used as the foundation for multiple comparisons across means using the Duncan method to see if there are any significant effects of the treatments, both the main factor and the interaction (SAS 2008). Using SAS software 9.1, this statistical analysis was carried out. Silage samples from four replicates were combined and analysed in duplicate for nutritional composition data. As a result, the nutritional composition data are given in a descriptive manner.

Result and Discussions

Nutritional composition of Elephant grass and Indigofera legume silage

The nutritional composition of Elephant grass and Indigofera legume silage added with shrimp shell, chitin and chitosan could increase the content of crude protein (CP), followed by an increase in neutral detergent insoluble crude protein (NDICP) and acid detergent insoluble crude protein (ADICP) contents (Table 1). Moreover, Indigofera legume has a higher protein content compared to Elephant grass. The crude fiber content of Elephant grass was higher than Indigofera legume. Table 1 showed that the nutritional composition of Elephant grass and Indigofera legume silage added with chitin and chitosan could increase the content of B3 fraction in Cornell Net Carbohydrate and Protein System (CNCPS).

High content of neutral detergent insoluble crude protein (NDICP) and acid detergent insoluble crude protein (ADICP) indicated that shrimp shells, chitin and chitosan contained protein matrix. A shrimp shell is the shell of a crustacean that consists of 20-30% chitin (Bakshi et al 2020). In chitin, the polysaccharide skeleton was strengthened and modified by a protein matrix that contains nitrogen molecules because it was a long-chain polymer of N-acetylglucosamine (Chaudhari et al 2011). Chitosan is a derivative of chitin which has a very functional amino group. The difference between chitosan and cellulose was that chitosan has an amine group (–NH₂) at the C-2 position, while cellulose has a hydroxyl group (–OH) (Anggraeni et al 2022).

The addition of chitosan in Elephant grass and Indigofera legume silage caused the increased content of NDICP and was considered slowly degraded crude protein or not degraded in the rumen. It was reported that chitosan (from crab shell containing 67 ± 0.3 mg g^-1 N) could not be degraded in the rumen of sheep with 96 h incubation times (Fadel El-Seed et al 2003). NDICP was an insoluble protein fraction in neutral detergents and was considered slowly degraded or not degraded in the rumen. The ADICP was a protein component insoluble in acid detergent and generally represents protein linked with lignin and heat-damaged protein (Pelletier et al 2010). As a result, this component was thought to be indigestible in ruminants' guts. The greater percentage of ADICP over CP suggests a feedstuff with low protein quality. The NDICP is a component of protein that was insoluble in neutral detergent and was thought to be poorly degraded or undegraded through the rumen.

The nutritional composition of Elephant grass and Indigofera legume silage added with chitin and chitosan could increase the content of B3, because NDICP was a component of ADICP, the B3 fraction was obtained by reducing NIDCP and ADICP, which can offer important data on the quantity of rumen by-pass protein (Jayanegara et al 2016; Higgs et al 2012). Furthermore, including chitin and chitosan in Elephant grass and Indigofera legumes silage could boost rumen by-pass protein capabilities. Adding chitin and chitosan reduced the rumen's degradable protein, indicating that chitin and chitosan are typically active in protein protection.

Silage quality profile of Elephant grass and Indigofera legume

Table 2 showed that Indigofera legume silage was a higher dry matter (DM) content than Elephant grass silage (p<0.01). DM loss in Elephant grass and Indigofera silages was lower in the treatment with chitosan additives (p<0.01). The pH value was significantly different for the forage species. The pH of Indigofera legume silage was higher than Elephant grass silage (p<0.01). The pH value of the chitosan additive was lower than the other treatments (p<0.01). Silage from Indigofera legume produced higher NH ₃ content than Elephant grass silage (p<0.01). The addition of shrimp shell, chitin and chitosan increase NH ₃ content of Elephant grass and Indigofera legume silage. The Indigofera legume silage had a higher concentration of acetic acid than the Elephant grass silage (p<0.01). The addition of chitosan additives affected the increase in C ₂ (p<0.05). Chitosan also tends to affect the increase in the concentration of propionic acid in the Indigofera legume silage. The concentration of butyric acid was not detected in silage additive treatments and forage species (Table 2).

Chitosan was more effective as a silage inhibitor additive than shrimp shell and chitin due to an active amine group as an antifungal and antimicrobial. It triggered a rise of lactic acid bacteria (LAB) so that the output of lactic acid increased, which impacted low pH and dry matter losses. The antimicrobial mechanism of chitosan can be through interactions on the cell's outer layer and external membrane via electrostatic charges or divalent cations, substitute of Mg⁺² and Ca⁺² ions, cell membrane destabilization and intracellular leakage resulting in cell death (Helander et al 2001; Dias et al 2017). DM loss in Elephant grass and Indigofera legume silage was reduced by adding chitosan than with shrimp shells and chitin. The previous study reported that adding chitosan additives to bagasse silage and whole soybean plants reduced DM loss (Gandra et al 2016; Gandra et al 2018; Pelletier et al 2010). The explanation was chitosan's ability to inhibit the growth of fungi and spoilage bacteria during the aerobic phase of the ensilage. Chitosan can be an antifungal and antimicrobial agent against various fungi and bacteria [28]. Generally, chitosan is predominant on the cell surface based on the microorganism species and chitosan molecular weight. Moreover, antimicrobial mechanisms of chitosan can be activated, such as DNA/RNA synthesis suppression or protein synthesis disruption (Verlee et al 2017).

Indigofera legume has a higher nutritional content than Elephant grass, especially in CP content. Indigofera legume has a CP of around 27 to 31% (Abdullah 2010), while Elephant grass contains a CP of 10.13-10.32% (Lounglawan et al 2014). It has been reported that legumes containing high CP have a higher buffer capacity than low CP forages such as grass (Kung et al 2018). Therefore, it is usually difficult for legume silage to lower a pH value of 4.5 or less (Jayanegara et al 2019), while grass silage can reach around 4.0 or less (Deaville et al 2010; Hapsari et al 2016). Adding chitosan additives can reduce the pH of silage due to an increase in the concentration of lactic acid. According to Gandra et al (2018), the application of chitosan in the whole soybean silage increased the amount of LAB and the concentration of lactic acid while reducing the number of molds and fungi. It was known that the bactericidal effect of chitosan is pH dependent with more significant activity at a pH value of around 4.5 (Şenel et al 2004).

Silage treated with chitosan had the highest acetic acid and the ability to retain dry matter (Gandra et al 2018; Gandra et al 2016; Harahap et al 2023). There was no butyric acid content observed after the application of the silage inhibitor additive, proving that clostridia fermentation declined. A reduction in butyrate concentration has also been confirmed when chitosan additives are added to alfalfa (Sırakaya & Beyzi 2022). The increase in acetic acidwas due to lactic acid bacteria's increased activity that breaks down carbohydrates in the silage fermentation. Moreover, increased NH ₃ concentration and increased butyric acid production were not expected in silage products, as the presence of NH ₃ was related to elevated protein degradation induced by a little pH reduction. Fungal activity was linked to butyric acid the following. When chitosan was added to silage, the maximum NH ₃ concentration value may be associated to CP chitin (chitosan precursor), which may achieve 10.8% (Manni et al 2009). Chitosan was found to be a weak base soluble in dilute acid solutions below its pKa (6.3), in which the glucosamine unit (NH₂) was transformed to the soluble protonated form (NH⁺³) (Goy et al 2009).

As a result of the protein protection mechanism, chitosan can suppress proteolysis in silage. According to Chiang et al (2009), the positively charged -NH²⁺groups of chitosan are able electrostatically with the negatively charged carboxyl groups of amino acids. Furthermore, the mechanism inhibiting proteolysis in silage is due to antimicrobial activity, especially in spoilage bacteria. According to Cazón et al (2017), the positive charge of chitosan is considered responsible for its antibacterial activity via interactions with cell membranes of negatively charged microbes.

In vitro rumen fermentation profile and digestibility of Elephant grass and Indigofera legume silage

It can be seen in Table 3 that the dry matter digestibility (DMD) and organic matter digestibility (OMD) of Indigofera legume silage were higher than that of Elephant grass silage (p<0.01) . Chitosan additives could increase DMD and OMD compared to other additives (p<0.01). The concentration of ammonia (NH₃), total volatile acid (TVFA), acetate (C₂) and propionate (C₃) were higher in the Indigofera legume silage than in the Elephant grass silage (p<0.01) of in vitro rumen fermentation profile. The concentration of TVFA and acetate (C₂) decreased in the addition of chitosan additives compared to other treatments (p<0.01). However, there was increased production of propionate (C₃) in addition to chitosan (p<0.01).

Indigofera legume silage had higher DMD and OMD than Elephant grass silage due to its low crude fiber content ranging from 3.96 to 4.10%; in comparison, the crude fiber content of Elephant grass silage ranges from 9.37 to 10.75%. Adding chitosan could increase the DMD and OMD of Elephant grass and Indigofera legume silage due to changes in the structure of the microbial population in the rumen. Chitosan reduces the number of protozoa that cause the reduced intensity of protozoan predation on bacteria. It caused an increased population of bacteria, which play an essential role in nutrient degradation and fermentation. The decrease in protozoa could increase the total bacterial population in the rumen because protozoa have predatory activity against bacteria in the rumen (Newbold et al 2015). The population of protozoa decreased through chitosan properties that changed the permeability of protozoan cells (Belanche et al 2015; Wencelová et al 2014). Chitosan caused the interaction between polycationic chitosan and electronegative charge on the surface of microbes (Muxika et al 2017). It caused an increase in DMD and OMD due to an increase in the population of bacteria which are highly responsible for nutrient degradation and fermentation.

The decrease in the concentration of NH ₃ added with chitosan additives indicates the ability of the protein by-pass through an electrostatic mechanism of cations of amine groups that bind to anionic amino acids. Previous studies reported that chitosan could reduce the concentration of NH ₃ in the rumen (Goiri et al 2010), similar to a meta-analysis study (Harahap et al 2020). The NH ₃ reduction may be associated with decreased amino acid degradation through a protective mechanism against rumen degradation through the positively charged chitosan -NH²⁺ group to electrostatically interact with the negatively charged carboxyl in amino acids under rumen pH conditions (Chiang et al 2009). Belanche et al (2015) reported that adding chitosan to feed increased the proportion of C ₃ and decreased the proportion of C ₄ in the rumen. Previous studies reported that adding chitosan increased the proportion of C ₃ and valerate (C₅) but decreased the total VFA concentration. It also decreased the proportion of C₂, the ratio between acetate and propionate (C₂:C₃) and branch chain volatile fatty acids (BCVFA) (Goiri et al 2009; Harahap et al 2022). Other studies have also confirmed an increase in C ₃ and a decrease in the proportion of C₂ with the addition of chitosan (Goiri et al 2009; Harahap et al 2022; Li et al 2013; Seankamsorn et al 2019). The increased proportion of C₃ with chitosan was also associated with decreased protozoan population. It has been reported that protozoan defaunation increases the molar proportion of C ₃ in the rumen and decreases the proportion of C₄ and C₂(Morgavi et al 2010). Another explanation is that the increase in propionic acid could be due to the carbohydrate fermentation profile of chitosan. Chitosan has a similar characteristic to glucose polymers such as beta-glucan, so it is related to the report that yeast-fermented rice (YRF) that contains 16% beta-glucan after seven days of anaerobic fermentation addition to basal diets affects the increase in propionic acid in the rumen fermentation profile. This increase is typically associated with water-soluble acts as energy sources for lactic acid bacteria. When YRF is added, it promotes the fermentation pathway, which leads to more propionic acid and less acetic acid (Phuong et al 2023).

In vitro rumen fermentation gas and methane production

Table 4 showed that Indigofera legume silage produced higher total gas than Elephant grass silage at an incubation time 24 hours (p<0.01). Adding chitosan additives could reduce the total gas and methane production at an incubation time 24 hours (p<0.01).

The mechanism of chitosan in the reduction of CH₄ indirectly inhibited the production of hydrogen gas as a raw material in the formation of methane. The ability of chitosan to reduce C₂ production in the rumen resulted in a decrease in the production of hydrogen gas (H₂), reducing methane gas formation activity. In this study, adding chitosan causes a decrease in CH₄, followed by a decrease in gas production, according to a previous study using a meta-analysis (Wencelová et al 2014). The in vitro rumen fermentation process produces total gases in CO₂, CH₄ and small amounts of H₂, N₂ and O₂. The total gas was produced from the degradation and fermentation of the substrate through rumen microbial activity. Among these macromolecules, carbohydrates are the primary nutrients that contribute significantly to total gas production compared to protein (Jayanegara et al 2018).

The decrease in CH ₄ production can be caused by inhibiting methanogenesis by reducing H₂as a substrate for the formation of CH ₄ (Janssen 2010). According to Harahap et al (2020), chitosan tends to reduce methanogenic archaea, the leading microbial group responsible for CH₄ formation. Another plausible explanation for the lower methanogenesis due to the addition of chitosan is reducing the protozoan population, particularly Entodinium spp. (Wencelová et al 2014; Harahap et al 2020). Many methanogens live in symbiosis with protozoa and take advantage of these faunas. Therefore, reducing the protozoan population was also expected to reduce methanogens and their possible methanogenic activity (Jayanegara et al 2021). Moreover, the replacement of acetate by propionate increases the demand for hydrogen, leaving less hydrogen available for methanogenesis, which results in lower methane emissions (Phuong et al 2023).

Conclusion

• Indigofera legume silage, which was high in protein, was more challenging to lower pH than elephant grass silage.
  
  
• Chitosan from shrimp shells impacted Elephant grass and Indigofera legume silage with lower dry matter loss than other additives.
  
  
• Chitosan additive increased the DMD and OMD of Elephant grass and Indigofera legume silage in the rumen in vitro.
  
  
• Chitosan additives in Elephant grass and Indigofera legume silage increased the C₃ content in the rumen but decreased the rumen NH₃ and C₂ concentration in the rumen in vitro.
  
  
• Chitosan additives can reduce CH₄ emissions, followed by a decrease in gas production.
  
  
• Chitosan additives can be used as a new alternative to silage inhibitor additives for Elephant grass, especially Indigofera legume with a high CP content.

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