Biochemical Assessment of Solid-State Fermented Elephant Grass and its Potential Incorporation in Broiler's Diets

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Introduction
The world's increasing population demands the utilization of existing livestock resources to meet the demand for animal protein, but this is only possible if optimal forage and fodder production is ensured (Weishaupt et al., 2020;Ezedom et al., 2022). Availability of quality feed and forage resources are essential for sustainable livestock production (Negawo et al., 2017;Naah et al., 2019;Bakare et al., 2020). One of the major challenges faced by livestock producers in tropical countries is providing proper nutrition for their animals, especially during the dry season when pasture and cereal residues are limited in quantity and nutritional quality Michalk et al., 2018;Kebede 2020).
Pennisetum purpureum Schumach, commonly known as elephant grass, is a monocot species belonging to the Poaceae family and Pennisetum genus. It is widely used as forage in tropical and subtropical regions and is a major source of livestock feed for cattle, dairy, and sheep in West Africa (Bakare et al., 2020). Due to the small and fragmented landholdings of most smallholder livestock producers, elephant grass is a preferred alternative to other feed options as it is a high-yielding forage that requires minimal inputs and acreage (Negawo et al., 2017;Ojo et al., 2022;Ndego et al., 2023).
There is potential for enhancing the nutritional quality of elephant grass through the application of advancements observed in other fodder crops (Simeão et al., 2021;Chandel et al., 2021). Improving the digestibility and nutritional value of elephant grass could significantly boost livestock productivity. However, the existing challenges in terms of palatability, nutritional quality, propagation via seed or vegetative organs, and diseases like elephant grass stunt and head smut significantly affect its production in some regions of Africa (Makini et al., 2019;Mutwedu et al., 2020;Egbune et al., 2022).
Improving the utilization of elephant grass requires the use of appropriate processing methods to enhance its nutritive value. Solidstate fermentation (SSF) is one such method used to improve the bioavailability of nutrients and increase the nutritive value of animal feed (Olukomaiya et al., 2020;Egbune et al., 2021a). The aim of this study is to enhance the nutritional value of elephant grass for use as animal feed and for the production of mannanase.
The study examines the biochemical evaluation of pre-treated ensiled elephant grass and its potential use in broiler diet.

Various pretreatment for elephant grass
To enhance the nutritional quality of elephant grass, various pretreatments were applied, including ensiling and solid-state fermentation with the addition of R. oligosporus and/or urea.
The ensiling process involves storing the elephant grass with 10% water in air-tight nylon bags for 21 days, followed by 7 days of solidstate fermentation. The solid-state fermentation process involves adding 10% water and/or 10% R. oligosporus and/or 1.2% urea to the elephant grass and then subjecting it to fermentation for 3 days, ensiling for 18 days, and then another 7 days of solid-state fermentation. After each process, the fermented mixture is dried and ground into small pieces. The following are various pretreatments for elephant grass, along with their respective acronyms: E: Elephant grass: Unfermented elephant grass: This does not involve any processing steps, and the elephant grass is fed as it is, without any fermentation or additives. fE: Ensiled elephant grass -In this pretreatment, 10% water is added to the elephant grass, and the mixture is ensiled for 21 days in air-tight nylon bags, followed by 7 days of solid-state fermentation. After that, the fermented mixture is dried and ground into small pieces. fEH: Ensiled elephant grass with R. oligosporus -Elephant grass + 10% R. oligosporus + 10% water: This process involves adding 10% water and 10% R. oligosporus to the elephant grass.
The mixture is ensiled for 21 days in air-tight nylon bags, followed by 7 days of solid-state fermentation. After that, the fermented mixture is dried and ground into small pieces. fEU: Ensiled elephant grass with urea -Elephant grass + 1.2% urea in 10% water: This process involves adding 1.2% urea to 10% water, and then adding the mixture to the elephant grass. The mixture is ensiled for 21 days in air-tight nylon bags, followed by 7 days of solid-state fermentation. After that, the fermented mixture is dried and ground into small pieces. fEHU: Ensiled elephant grass with R. oligosporus and urea -Elephant grass + 1.2% urea in 10% water: This process involves adding 1.2% urea to 10% water, and then adding the mixture to the elephant grass. The mixture is ensiled for 21 days in air-tight nylon bags, followed by 7 days of solid-state fermentation. After that, the fermented mixture is dried and ground into small pieces. fsEH: Solid state fermented and ensiled elephant grass with R. oligosporus -Elephant grass + 10% R. oligosporus + 10% water: In this process 10% water and 10% R. oligosporus are added to the elephant grass. The mixture is then subjected to solid-state fermentation for 3 days, ensiled for 18 days, and then subjected to another 7 days of solid-state fermentation. fsEHU: Solid state fermented and ensiled elephant grass with R. oligosporus and urea -Elephant grass + 10% HPY + 1.2% urea in 10% water: This process involves adding 10% R. oligosporus, 1.2% urea, and 10% water to the elephant grass. The mixture is then subjected to solid-state fermentation for 3 days, ensiled for 18 days, and then subjected to another 7 days of solid-state fermentation.

Biochemical parameters
Total soluble protein was determined using the procedure described by Gornall et al. (1949) with bovine serum albumin as the standard. The glucose content was estimated using the Randox glucose kit and following the manufacturer's instructions. Reducing sugars were estimated using the 3,5-dinitrosalicylic acid (DNS) colorimetric technique (Miller 1959). The total phenol content was determined by the procedure described by Singleton and Rossi (1965), using catechin as the standard. The total flavonoid contents were determined by colorimetry following the assay procedure described by Jia et al. (1999). The study utilized the DPPH assay, as described by Hatano et al. (1988), to determine the antioxidant activity of elephant grass. The reducing power of the extracts was examined using the procedure described by Benzie and Strain (1996). The modified methods of Araujo and Ward (1990) were used to determine the mannanase activity.
Experimental design, feed formulations and trial In this study, eight different broiler starter feed diets were formulated by substituting 25% of maize with pretreated elephant grass while keeping all other ingredients constant (see Table  1. for detailed diet compositions). The study involved a total of forty newly hatched Cornish White x White Plymouth Rock broiler chicks, which were divided into eight groups of eight chicks each and fed the respective diets for a duration of four weeks. Throughout the trial, the chicks had free access to feed and water. To determine their weight gain, the chicks' weights were recorded on the first day of the experiment and at the end of the fourth week, and the difference in weight (g) between the two measurements was calculated. Venous blood was collected from the chicks' wings and/or legs using sterile syringes and needles, and serum was obtained by centrifuging the clotted blood at 2000 g for 10 minutes. The serum was stored at 4°C for subsequent biochemical analyses.

Weight gain measurement
Each chick's weight was measured at the start of the experiment and after four weeks to calculate their weight gain. In an effort to develop a costeffective, nutritious, and healthy feed, 25% of the maize content in the broiler starter feed was substituted with pretreated elephant grass.

Evaluation of the biochemical effect (s) of feed formulations
The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were determined as biochemical indices. The assays for these indices were conducted following the instructions provided in their respective RANDOX assay kits (Randox Laboratories Ltd, UK).

Statistical analysis
Data obtained were subjected to statistical analysis using one-way ANOVA (analysis of variance) and Fischer's test of least significance (LSD); values are presented as Mean ± Standard deviation. A significance level of 0.05 (p<0.05) was used to determine the statistical significance of the results with a 95% confidence level.

Results
The pre-treated ensiled elephant grass showed a significant (p<0.05) increase in soluble protein concentration following fermentation compared to the control, as depicted in Figure 1. The solidstate fermented and ensiled elephant grass using R. oligosporus exhibited the highest concentration of soluble protein, which was measured at 26.4±0.6mg/g. Figure 1 presents the glucose concentration of pre-treated ensiled elephant grass, which decreased significantly (p<0.05) due to fermentation when compared to the control. Similarly, Figure 1 shows a significant decrease (p<0.05) in the reducing sugar concentration of pre-treated ensiled elephant grass compared to the control. show significant differences (p > 0.05) from the control, whereas values with the letters "b", "c", "d", and "e" demonstrated significant differences (p < 0.05).
In this study, various pre-treatments were examined to determine their effect on the total phenolic and flavonoid content of ensiled elephant grass. As indicated in Figure 3, the solid state fermentation and ensiling of elephant grass with R. oligosporus and urea resulted in the highest total phenolic content (32.8±0.6 μg/ml), which was significantly different (p<0.05) from the control (25.1±0.5 μg/ml) and other pre-treatments. All pre-treatments showed a significant increase (p<0.05) in total phenolic content compared to the control.    Urea. The results indicated that values with the letter "a" did not show significant differences (p > 0.05) from the control, whereas values with the letters "b", "c", "d", and "e" demonstrated significant differences (p < 0.05).
The results presented in Figure 5 show that there was no significant difference in weight gain observed in the birds that were maintained on ensiled elephant grass, ensiled elephant grass with R. oligosporus and solid state fermented and ensiled elephant grass with R. oligosporus and urea. However, birds that were maintained on all other diets exhibited a significant reduction.   Figure 6 presents the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) activities (measured in U/L) of the birds. The findings revealed no significant difference in the activities of these enzymes between the control diet and the experimental diets. However, there was a significant increase (p<0.05) in the alanine aminotransferase (ALT) levels of birds fed ensiled elephant grass with urea compared to the control diet. Fig 6: AST, ALT and ALP activity in the serum of broiler chicks fed with formulated diet Each bar is an expression of mean ± SD, an equivalent of five replications (n = 8), and designation of each bar with different letters showed marked significant difference at p < 0.05. Each bar in the graph represents the mean value ± standard deviation of five replications (n=5). Different letters used to label the bars indicate significant differences at p<0.05. The different treatments are represented as follows: E (Elephant grass), fE (Ensiled Elephant grass), fEH (Ensiled Elephant grass with R. oligosporus), fEU (Ensiled Elephant grass with urea), fEHU (Ensiled Elephant grass with R. oligosporus), fsEH (Solid state fermented and ensiled elephant grass with R. oligosporus), and fsEHU (Solid state fermented and ensiled elephant grass with R. oligosporus and Urea).

Discussion
During the solid-state fermentation and ensiling of elephant grass with R. oligosporus, there was a significant increase in the concentration of soluble proteins compared to the control. This process of solid-state fermentation has numerous benefits, including increased amino acid and soluble protein content, decreased mycotoxins, and prevention of oxidative rancidity.
Additionally, changes in the concentrations of protein, fat, and carbohydrates indicate that the enzymatic activity of microorganisms alters the texture, flavor, and nutritional composition of the fermented product (Londoño-Hernandez et al., 2018). Similar results have been observed in The increased protein content in the fermented maize cob is believed to be due to the secretion of extracellular enzymes, such as amylases, linamarase, and cellulase, by the fermenting organisms as they utilize cassava starch as a carbon source. These findings are consistent with the results of Anigboro et al. (2020).
In this study, the concentration of glucose in pre-treated ensiled elephant grass was significantly lower after fermentation than in the control group (p>0.05). This is in contrast to a study by Anigboro et al. (2020), which found an increase in glucose content in maize cob after fermentation with R. oligosporus. The findings of Anigboro et al. (2020) indicate that solid-state fermentation can increase glucose concentration at different inoculum sizes. During the fermentation process, the fungus absorbs simple sugars and starts breaking down starch chains into multiple pieces through the action of enzymes such as amylase, which increases the sugar level. This hydrolysis of starch leads to the release of glucose, which is the main reducing sugar and causes an increase in this type of sugar for all tested treatments, while the fungus is growing in the substrate. As a result, microorganisms produce specific extracellular enzymes that break down complex polysaccharides to glucose throughout the fermentation process, leading to an increase in glucose concentration.
The concentration of reducing sugar in pretreated ensiled elephant grass decreased significantly (p>0.05) compared to the control. The breakdown of starch during fermentation caused the reducing sugar in the fermented product to decrease. Previous studies have shown that higher levels of reducing sugars increase the susceptibility of starch to hydrolysis, which suggests that consuming fermented agricultural byproducts may be beneficial for people with diabetes (Budhwar et al., 2020;Baniwal, et al., 2021;Qin et al., 2022).
The study found that pre-treated ensiled elephant grass had significantly higher DPPH free radical-scavenging ability than the control. These results align with earlier research that has shown solid-state fermentation (SSF) to be a promising technology for enhancing the antioxidant potential of fermented agricultural byproducts, such as cassava stems, due to their rich polyphenolic compounds that can neutralize reactive oxygen species (ROS) generated during the lipid peroxidation process (Jogawat et al., 2021;Sharma et al., 2021). The polyphenolic compounds in the fermented elephant grass may act as proton donors to ROS, terminating the lipid peroxidation chain by forming more stable and less reactive compounds in the presence of the R. oligosporus strain.
Compared to the control sample, the pre-treated ensiled elephant grass that was solid-state fermented, ensiled, and combined with R. oligosporus and urea had a significantly higher total phenolic content (p<0.05). Similar results were observed for other pre-treated ensiled elephant grass samples (p<0.05), but there was no significant difference in flavonoid concentration between the pre-treated ensiled elephant grass and control samples (p>0.05). However, the pre-treated ensiled elephant grass that was solid-state fermented and ensiled with R. oligosporus and urea had a significantly higher total phenolic content (p<0.05) than the sample that was only solid-state fermented and ensiled. This is consistent with previous research that has demonstrated the ability of phenolic compounds to quench oxygen radicals (de Siqueira et al., 2019;Liu et al., 2022). The polyphenolics in the fermented media likely served as effective electron and hydrogen atom donors, preventing the chain reaction of free radicals by converting them to more stable molecules (Egbune et al., 2022b).
The findings of the study are in agreement with earlier research on fermented seeds, which showed that fermentation increased the phenolic content of the seeds (Osete-Alcaraz et al., 2019;Inada et al., 2020Santos et al., 2021. Phenolic compounds are typically bound to sugar, reducing their bioavailability to the body. During fermentation, proteolytic enzymes break down phenolic compounds into soluble-free phenols and other less complex but more physiologically active phenols that are readily absorbed (Gabriele and Pucci 2022;Rasera et al., 2023). The study also found that the phenolic concentration decreased as fermentation time increased. This is likely due to the diffusion of phenolics in cell liquids and the oxidation of disseminated phenolics by polyphenol oxidase, as reported by Lasekan and Shabnam (2013).
The study found that birds fed diets comprising solid state fermented elephant grass, ensiled elephant grass with R. oligosporus, and ensiled elephant grass with R. oligosporus and urea did not exhibit any significant differences in weight gain when 25% of their maize diet was replaced. However, birds on other diets experienced significant weight loss. The biochemistry behind these results can be attributed to the nutritional composition of the diets. Ensilage is a method of preserving forages that involves anaerobic fermentation, which can improve the nutrient profile of the feed (Wilkinson and Rinne 2018;Egbune et al., 2021b). The addition of R. oligosporus to the ensiled elephant grass can further enhance its nutritional quality by breaking down antinutrients and increasing protein content . Solid state fermentation of elephant grass also has the potential to improve its nutritional value by breaking down fiber and increasing the availability of nutrients (Debnath et al., 2022;Anigboro et al., 2022). Urea is a source of non-protein nitrogen that can be used to supplement low-protein feeds and improve the nitrogen balance in the diet (Lopes et al., 2020). Therefore, the diets containing these forages were able to provide the necessary nutrients for the birds to maintain their weight, resulting in no significant difference compared to the control diet. Overall, the use of forages in broiler feeding can have economic benefits, as they can reduce the volume and cost of feed used in production (Jørgensen et al., 2022). Additionally, the nutritional quality of the forages can contribute to maintaining weight and improving the overall health of the birds. The broiler chicks were able to consume and metabolize the experimental diets as well as the control feed with equal efficiency. These findings are consistent with previous studies conducted by Avwioroko et al. (2016), Tonukari et al. (2016), Svihuset al. (2004). Using forages in broiler feeding could lead to a complete reduction in feed volume and cost, resulting in substantial cost savings. Furthermore, the study examined the effects of the experimental diets on liver health by measuring serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) activities in the birds. The results showed no significant differences in these enzyme activities between birds fed the control diet and those fed the experimental diets, except for birds fed elephant grass and urea, whose ALT levels increased considerably (but not significantly) compared to the control. According to Koranteng et al. (2022), there was no difference in these enzyme activities when birds were fed fermented maize cob compared to the control. The ALT/AST activities observed in this study were within the normal range, and significant elevation of these enzyme activities could indicate liver damage, inflammation, or cell death. ALT and AST are involved in the metabolism of carbohydrates and proteins by transaminating key chemicals. Therefore, it can be concluded that the experimental diets were equally safe for the liver as the control diet, as there were no significant differences (p>0.05) in serum ALT and AST activities in birds fed the experimental diets compared to those fed the control diet.

Conclusion
Pre-treated and ensiled elephant grass can be a valuable ingredient in animal feed production, thanks to its significant increase in protein content and antioxidant activity. This study highlights the potential usefulness of mannanase in the animal feed industry, particularly for incorporating elephant grass into feed formulations. The results of the study suggest that adding pre-treated ensiled elephant grass to broiler starter diet does not have any adverse effects on blood biochemical profiles. References Ezedom, T., Egbune, E., Ehikordi, M., Ezeugo, N., Eledu, F., Esiete, J. and Tonukari, N. (2022). Biochemical evaluation of autoclaved and solid state fermented tropical pasture grasses. J.