sensory and chemical evaluation of laboratory ensiled hybrid Napier grass prepared using Lactobacillus plantarum and propionic acid as additives #

Sensory

Livestock plays an important role in Indian rural economy (Hosure et al., 2022) and maximisation of returns from dairy enterprises requires optimum utilisation of resources . Ensuring the availability of green fodder throughout year has been identified as a major constraint in economizing dairy cattle production in India (Kumar et al., 2015). Silage making is one of the economical methods of fodder preservation that could help to meet green fodder requirements during lean seasons (Stewart, 2011). Loss of nutrients during ensiling has been identified as a major challenge for effective silage production. Thus, strategies need to be optimized for preserving the nutritive value of forages while ensiling (Balehegn et al., 2022). Use of silage additives to modify fermentation process and to minimise nutrient losses from preserved fodder has been widely acknowledged.
Bacterial inoculum and organic acids can be used as silage additives. The technical and research innovations for identifying an ideal additive for silage preparation need to be explored. Quality of silage additives can be initially screened by laboratory ensiling of small quantity of fodder. Hence, the present study has been under taken to evaluate silage made from hybrid Napier grass ensiled in the laboratory prepared with or without additives.

Laboratory ensiling
Laboratory ensiling of fodder grass was undertaken at Department of Animal Nutrition, College of Veterinary and Animal Sciences, Mannuthy. Grass fodder ensiled with or without additives was evaluated for its physical and chemical characteristics and the silage adjudged to have highest nutritional quality was identified during the course of this study.
Hybrid Napier grass (Pennisetum purpureum) harvested at 45 days of maturity was procured from University Livestock Farm Fodder Research Development Scheme (ULF & FRDS), Mannuthy. L. plantarum and propionic acid (99 per cent pure) were used as silage additives for laboratory ensiling during the present study.
The chopped grass (2 to 3 cm length) was filled into a strong high-density polythene bag of size (45×60 cm) having a capacity to hold 2 to 6 kg of green fodder. Polythene bags used were clean, transparent and without any holes. During the study, grass fodder was ensiled without additives and with additives namely bacterial inoculum of L. Plantarum at a concentration of 1 x 10 5 CFU per gram of green fodder, propionic acid at the rate 0.45 kg per ton of green fodder, and a combination of both, as detailed in the Table 1. All four silages were prepared in three replicates. Ensiled mass was then gently and firmly squeezed by hand to expel air, and while compressing, the neck of the bag was twisted, turned over and tied with twine. Polythene bags containing chopped fodder were then inverted into another empty clean bag, which was also later closed and tied. The polythene bags were appropriately labelled showing details of date of packing and due date for opening. Sealed bags were then carefully placed in a plastic bucket to ensure protection from vermin and were stored at room temperature.

Evaluation and selection of ensiled grass fodder
The ensiled materials were evaluated for their physical and chemical characteristics at 21 st , 30 th and 45 th day of ensiling. A score card was devised for evaluating the physical and chemical characteristics of silages obtained from laboratory ensiling. Total score of silage was calculated by taking the sum of assigned scores.

Physical characteristics of silage
Silage odour was measured using score card devised. The sweet aroma, pungent and putrid odours were assigned +1, -1, and -2 scores, respectively.
Silage colour was evaluated using the colour scores table for silages developed by Tahuk et al. (2021). The natural green or yellowish green colour was given score of +2, dark green or yellowish colour was awarded +1 and brown to black colour was given score of -1, respectively.
Presence or absence of extraneous material as fungus or worms were observed and evaluated using score card. The absence of extraneous material was given a +1 score and their presence was given a -1 score, respectively.

Chemical characteristics of silage
Silage pH was evaluated using the score card. The pH of silage was estimated by method described by Australian Fodder Industry Association Laboratory Methods Manual (2011).
Silage samples collected at the end of ensiling period were used for the estimation of lactic acid (g/100g fresh silage) following the procedure given by Wilson and Wilkins (1972), using a spectrophotometer (Avi Scientific India, Thane, Mumbai).
The proximate composition, fibre fraction and acid insoluble ash were analysed by AOAC (2016). The toluene dry matter (DM) of ensiled material was calculated using formula given by Haigh (1979).

Sensory characteristics of silages
The sensory and chemical characteristics of various silages at 21 st , 30 th and 45 th day of ensiling are presented in Tables  2, 3 and 4, respectively. GS had an undesirable pungent odour at 21 st , 30 th and 45 th day of sampling. A dark green colour was observed on 2 st day of ensiling while brown to black colour was recorded on 30 th and 45 th day of ensiling. Fungal growth was observed on the 45 th day of ensiling. GSL produced a desirable sweet aroma in all samples collected at 21 st , 30 th and 45 th day of ensiling. A yellowish green colour was observed at the 21 st day of ensiling but the colour changed to dark green towards the 30 th and 45 th day of ensiling. Presence of fungus or worms was not observed while using L. plantarum as additive during various periods of ensiling. GSAhad an undesirable pungent odour and dark green colour at all three periods of sampling. The silage material was devoid of the presence of any fungus or worms at 21 st , 30 th and 45 th day of sampling. GSLA had a sweet aroma at 21 st and 30 th day of ensiling while it had an undesirable pungent odour at the 45 th day of sampling. A dark green colour was observed at each ensiling period and the ensiled materials were devoid of the presence of fungus or worms. Based on the results, GSL was found to be better in preserving the nutritive value of grass fodder. Similar observations were made by Obrien et al. (2008), who noted that the excellent quality silages had fresh texture and should not contain any fungal organisms in it. In a similar study, Randa et al. (2018) observed a light yellow to greenish colour and an acidic pungent smell for Napier grass ensiled for 45 days. They opined that good silage should possess moderately sweet aroma and this observation was similar to the present study. Habib et al. (2022) found that the colour of Napier grass silage as straw yellowish with a sweet fruity aroma and his observation was in collaboration with present the present study.

Silage pH
The pH value of GS ranged from 5.6 to 5.9. However, the pH of GSL silage at 21 st , 30 th and 45 th day of ensiling were found to be 4.3, 4.4, and 4.6 respectively. The pH of GSA ranged from 5.2 to 5.8; while that of GSLA ranged from 4.4 to 4.9. Kung et al. (2018) stated that pH values of good quality silage ranged between 4.3 and 4.7. Johnson et al. (2005) observed that the pH of the silages prepared with and without bacterial inoculum were 3.94 and 4.21.
These observations were in accordance with the present study.  Oliveira et al. (2017), who noted that the DM content of silages made from tropical grasses ranged between 24.85 to 31.80 per cent.

Chemical composition of silages
After laboratory ensiling, GS had the CP content of 6.40 to 6.90 per cent and GSL had CP content of 9.72 to 9.73 per cent. The CP content ranged from 8.37 to 8.60 per cent, in GSA whereas GSLA had CP content of 8.45 to 8.88 per cent, respectively. In accordance with the values observed in this study, Yammuen et al. (2020) observed that L. plantarum as additive had resulted in silage having a CP content of 6.80 per cent. Similarly, Xiong et al. (2022) reported that when L. plantarum inoculated hybrid Napier silage had a CP content of 8.20 per cent.
During lab enisling, all the four silage combinations had a CF content that ranged between 28.43 to 29.63 per cent. GS had the EE content of 1.72 to 1.75 per cent, while GSL had 2.10 to 2.98 per cent EE. Similarly, the EE content in GSA and GSLA were 1.88 to 2.26 and 2.28 to 3.23 per cent, respectively. In accordance with the results of present study, Srisaikham (2022) observed that CF content and EE content were 26.85 and 2.48 per cent, respectively in Napier grass ensiled for 21 days. Similarly, Mbuthia and Gachuiri (2003) reported that ensiling had resulted in a reduction of NDF value from 52 to 61.30 per cent in Napier grass silage and Bureenok et al. (2012) reported that the NDF content ranged between 62.30 to 72.60 per cent when grass was preserved with Lactic Acid Bacterial (LAB) inoculum.

Lactic acid concentration (g/100g fresh silage)
The lactic acid concentration was found to be in the range of 0.11 to 0.22 for GS. For GSL silage at 21 st , 30 th and 45 th day of sampling the lactic acid concentrations were 5.47, 4.22 and 2.39 respectively ( Table 8).
The lactic acid concentration of GSA ranged between 0.13 to 0.51 and the values for GSLA 520 Sensory and chemical evaluation of laboratory ensiled hybrid Napier grass ___________________________   Boonkoed et al. (2018) reported that lactic acid concentration of Napier silage was 4.45 per cent of total volatile fatty acid. Based on the total of various grass silages, it could be concluded that use of additives had improved the sensory attributes and chemical characteristics of silage. Hence GSL at 21 st day of ensiling was adjudged to be the best quality silage.

conclusion
Based on the results of laboratory ensiling, it was found that the Hybrid Napier grass ensiled with L. plantarum as additive at the rate of 1 x 10 5 CFU per gram of fresh forage had superiorly preserved the nutritive value of green grass than other three groups. L. plantarum helps in rapid reduction of silage pH and increased lactic acid production that enhances the keeping quality of ensiled material. Hence, use of L. plantarum had resulted in production of superior quality silage with acceptable acidic pH, greenish yellow colour, desirable aromatic smell, and higher lactic acid concentration. Observations made in the current study also suggested that combination of additives (L. plantarum and propionic acid) also resulted in production of good quality silage. However, use of propionic acid alone could not result in production of good quality silage. From the overall results, it could be concluded that hybrid Napier grass, ensiled for 21 days using L. plantarum was effective in preserving its nutritive value,