Biological enrichment of mustard oil cake by solid-state fermentation using lactic acid bacteria and its impact on rumen fermentation

Introduction

Mustard oil cake (MOC) is a by-product of mustard oil extraction and is a nutritionally rich agro-industrial residue with appreciable levels of protein, fibre, and minerals. However, its direct use in animal feed or value-added products is limited by the presence of antinutritional factors, such as glucosinolates, phenolic compounds, and complex fibre fractions, which reduce digestibility and palatability (Singh et al., 2025) [24] . In recent years, there has been growing interest in biotechnological approaches to enhance the nutritional quality and bioavailability of such agro-residues, promoting sustainable feed resources and reducing environmental burden (Barcelos et al., 2020) [2] . Solid state fermentation (SSF) is a costeffective bioprocess technique that involves the growth of microorganisms on moist solid substrates in the absence or near-absence of free water. SSF has shown considerable promise in improving the nutritional profile of various agro-wastes by degrading complex polymers, increasing protein content, and reducing anti-nutritional compounds (Pandey, 2003; Soccol et al., 2017) [21,25] . The unique ecological niche provided in SSF closely mimics natural decomposition and allows selected microbial strains to produce extracellular enzymes such as cellulases, proteases, and phytases, which enhance substrate digestibility (Llimós Turet, J., 2022) [14] . Lactic acid bacteria (LAB), particularly strains of the genus Lactobacillus, are widely recognized for their safe status (GRAS) and their ability to ferment a broad range of substrates, producing organic acids, vitamins, and antimicrobial compounds (Hammes & Vogel, 1995) [11] . Their application in SSF of oilseed cakes has been associated with improved nutrient release, reduction of anti-nutritional factors, and increased probiotic potential (Barcelos et al. 2020; Bintsis et al., 2018) [2,4] .

Despite extensive studies on bacterial and fungal fermentations of various oilseed cakes, there is limited research specifically targeting SSF of mustard oil cake using Lactobacillus strains for nutrient enhancement. In this context, the present study investigates the potential of selected Lactobacillus strains to biologically enrich mustard oil cake through solid state fermentation. We hypothesise that SSF with Lactobacillus will improve the nutritional availability, decrease anti-nutritional components, and enhance the overall suitability of MOC for use in animal feed and other value-added applications. Findings from this study may contribute to more sustainable utilization of oilseed residues and offer insights into microbial treatments that enhance agro-industrial by-products. Solid-state fermentation (SSF) can be used to valorize agro-industrial by-products such as brewer's spent grains, olive pomace, grape mark, vine-shoot trimmings, wheat bran, rice bran, using an eco-friendly approach with low environmental impact compared to conventional submerged fermentation (Zhao et al., 2017; Ferreira et al., 2020) [31,10]

Materials and Methods

The current research was conducted at the Rumen Biotechnology Laboratory, Animal Nutrition Division, ICAR-National Dairy Research Institute, Karnal, Haryana. All media, chemical ingredients, and reagents utilized during the investigation were procured from M/S Hi Media Laboratories Private Ltd. (Mumbai, India).

Mustard oil cake fermentation 2.1.1 Inoculum preparation

The inoculum preparation method described by Zhang et al., (2014) [30] , with slight modification was used. The selected strain of Lactobacillus consortia which contain Lactobacillus plantarum and Pediococcus acidilactici was grown in MRS broth with (peptone 10 g/L, yeast extract 5 g/L, beef extract 10 g/L, sodium acetate anhydrous 5 g/L, ammonium citrate tribasic 2 g/L, K 2 HPO 4 2g/L, MgSO 4 .7H 2 O 0.1 g/L, MnSO 4 .H 2 O 0.05 g/L, tween-80 1 g/L, pH 6.8) at 37 °C for 20 h. The cells were then harvested and resuspended in sterilized physiological saline and adjusted to 10 8 CFU/mL. This cell suspension was ready to serve as an inoculum for MOC fermentation.

Preparation of fermented mustard oil cake

Fermented mustard oil cake was prepared using a method adapted from Dai et al., (2017) [8] with slight modifications. Mustard oil cake was first ground into a powder using a 40mesh sieve before fermentation. About 50 g of each mustard oil cake was mixed with 50 mL of water and autoclaved at 121 °C for 15 minutes. After cooling, the mixtures were inoculated with Lactobacillus at a 2 McFarland concentration and incubated at 37 °C for 72 hours to obtain the fermented mustard oil cake.

Preparation of fermented meal hydrolysate extract

Fermented feed extract was prepared according to the method described by Ye et al., (2003) [29] with slight modifications. About 5 g of fermented feed sample was mixed with 50 ml of distilled water, homogenized for 1 minute and incubated at 37 °C for 60 minutes. The incubated mixture was centrifuged at 10,000 rpm for 4 minutes, and the residue was washed with 20 ml distilled water, centrifuged again at the same speed and time, and the combined supernatant was freeze-dried and stored at (-) 20 °C until further use.

The determination of pH

Approximately 10 g of fermented mustard oil cake was homogenized in a blender with 90 mL of distilled water for 30 seconds. The pH value of the resulting suspension was then measured using an EUTECH pH meter.

Determination of lactic acid in fermented feed

The estimation of lactic acid in mustard oil cake and fermented mustard oil cake samples was done as per the method of Barker and Summerson (1941) [3] . In all the test tube containing samples and standards, 0.05 mL 4% CuSO 4 was added, subsequently 6 mL concentrated H 2 SO 4 was added drop by drop at the center of the liquid with continuous shaking. The tubes were kept in boiling water bath for 5 min cooled down to room temperature, and added with 0.1 mL p-hydroxy diphenyl reagent drop by drop. The contents were mixed immediately with vigorous shaking followed by incubation at 30 °C for 30 min with periodic shaking. Again, the tubes were kept in boiling water bath for 90 seconds, remove and cooled to room temperature. Finally, the colour of the solution was read at 560 nm against a reagent blank. The value obtained from the standard curve is multiplied by the dilution factor 10 to get the lactic acid content in µg/mL water extract of fermented feed.

Chemical analysis of collected samples (AOAC, 2005) 2.2.1 Dry matter (DM)

A measured quantity of sample in a dry aluminum moisture cup or aluminum tray was taken and placed in hot air oven to dry at 100±5 °C for 24 h. The loss in moisture content after drying was estimated and the dry matter was calculated as follows:

(wt. of moisture cup + sample after drying-Wt. of moisture cup) DM (%) = *100 wt. of sample 2.2.2 Total ash (TA) 3g sample was taken in pre-weighed silica crucible. After charring the sample on heater (till the smoke disappeared), the crucible was kept in the muffle furnace for ignition at 550 °C for 2-3 h. The crucible was removed on cooling and kept in a desiccator and weighed again to find out weight of ash. The ash content was calculated as given below.

(wt. of crucible + ash after cooling-wt. of crucible) Total ash (%) = *100 Wt. of fresh sample

Organic matter (OM)

The organic matter was determined by subtracting the total ash content from 100 i.e.; OM (%) = 100-Total ash (%)

Crude Protein (CP)

1.0 g sample was taken in Kjeldahl flask and digested with 30 mL concentrated H 2 SO 4 in the presence of 2-3 g of digestion mixture, till the solution became colourless. After digestion, the contents were cooled and volume was made to 100 mL, 10 mL of aliquot was distilled in Kjeldahl distillation apparatus (KELPLUS Nitrogen Analyzer) after adding 10-15 mL of 40% NaOH solution. About 60-75 mL of distillate (light green colour) was collected into an Erlenmeyer flask containing 10 mL of 2% boric acid indicator solution. The distillate was then titrated against N/100 H 2 SO 4 solution, and the end point was recorded when the colour changed to a slight pinkish. The volume of N/100 H 2 SO 4 solution used in titration was measured and recorded.

(0.014x volume of N H 2 SO 4 x volume made (ml) N (%) =

x 100 (Aliquot taken x wt. of sample)

The crude protein (%) of the sample was calculated by multiplying the N-content with factor 6.25. This was based on the principle that protein contains 16% nitrogen.

Ether extract (EE)

For ether extract analysis 3g sample was taken in a cellulose thimble and extracted for 8 h with petroleum ether (40-60 °C) in Soxhlet's extraction apparatus attached to a pre weighed oil flask. The oil flask was removed, and after evaporating the excess of ether, it was dried overnight in a hot air oven (temperature 100±5 °C). The flask was cooled in a desiccator and weighed to a constant value. The ether extract was estimated as the difference in the weight of oil flask with and without oil. To this, 100 mL of neutral detergent solution (Sodium lauryl sulphate 30.00g, Disodium ethylene diamino tetra-acetate (EDTA) 18.61 g, Sodium borate decahydrate 6.81 g, Disodium hydrogen phosphate (anhydrous) 4.56, Triethylene glycol10 mL, Distilled water 990 mL) was added. Afterwards, 0.5 g of anhydrous Sodium sulphite was added. The contents of the spoutless beaker were refluxed for an hour. One hour after the initial onset of boiling, the sample was filtered through pre-weighed 50 mL sintered glass crucible grade-1 using an oil-free vacuum pump. The material was washed with hot boiling water and then acetone to remove all salts. The sintered crucible containing residue was dried in a hot air oven (100±5 °C) and weighed again. The NDF was calculated as follows:

(wt. of crucible with residue-wt. of empty crucible) NDF (%) = *100 wt. of sample taken

Measurement of in vitro Rumen Fermentation Parameters 2.3.1 Substrate Preparation and Experimental Treatments

The substrate feed was prepared containing 60% roughage and 40% concentrate mixture. The concentrate mixture was composed of maize grain (33%), cotton seed cake (11%), ground nut cake (11%), soybean meal (11%), wheat bran (20%), De-oiled rice bran (6%), Bajra (5%), mineral mixture (2%), and common salt (1%). Roughage feed contains green maize fodder and wheat straw fodder in a 60:40 ratio. Both components were thoroughly mixed. The substrate feed was dried at 60 °C for 24 hours to ensure consistent dry matter content. Four experimental treatments were evaluated: control T 0 (only TMR), T 1 (22% mustard oil cake in concentrate of TMR), T 2 (11% mustard oil cake +11% fermented mustard oil cake in concentrate of TMR), T 3 (22 % fermented mustard oil cake in concentrate of TMR). All treatments were conducted in triplicate.

Incubation Procedure

The incubations were performed in 100mL calibrated glass syringes, by the method given by Menke and Steingass (1988) [18] with slight modifications. For each incubation syringe, 200 mg of the T 0 , T 1 , T 2 and T 3 were precisely weighed into each syringe. The piston was coated with petroleum jelly and pushed inside the syringe. The syringes were incubated at 39 °C in a water bath after adding in vitro solution and removal of air from the syringe. The in vitro solution consisting of distilled water (500 mL), rumen buffer solution (250 mL), resazurin solution (1.25 mL), micro mineral solution (0.124 mL), and macro mineral solution (250 mL) was prepared separately. Rumen liquor was added once the mixture had turned colorless after being preheated to 39 °C and fizzed with CO 2 . Following adequate mixing, 30 mL of this mixture was injected into syringes via an auto dispenser, gently shaken, the outlet closed, and then incubated for 24 hours at 39 °C for later use. Syringes were shaken every 30 minutes, and the piston level was verified for the initial reading.

Measurements and Analytical Procedures 2.4.1 Total gas production

In vitro gas production was measured at 24 hours after incubation by reading the volume on the calibrated syringes. The total gas produced was expressed as the volume of gas accumulated (mL/200 mg DM).

Digestibility parameters

After end of 24 h of incubation, fermentation was arrested by chilling at 4 °C, followed by the collection of a suitable aliquot of gas for methane estimation; the syringe contents were then transferred to centrifuge tubes. The tubes were centrifuged at 3000 rpm for 10 min, and the resulting pellets were used to determine in vitro dry matter (DM) and organic matter (OM) digestibility. Pellets were dissolved in 100 mL neutral detergent solution (NDS) in 500 mL spoutless beakers and refluxed at 100 °C for 1 h. The contents were vacuum-filtered through pre-weighed (G1) crucibles, rinsed with hot water, oven-dried at 65 °C to constant weight, and ashed at 550 °C. in vitro dry matter digestibility (IVDMD) and true organic matter digestibility (IVTOMD) were determined from DM and OM disappearance, respectively. In vitro organic matter digestibility PF (mg/mL) = Net gas production (mL)

Estimation of Total and Individual Volatile Acid Production

For determining individual volatile fatty acids, 5 mL of the sample (from IVGPT) was taken in a beaker, and 1 mL of 25 % metaphosphoric acid (prepared in 5 N H

Results and Discussion

Change in pH of fermented Mustard oil cake

The pH of mustard oil cake decreased significantly (p<0.05) during solid-state fermentation with lactic acid bacteria. The initial pH of raw MOC was 5.66, which decreased to 5.31 in the control after 72 h and further declined to 4.35 in the fermented treatment. The significant reduction in pH in the fermented mustard oil cake indicates active fermentation and organic acid production by lactic acid bacteria. The decrease in pH is primarily attributed to the production of lactic acid and other organic acids during microbial fermentation of soluble carbohydrates (McDonald et al., 2011; Kung et al., 2018) [17,13] . Lactic acid bacteria convert fermentable sugars into lactic acid, resulting in acidification of the substrate and improved preservation and microbial stability (Soccol et al., 2017) [25] . Lower pH also inhibits the growth of spoilage and pathogenic microorganisms, thereby improving feed safety and quality (Niba et al., 2009) [20] . Similar reductions in pH during lactic acid bacterial fermentation of oilseed cakes and plant protein sources have been reported by Mukherjee et al. (2006) [19] , indicating effective fermentation and improved feed quality. The observed decrease in pH in fermented mustard oil cake confirms successful lactic acid fermentation and enhanced preservation and nutritional value. [13,25] . The higher lactic acid production in fermented mustard oil cake reflects enhanced microbial fermentation and improved substrate utilization. Increased lactic acid concentration also helps reduce undesirable microorganisms and improves feed stability and safety (McDonald et al., 2011) [17] . Similar increases in lactic acid production during LAB fermentation of plant-based feed substrates have been reported by Mukherjee et al. (2006) [19] and Niba et al. (2009) [20] , indicating improved fermentation efficiency and feed quality.

Organoleptic evaluation of fermented MOC

After 72 hours of fermentation, mustard oil cake (MOC) exhibited a lighter texture, darker color, and a stronger sour aroma compared to its unfermented MOC. In a similar study, Ma et al. (2022) [15] observed that after 3 and 30 days of fermentation, the ZZUPF95 and ZZUPF95+protease groups consistently maintained a fluffy texture, brown coloration, and sour aroma, distinguishing them from other groups after just 3 days of fermentation.

The lighter texture of the fermented mustard oil cake (MOC) is likely a result of the breakdown of complex macromolecules into simpler forms, such as proteins and carbohydrates, during fermentation. The enzymatic activity of lactic acid bacteria (LAB), coupled with the production of organic acids, helps loosen the substrate structure, contributing to the fluffy appearance (Ma et al., 2022) [15] . The darkening of color is primarily due to Maillard reactions and the oxidation of phenolic compounds during fermentation. Additionally, microbial growth and the accumulation of fermentation by-products, such as organic acids, contribute to these colour changes. The darker coloration enhances the visual appeal, particularly for animal feed formulations. The sour aroma is a consequence of the production of lactic acid, volatile organic compounds, and other metabolites by LAB.

Proximate analysis of fermented mustard oil cake

The proximate composition of mustard oil cake (MOC) before and after fermentation with lactic acid bacteria (LAB) is shown in [6,25] . Microbial fermentation enriches protein content through microbial cell proliferation and synthesis of single-cell protein, thereby improving the nutritional quality of the substrate (Niba et al., 2009) [20] . Crude fiber (CF) content decreased significantly from 5.86% in MOC to 4.72% in FMOC, indicating partial degradation of structural carbohydrates during fermentation. Lactic acid bacteria and associated microorganisms produce fibrolytic enzymes that degrade cellulose and hemicellulose, resulting in reduced fiber content and improved nutrient availability (Van Soest, 1994; Kung et al., 2018) [27,13] . Ether extract (EE) content also decreased significantly in FMOC (7.16%) compared to MOC (8.52%), which may be due to microbial utilization of lipids as an energy source during fermentation (Soccol et al., 2017) [25] . Similar reductions in ether extract and fiber content following microbial fermentation have been reported in fermented oilseed cakes and plant protein sources (Kumar et al., 2015) [12] . Dry matter (DM) content decreased significantly in fermented samples compared to raw MOC, which may be attributed to moisture addition and microbial metabolism during solid-state fermentation (Mukherjee et al., 2006) [6] . Total ash (TA) content did not differ significantly among treatments, indicating minimal effect of fermentation on mineral concentration. Overall, the results indicate that solid-state fermentation of mustard oil cake with lactic acid bacteria improved its nutritional quality by increasing crude protein and reducing fiber and ether extract content, thereby enhancing its suitability as a protein source for ruminant feeding.

In vitro gas production technique for evaluation of Total gas production, IVDMD, IVOMD, and partition factor of fermented soybean meal

The total gas production (TGP), neutral detergent fibre (NDF), in vitro dry matter digestibility (IVDMD), in vitro organic matter digestibility (IVOMD), and partitioning factor (PF) for the various samples are presented in Table 4. Net gas production differed significantly (p<0.01) among treatments. The highest gas production was recorded in T 3 (34.00 mL), followed by T 2 (28.33 mL), whereas T 0 (25.33 mL) and T 1 (26.00 mL) showed significantly lower and statistically similar values. The increase in gas production with fermented mustard oil cake indicates enhanced microbial fermentation and improved availability of fermentable nutrients. Solid-state fermentation by lactic acid bacteria improves substrate degradability by [6,25] . LAB fermentation also reduces anti-nutritional factors such as glucosinolates present in mustard oil cake, thereby improving microbial accessibility and fermentation efficiency (Tripathi and Mishra, 2007) [26] . Increased gas production reflects enhanced microbial activity and improved fermentability of fermented substrates (Menke and Steingass, 1988) [18] . Similar findings have been reported where microbial fermentation of oilseed cakes significantly increased in vitro gas production due to improved nutrient availability and microbial utilization (Kumar et al., 2015; Denek and Can, 2006) [12] . The NDF content decreased significantly (p<0.01) with increasing inclusion of fermented mustard oil cake. The highest NDF was observed in T 0 (38.61%), followed by T 1 (36.62%), while T 2 (34.25%) and T 3 (32.94%) showed significantly lower values. The reduction in NDF indicates partial degradation of structural carbohydrates during solid-state fermentation. Lactic acid bacteria and associated microbial populations produce cellulolytic and hemicellulolytic enzymes that degrade plant cell wall components, resulting in reduced fiber content (Sharma et al., 2016; Soccol et al., 2017) [25] . Fermentation also disrupts lignocellulosic bonds, increasing accessibility of carbohydrates to rumen microbes (Kung et al., 2018) [13] .

Reduction in NDF improves rumen fermentation efficiency by enhancing microbial colonization and digestion (Van Soest, 1994) [27] . Similar reductions in fiber fractions following microbial fermentation of oilseed cakes and agro-industrial byproducts have been reported by Chiou et al. (2000) [7] and Xu et al. (2012) [28] .

The IVDMD increased significantly (p<0.05) with increasing inclusion of fermented mustard oil cake. The lowest digestibility was observed in T 0 (52.41%), followed by T 1 (55.35%), while T 2 (62.78%) and T 3 (66.08%) showed significantly higher digestibility. The highest digestibility observed in T 3 indicates superior nutritional quality of fully fermented mustard oil cake. The improvement in digestibility is attributed to microbial hydrolysis of complex organic compounds during fermentation. LAB fermentation converts complex proteins and carbohydrates into simpler forms such as peptides, amino acids, and soluble sugars, which are readily utilized by rumen microorganisms (Niba et al., 2009) [20] . Fermentation also reduces anti-nutritional factors, improving nutrient availability and digestibility (Tripathi and Mishra, 2007) [26] . These results are consistent with previous findings where microbial fermentation improved dry matter digestibility of oilseed cakes and plant-based feed ingredients (Kumar et al., 2015; Canibe and Jensen, 2012) [12,6] . IVOMD increased significantly (p<0.05) with increasing levels of fermented mustard oil cake. The values increased from 54.47% in T 0 to 68.27% in T 3 . The significant improvement in organic matter digestibility indicates enhanced microbial degradation and fermentation efficiency. Improved organic matter digestibility is associated with microbial breakdown of structural carbohydrates and increased availability of fermentable substrates (Van Soest, 1994) [27] . LAB fermentation enhances enzymatic hydrolysis and nutrient solubilization, resulting in improved rumen fermentation (Soccol et al., 2017) [25] . Enhanced digestibility leads to increased production of volatile fatty acids, which are major energy sources for ruminants (McDonald et al., 2011) [17] . Similar improvements in organic matter digestibility following microbial fermentation have been reported in fermented soybean meal and oilseed cakes (Chiou et al., 2000; Xu et al., 2012) [7,28] .

The partitioning factor differed significantly (p<0.01) among treatments. The highest PF values were observed in T 1 and T 2 , while T 3 showed the lowest PF value (2.00 mg/mL). The lower PF value in T 3 may be attributed to increased gas production relative to substrate degradation. Partitioning factor reflects the efficiency of microbial biomass production relative to gas production (Blümmel et al., 1997) [5] . Lower PF values in highly fermentable substrates indicate increased fermentation rate and gas production. Although microbial biomass production may be slightly reduced relative to gas production, overall fermentation efficiency and nutrient availability are improved (Makkar, 2004) [16] . [6,20] . The NH₃-N values observed were within the optimal range required for efficient microbial growth and rumen fermentation (Satter and Slyter, 1974). Total volatile fatty acid (TVFA) concentration increased significantly (p<0.05) with fermented mustard oil cake inclusion, with the highest concentration in T 3 (89.87 mM/L), followed by T 2 (85.75 mM/L), whereas T 0 (80.40 mM/L) and T 1 (82.12 mM/L) showed lower values. Individual VFA concentrations, including acetate, propionate, and butyrate, also increased significantly (p<0.05) in fermented treatments. The increase in VFA concentration reflects improved microbial fermentation and enhanced degradation of organic matter. LAB fermentation improves nutrient availability by breaking down complex carbohydrates and reducing antinutritional factors, thereby promoting rumen microbial activity and VFA production (Van Soest, 1994; Soccol et al., 2017) [27,25] . Acetate concentration increased significantly from 52.51 mM/L in T 0 to 57.40 mM/L in T 3 , indicating enhanced fiber fermentation, while propionate increased from 19.49 to 22.60 mM/L, suggesting improved fermentation of soluble carbohydrates. Butyrate concentration also increased significantly, indicating enhanced overall fermentation efficiency. Similar increases in VFA production following microbial fermentation of feed substrates have been reported by Kung et al. (2018) [13] and Kumar et al. (2015) [12] . The results indicate that solid-state fermentation of mustard oil cake using lactic acid bacteria significantly improved rumen fermentation characteristics by increasing NH₃-N and VFA production. This improvement can be attributed to enhanced protein degradation, increased nutrient availability, and improved microbial activity due to fermentation (Mukherjee et al., 2006; Van Soest, 1994) [6,27] . The highest improvement observed in T 3 suggests that complete fermentation of mustard oil cake maximizes rumen fermentation efficiency and nutrient utilization.

Ammonia

Conclusion

Solid-state fermentation of mustard oil cake using a Lactobacillus consortium significantly improved its nutritional composition and rumen fermentation characteristics. Fermentation reduced pH and increased lactic acid concentration, confirming effective microbial activity. Fermented mustard oil cake showed higher crude protein and lower fiber and ether extract, indicating improved nutrient availability. In vitro studies demonstrated significant increases in gas production, dry and organic matter digestibility, ammonia nitrogen, and volatile fatty acid concentrations, reflecting enhanced microbial fermentation and substrate utilization. The greatest improvement was observed with complete replacement of mustard oil cake by fermented mustard oil cake in the total mixed ration. Overall, lactic acid bacteria-mediated fermentation is an effective and sustainable strategy to enhance the feeding value of mustard oil cake and promote efficient utilization of agro-industrial by-products in ruminant nutrition.

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