Strategic Nutrient Enhancement of Mustard Oil Seed Cake by Briquetting and Koji Fermentation for Ruminants Feed

Edible oil industries are shifting to the increased production of cold pressed oils in order to preserve some of the vital nutrients in the oil. Consequently, the seed cake residue would lack significant nutrients that are otherwise retained in the oils, thus making the quality of residue inferior when applied as a cattle feed. In this study, Mustard Oil Seed Cake (MOSC) was employed as a substrate to enhance the feed quality using koji strains namely Aspergillus oryzae and Aspergillus niger separately and suitable process parameters such as solid to liquid ratio, pH, incubation time and inoculum quantity were optimized for the maximum nutritive enhancement of cold pressed MOSC. Changes in the physico-chemical properties were analyzed by SEM, EDS and FTIR along with feed functional properties to analyze the quality. Free Amino Acid (FAA) and Reducing Sugar (RS) were chosen as a critical indicators of enrichment. A. oryzae resulted in 20.74-fold and 19.07-fold increase in FAA and RS respectively, whereas, A. niger showed 13.24-fold and 3.04-fold increase. Critical parameters such as solid to liquid ratio, pH, incubation time and inoculum volume were selected. Briquetting resulted in efficient mycelia coverage as evident from SEM images and the EDS analysis indicated the enhancement of the essential elements in the MOSC and also the functional properties after fermentation indicated an effective biotransformation of MOSC. These fermented MOSC can be applied as potent cattle feed thereby adding value to this agro industrial waste by establishing effective solid state fermentation strategy.


Introduction
Dairy industries play an extremely important role in the Indian economic development, contributing to about 33% of the gross income. In India, milk and its products provide an immense value in the agricultural and food sector. About 4% of India's GDP is contributed by the livestock sector and the majority of it has been shared by the dairy industries. This brings the need for the production and maintenance of healthy dairy cattle and developing an effective feed that can provide all the vital nutrients [1].
Dairy farming is especially important for the marginal farmers. Dairy farms contribute to about 60-65% of total income for the small-scale farmers. The dairy industry also plays a major role in improving the socio-economic development by positively influencing the lives of the people who are involved in this business [2]. The need of efficient animal feed sources brought the attention towards pressed oilseed cakes as an alternative to be used as a feed which is a by-product produced after the oil has been extracted. The consumption of edible oils in India has risen dramatically, making it the second largest consumer and the largest being China [3]. Many different types of oilseed crops are produced in a wide range of climatic conditions of India. India is the fourth largest edible oil producer and contributes to 1 3 about ten percent of the world oilseed production. Groundnut, soybean, mustard and rapeseed are the major oilseeds produced in India, accounting for about > 88% of total oilseed output. Mustard and the rapeseed are the third most important oilseed crop next to soya bean. The oilseed crops are majorly grown in Rajasthan, Haryana, Madhya Pradesh, West Bengal, Uttar Pradesh and Gujarat, where the estimated production is 7.1 million tonnes, accounting for about 93% of the total production in the country [4]. Unlike other conventional extraction techniques, aqueous enzyme used for oil extraction from oilseeds are found to be highly potential, however high cost of enzymes, longer incubation time and additional de-emulsification step hampers the commercial application [5]. On the other hand, cold press is the mostly widely used technique for oil extraction as it requires less energy and more environment-friendly [6]. High quality oils can be extracted when performed under less harsh conditions and low temperature using this cold press method. Thus, the oil obtained are found to be more nutritious, possess heat labile components like carotenoids that were otherwise destroyed when extracted by hot press method. An increase in the awareness among the population about the health benefits of these cold pressed oils would possibly increase the production of the cold pressed seed cakes to a large amount in the near future. Therefore, utilizing these cold pressed oilseed cakes as a feed would serve as an efficient way for utilization of this agro industrial waste.
The koji moulds namely Aspergillus oryzae and Aspergillus niger are prominently known for the industrial production of several intracellular and extracellular enzymes namely cellulase, amylase, glucoamylase, lipase and protease that are involved in the hydrolysis of various compounds like carbohydrates (amylose, maltose, cellulose and xylose), starch, polypeptides and to some extent lipids as well [7][8][9]. The products such as sugars, amino acids and lower molecular weight peptides are needed for the soya sauce production. However, the moulds need to be inspected carefully and check for the presence of aflatoxins and other mycotoxins like kojic acid under certain conditions [10]. In line with the aforementioned advantages of koji fermentation, there are lot of studies that are reported on its application to agro-byproducts other than conventional raw materials such as walnut meal [11], rice dreg protein [12] and millet powder [13].
Mustard oilseed cake (MOSC) has a good amount of protein and sulphur-rich amino acids which often serves as a limiting factor for cattle growth and development. MOSC, even though is rich in protein yet contains Rumen undegradable protein (RUP). Cold-pressed MOSC contains 460 g per kg RUP [14]. However, the RUP content differs depending upon various conditions like maturity during the harvest, techniques used for sample preparation and the conditions during the extraction procedures. MOSC could serve as an alternative feed source, however not much information on the percentage of feed to be given is available. It contains high amount of bioactive compounds like phenolics, which maybe present in free or esterified form. It may also be conjugated with insoluble compounds. However, the bitter taste contributed by the tannins makes it less palatable to the ruminants [3]. Therefore, such Anti-nutritional factors (ANFs) need to be alleviated and increase easy assimilable sugar and essential amino acids in MOSC to serve as a potent cattle feed. Solid state fermentation (SSF) is highly preferred for agro-industrial waste processing owing to its several advantages such as operating in absence or near absence of water, low maintenance, high product yield, easy downstream processing with low energy requirements. Therefore, SSF is identified as a potential strategy for the large-scale biomass conversion [15].
MOSC is one of the major agro-industrial residues that remain unutilized to its full scale despite its nutritional benefits. Complex nature of nutritional components such as carbohydrates and proteins are not readily assimilable by the cattle rumens, thereby a preliminary feed processing strategy is needed. To the best of authors' knowledge, this is the first study to establish a fermentation strategy for a cold pressed MOSC to improve its nutrient content using koji strains. In this study, koji fermentation using GRAS (Generally Regarded As Safe) microbes such as A. oryzae and A. niger were employed by considering the effective release of fungal enzymes that breakdown the macromolecules into simpler forms such as amino acids and assimilable sugars which makes it suitable for cattle feed application. The effect of koji strains on improving the levels of Free amino acids (FAA) and Reducing sugar (RS) content of the cold pressed MOSC and process parameters like solid to liquid ratio, incubation time, pH, inoculum volume and moisture content were screened and optimized for maximum nutrient enrichment. Further, changes in functional properties, levels of ANFs, morphology and functional groups were also investigated, before and after fermentation thereby taking a step forward to establish it as a feed.

Microorganisms and Raw Material
Aspergillus oryzae MTCC 3107 and Aspergillus niger were procured from the Institute of Microbial Technology (IMTECH) culture collection bank, Chandigarh, India. These strains were subcultured in Potato dextrose agar (PDA) media for its subsequent use as an inoculum in koji fermentation. Both hot and cold pressed MOSC were obtained from local oil extraction units, Chennai, Tamil Nadu, India. The substrates were ground in a ball mill to reduce and sieved to obtain the particle size of 0.5 mm.

SSF of MOSC with Koji Technique
In lab scale, SSF was performed in petriplates with the briquettes of cold-and hot-pressed MOSC as the substrates which provides wider surface area (15 briquettes per plate) inoculated with the fungus (Supplementary Appendix A- Fig. 1). Media components such as KH 2 PO 4 (0.02 g), MgCl 2 (0.01 g), NaCl (0.01 g) were taken in 10 mL of water to utilize as the basal media for fermentation. The prepared media was mixed to the MOSC in minimum amount to make it moist. The solid substrates should not have any free-flowing liquid. After autoclaving at 121 °C, about five pieces of ~ 1 cm 2 agar cubes of koji strains (spore count for A. niger and A. oryzae were found to be 1.714 × 10 6 spores/mL and 9.86 × 10 5 spores/mL respectively) were cut and dispersed into the plates containing the substrates. The plates were then left for incubation at 35 ºC and growth of the fungus was monitored. After five days of growth the fungal mycelia had effectively penetrated the inside of the solid substrate voids.

Briquetting Process
Briquetting process was done to increase the surface area of the oilseed cake for uniform fungal growth and subsequent mycelial encroachment. About 5 g of MOSC was taken in a 100 mL glass beaker and minimal media was added to it. After that, oilseed cakes were inoculated with both the fungal species of A. oryzae and A. niger and briquettes were made using a hand-held extruder.

Biochemical Characterization of MOSC
The characterization of nutrients such as estimation of carbohydrate content [16], RS content [17], protein content [18] and FAA content [19] were performed for the MOSC.

Extraction and Estimation of FAA Content using Ninhydrin Assay
About 500 mg of the MOSC was ground to a thick paste using 80% (v/v) ethanol. The supernatant obtained after centrifugation was tested for the presence of FAA using standard ninhydrin assay which involves addition of 1 mL of 8% ninhydrin solution in acetone to the diluted sample. This is further followed by heating in a boiling water bath along with some marble chips for preventing evaporation related losses for about 15 min. After which 1 mL of 50% (v/v) ethanol was added to the heated sample in order to halt the ongoing reaction thereby preventing any change in the Optical density (OD) taken. The OD was then taken at 570 nm while keeping water as the blank.

Selection of Process Parameters for Optimal SSF
The conventional One variable at a time (OVAT) approach was used to select the significant parameters and the initial test range of the four variables i.e., solid to liquid ratio, time (days), inoculum volume (mL) and pH for fermenting cold pressed MOSC inoculated with A. oryzae and A. niger. The effect of these parameters on the nutrient enrichment was checked by varying one parameter at a time and keeping the other parameters and process conditions constant. RS and FAA content were kept as an indicator for nutrient enrichment. All the experiments were performed in triplicates and the mean and standard deviation were calculated through Microsoft Excel 2013.

Analysis of Functional Properties
All the functional properties were calculated according to the methods given by Sadh et al. [20].

Bulk Density
MOSC was filled in a 10 mL cylinder and tapped gently. The powder was weighed and bulk density was calculated as mass of sample per unit volume of sample.

Water Binding Capacity
About 1 g of MOSC was mixed with 15 mL of deionized water and was held for 30 min. Centrifugation was done at 3000 rpm for 10 min. Water binding capacity was expressed as gram of water retained per gram of sample.

Oil Binding Capacity
About 1 g of MOSC was mixed with 15 mL of coconut oil and was held for 30 min. Centrifugation was done at 3000 rpm for 10 min. Oil binding capacity was expressed as gram of oil retained per gram of sample.

Emulsifying Capacity (EC)
About 1% (w/v) fermented sample was sonicated with 25 mL of coconut oil with 5 s pulse rate for 15 min. Emulsions were centrifuged at 11000 rpm for 5 min. The formula for calculating the EC is given in Eq. 1.
(1) EC% = Height of emulsified layer Height of total content in the tube * 100 1 3

Emulsifying Stability (ES)
Emulsion was heated at 80 ºC for 30 min and centrifugation was done at 11000 rpm. The formula for calculating ES is given in Eq. 2.

Foaming Activity (FA)
About 3% (w/v) solution of MOSC sample was mixed with deionized water for 45 min on a magnetic stirrer followed by blending. Volume (mL) was measured before and after stirring. The formula for calculating the FA is given in Eq. 3.
All the experiments were carried out in triplicates (n = 3) and the values plotted in the graphs/figures is average ± SD.

Scanning Electron Microscopic (SEM) and Fourier Transform Infrared (FTIR) Spectroscopy Analysis
SEM analysis was performed to provide high resolution imaging of unfermented as well as fermented briquettes of cold pressed MOSC and to investigate the morphology of briquettes after inoculation with A. oryzae and A. niger. The briquettes were ground well before being analyzed. The samples were studied using FEI Quanta 200 FEG -High Resolution Scanning Electron Microscope. SEM-Energy Dispersive X-Ray Spectroscopy (EDS) analysis was also performed to obtain the elemental composition of the fermented and unfermented MOSC. FTIR analysis was done to analyze the changes in the organic and polymeric functional groups present in the fermented and unfermented MOSC. The infrared spectra of the samples were analyzed using Fourier Transform Infrared Spectrometer (M/s. IRTRACER 100). The sample was loaded on the sample holder and it was scanned from 4000 to 500 cm −1 wave range.

Estimation of ANF Component in Terms of Tannin Content
The extraction and estimation of tannins was performed before and after SSF for both A. niger and A. oryzae. For extracting tannins, about 50 mg of the powdered sample was boiled with 7.5 mL of distilled water for about 30 min followed by centrifuging for about 10 min at 3000 rpm. The supernatant obtained was analyzed using the standard Folin-Denis spectrophotometric method which involves addition of 0.5 mL of Folin-Denis reagent and 1 mL of filtered sodium Height of emulsified layer after heating Height of emulsified layer before heating * 100 Final volume − Initial volume Initial volume * 100 carbonate solution to 1 mL of sample in a covered glass test tube. This was further diluted to make the total volume to 10 mL. The test tube was then shaken properly and the absorbance was measured at 700 nm.

HPLC Analysis of FAA
The total FAA composition of the protein concentrates of koji fermented MOSC was analyzed by Agilent 1100 HP-HPLC analyzer, reaction temperature − 40 °C, 0.5 mL per minute flow rate at 338 nm. One gram of the fermented MOSC sample were treated with phosphate buffer (pH 7.0) followed by centrifugation at 3000 rpm for about 20 min at 4 °C. Then, the sample was precipitated by treating with 10% (w/v) of sulphosalicylic acid and the supernatant was subjected to the FAA analysis by HPLC. Similarly, the deposits containing the insoluble proteins are treated with 1 N of NaOH (5 mL) and centrifuged at 3500 rpm for 20 min at 4 °C. Further, the centrifuged deposits are hydrolyzed with 6 mL of 6 N HCl in a boiling water bath and the supernatant obtained were neutralized with 1 N NaOH before proceeding for amino acids estimation by HPLC analysis.

Characterization of MOSC
MOSC are typically yellow to brown in color. It has a pleasant smell and characteristic odor as reported in earlier studies [21]. The intensity of this odor varies in different type of mustard. It was observed that the carbohydrate, RS and FAA concentration was lower in case of cold pressed MOSC than in hot pressed MOSC (Table 1). However, the protein concentration was found to be higher in case of cold pressed MOSC (13.08 mg/g of biomass). This can be directly related to the difference in the production processes and health benefits of cold pressed over hot pressed oils. In cold pressed oils, the heat-labile components like proteins and vitamins are not degraded [22]. In general, cattle need amino acids for protein synthesis required for proper metabolism, growth, lactation and reproduction. Mostly ruminants depend on microbial proteins synthesized in rumens and from dietary feed supplementation that are undegraded in the rumen. Inspite of these routes, dependence of production of microbial protein in rumen is insufficient to supply required quantity of amino acids for optimal metabolism [23]. Therefore, the cattle feed should adequately have constituted with these FAA thereby the limiting amino acids shall be supplied through consumption patterns. The objective of this study is to enrich such FAA through adoption of the koji fermentation.

Verification of Koji Fermentation on the Hot Pressed and Cold Pressed MOSC
From Fig. 1a, it was observed that the carbohydrate concentration decreased after fermentation of the MOSC (hot pressed) with both the Aspergillus sp. A subsequent increase in the concentration of RS was observed after fermentation.
A. oryzae showed a significant increase in RS concentration (11.72 mg/g of MOSC) as compared to A. niger (2.5 mg/g of MOSC) after 5 days fermentation of the oilseed cake (Fig. 1c). This can be attributed to the fact that A. oryzae MTCC 3107 is an efficient producer of amylase [24] and therefore hydrolyze the starch into simpler sugars, in addition to the lignin and cellulose breakdown. A. niger is a well-known ligninase and cellulase producer and thereby degrades the lignin to make the cellulose accessible to the enzymes [25]. From Fig. 1b, it was observed that the protein concentration decreased after fermentation of the MOSC (hot pressed) with both Aspergillus sp.
A subsequent increase in the concentration of FAA was observed (Fig. 1d) after fermentation. Being an efficient protease producer, A. oryzae showed a significant increase in FAA concentration (2.88 mg/g of MOSC) as compared to A. niger (1.634 mg/g of MOSC) after fermentation of the oilseed cake [24]. Briquetting of the cold pressed MOSC was done using hand-held extruder as shown in the Supplementary Appendix A- Fig. 1. This resulted in uniform growth of the mycelia around the briquetted oilseed cake as well as inside the briquettes when compared to the powdered form of the substrate. Earlier, non-uniform growth of the mycelia in powdered MOSC was observed as there was no solid support for fungal attachment and growth. Thus, briquetting of the oilseed cake resulted in uniform growth of the mycelium around the cold pressed MOSC that eventually, ease in feeding the cattles, packaging and transportation of the oilseed cake in large scale field application. The dimension of each briquette was measured and the measurements were found out to be as follows: diameter = 1.3 cm; height = 0.9 cm; surface area = 6.34 cm 2.
A significant increase in the concentration of RS and FAA deciphers the breakdown of polymers into monomers or simpler units by the action of enzymes released by Aspergillus sp., [26]. Other authors who employed koji fermentation in agro-byproducts have used increase in sugar and amino acids as a critical indictor of the process efficiency [11,12]. Hence, FAA and RS were chosen as the dependent factors for the selection of process parameters using two

Selection of Process Parameters for SSF
Four process parameters (solid to liquid ratio; incubation time; pH; inoculum volume) that affect fungal growth and fermentation were chosen and the selection of the optimum values for each of these process parameters were done using OVAT approach. The effects of different parameters have been schematically represented in Fig. 2a-h.

Effect of Solid to Liquid Ratio
Solid to liquid ratio indicating the moisture content plays a critical role in the growth of any microorganism. Alteration in the moisture content can lead to changes in the endproduct. Higher moisture content would lead to increased humidity whereas, lower moisture content would result in dry conditions inadequate for fungal growth [27]. Figure 2a and b show the effect of varying moisture content on SSF of cold pressed MOSC using A. oryzae and A. niger respectively. For A. oryzae the maximum production of FAA (5.759 mg/g of MOSC) and RS (4.583 mg/g of MOSC) were observed with 2.5:1 solid to liquid ratio that are found to be 8.22 and 2.29 fold increase as compared to control respectively. Similarly, for A. niger, the maximum production of RS (6.6 mg/g of MOSC) and FAA (1.928 mg/g of MOSC) were observed with 2.5:1 solid to liquid ratio with 3.3 and 2.82 fold higher than the unfermented MOSC. Thus, 2.5:1 solid to liquid ratio were chosen as the optimum for both the organisms. The comparison also elucidated the potential of A. oryzae and A. niger in proteolytic and saccharolytic properties respectively.

Effect of pH
pH is being considered as the important process parameter as it affects the growth and sporulation of the microorganism. From Fig. 2c and d, it can be clearly deciphered that there is a gradual increase in FAA from initial pH of 6 to 7 (Fig. 2c) and there was a maximum yield at 7.5 and 8 for A. oryzae and A. niger respectively which indicates that both the strains prefer the neutral to slightly alkaline range for followed which there was steep decline. Whereas, for A. oryzae (Fig. 2d), the maximum production of RS (5.44 mg/g of MOSC) was observed at pH 7. Similarly for A. niger, the maximum production of RS (5.45 mg/g of MOSC) was observed at pH 8. Thus, pH 7 for A. oryzae and pH 8 for A. niger were chosen as the optimum pH as the enzymatic (protease, amylase, cellulase, etc.) activity was found to be highest at these pH as reported by Abubakar et al. [28].

Effect of Incubation Time
Incubation time plays an important role in sporulation and enzymatic activity of fungi. Ideally, fungal growth and enzyme production is optimum between 2 and 5 days [29] which forms the basis for choosing the range in the present investigation. Figures 2e and f show the effect of incubation time on SSF of cold pressed MOSC using A. niger and A. oryzae respectively. For A. oryzae, the maximum production of FAA (13.35 mg/g of MOSC) and RS (5.416 mg/g of MOSC) were observed on the 4th day of incubation which is 2.72 and 19.02 fold respectively higher as compared to 0th day. Similarly, for A. niger, the maximum production of RS (6.042 mg/g of MOSC, 2.72 fold increase) and FAA (9.272 mg/g of MOSC, 13.11 fold increase from 0th day) were also observed on the 4th day. The fold increase in A. niger was however found to be less as compared to A. oryzae. Thus, the 4th day of incubation was chosen as the optimum time of incubation for both the organisms. Longer incubation time at elevated temperature might lead to interreaction between the RS and FAA in an aqueous condition like Milliard reaction [11] that might be the plausible reason for decrease when incubated for 5 days.

Effect of Inoculum Volume
Inoculum volume or the spore count, plays a very important role in the process of fermentation. Higher inoculum volume can lead to scarcity of oxygen and nutrients in the medium whereas lower inoculum volume leads to lesser biomass formation. Figure 2g and h exhibit the effect of varying inoculum volume on SSF of cold pressed MOSC using A. oryzae and A. niger, respectively. For A. oryzae, the maximum production of FAA (14.52 mg/g of MOSC) and RS (4.652 mg/g of MOSC) were observed with 3 mL of inoculum which amounts to 20.74 and 2.32 fold higher as compared to control. For A. niger, the maximum production of RS (8.159 mg/g of MOSC, 4.07 fold increase) and FAA (4.336 mg/g of MOSC, 6.19 fold increase) was observed with 2.5 mL of inoculum. Thus, 3 mL for A. oryzae and 2.5 mL for A. niger were chosen as the optimum inoculum volume. From the overall effect of parameters, it can be clearly observed that the fold increase in nutrient content found to elevate with changes in the process parameters. Further, statistical optimization is required from the range chosen from this study to arrive at the exact combination of effective parameters towards maximum nutrient enhancement.

Functional Properties of Fermented Cold Pressed MOSC
Since MOSC is relatively cheaper than most other oil seed cakes like peanut and soyabean oil seed cakes and produces lesser aflatoxins than groundnut oil seed cake, the functional properties of MOSC were tested to check if fermentation can improve the functional properties to be more easily digestible by the cattle. The functional properties of control, A. niger and A. oryzae fermented MOSC are tabulated in Table 2.

Bulk Density of Fermented MOSC
The bulk density of powder sample influences the texture, and the amount and strength of packaging material required for its distribution [30]. It has been recommended to reduce the bulk density of the feed. After subjecting to SSF, there was a reduction in bulk density to about 27.59% and 22.41% as compared to the unfermented (control) with A. niger and A. oryzae respectively ( Table 2). The advantage of decreased bulk density of the fermented sample is in better packaging as well as low bulk feed material [31].

Water and Oil Binding Capacity
Water binding capacity significantly affects the inter-meal gap in cattle whereas, the oil binding capacity plays an important role in flavor retention and texture of the feed. Both of these properties are in inverse correlation where a feed is expected to be in decreased water binding and increased oil binding ability which is in well correlation with the fermented MOSC. From Table 2, the hierarchy amongst the analyzed samples for water binding capacity was found to be Control > A. niger > A. oryzae whereas for oil binding it was vice versa. Therefore, A. oryzae found in superior quality as compared to A. niger fermented in terms of feed digestibility. Fermentation causes unfolding and modification of macromolecules of the products, the unfolding exposes the hydrophilic domains of the amino acid residues of proteins and other macromolecules which have a higher affinity for the aqueous medium. Higher value of water binding capacity indicates that fermentation process resulted in an increased number of exposed hydrophilic interactions as compared to oil binding capacity and unfermented sample. Therefore, the fermented product is easily digested in comparison to unfermented product. These factors significantly influence the composition, physical structure, porosity, and particle size of the dried cake powder.

Foaming Activity
Foam formation and stability are dependent on properties like pH, viscosity, surface tension and the processing methods employed which is directly related to the presence of surface soluble proteins [20]. The foaming property is decreased from 11.26% (control) to 5% and 6.70% with A. oryzae and A. niger respectively, because of the protein content also decreased after the fermentation of sample as indicated from the SSF potential studies.

Emulsifying Capacity (EC) and Emulsifying Stability (ES)
EC and ES were found to be increased in the fermented MOSC, thus indicating improved digestibility of fats. EC signifies the maximum quantity of oil that can be emulsified through dispersion, whereas ES elucidates the ability of an emulsion with a certain composition to remain unchanged. The fungal proteolytic activity might have exposed hydrophobic groups which resulted in the change of Hydrophilic-lipophilic balance (HLB) that eventually favored emulsification [32]. High HLB surfactant are generally water-soluble, whereas low HLB surfactant is oil soluble. Enzymatic hydrolysis generally results in improving emulsifying activity by producing lower molecular weight peptide that easily migrates into the oil-water interface.

Morphology Characterization of Fermented MOSC by SEM
The unfermented and fermented briquettes were analyzed by SEM for the better understanding of fungal coverage morphology and observe the growth of the mycelium inside the briquetted oilseed cake. Figures 3a-g depict the SEM images of the fermented briquettes at different magnifications and control (unfermented briquette). The dense growth of mycelium of A. oryzae was observed that percolated even inside the briquettes. The presence of distinct spores of A. niger was observed even inside the briquettes. While performing briquetting process the MOSC moistened with media and mixed well with the spores and extrudated to form briquettes. This ensures uniform microbial distribution in the solid substrate where inoculum dispersion is considered as one of the bottlenecks of SSF while using powdered biomass.

Elemental Analysis by SEM-EDS
SEM-EDS analysis of the unfermented and fermented briquettes was performed to understand the change in the elemental composition of the briquetted oilseed cake. Figure Fig. 4 a, b and c, it has been observed that calcium, phosphorus, sulfur, sodium, nickel and aluminum are present in the cold pressed MOSC after fermentation with Aspergillus sp. Calcium and phosphorus play a very important role in the development of the skeletal system and lactation in cattle. Deficiency in either or both causes a decrease in ability to gain weight and formation of weak bones. Cows provided with a diet richer in calcium tends to provide superior quality milk than the ones lacking it. Sulfur acts as a precursor for the formation of cysteine and methionine, which inturn promote lactation in cows. Sodium plays a vital role in pH regulation, water absorption and proper functioning of the nervous and muscular systems. Sodium deficiency in cattle may lead to decrease in weight gain and appetite [33]. Nickel in cattle feed supplement improves feed efficiency and ruminal urease activity in ruminants [34]. Aluminum is known to alter the metabolism of other minerals as well as reduction of toxicosis in ruminants [35]. Since MOSC fermented feed supplement has all these essential macronutrients, the above mentioned problems due to elemental deficiencies can be alleviated by using SSF approach.

Functional Group Analysis by FTIR
The unfermented and fermented cold pressed MOSC were analyzed by FTIR to observe the changes in the chemical structure and functional groups, before and after fermentation. From the FTIR spectra as shown in Fig. 5a-c, there were clear differences in control and fermented samples. FTIR analysis shows some prominent features, indicating some significant conversions during the process of fermentation. Appearance of peak at 1220-1250 cm −1 shows formation of ether after the process of fermentation. A similar observation was also noted by Shi et al. [36] which stated an increase in the concentration of the ether extract with increase in incubation time. Change in the appearance of the peak at 2890-2925 cm −1 from sharp, strong to broad shows that there has been a conversion from methylene to methine group after fermentation. Disappearance of the peak at 1745 cm −1 indicates that there is a possibility of utilization or conversion of the esters by the microorganisms. Appearance of the peak at 500-550 cm −1 shows the formation of chloroalkanes. This is further supported by the study conducted by Shi et al. [36], where an increase in the concentrations of Trichloro-acetic acid soluble protein nitrogen (TCA-SP) was observed after fermentation. It is assumed the TCA-SP consists of small peptides and FAA. This is further supported by our results, which shows an increase in amino acid concentration with fermentation. The ester-carbonyl group was seen after fermentation [37]. External aquaphobes are related with the content of alpha-helix and in case of fermentation, improved oil binding capacity and decreased water binding capacity significantly revealed the balance of hydrophilic and hydrophobic domain, as can be inferred from Aryee et al. [32].

Analysis of ANFs (Tannins) in Cold Pressed MOSC
ANFs are compounds that are produced in human and animal feed by normal metabolic processes that interfere with nutrients uptake. This leads to a decreased metabolic performance in animals. The primary ANFs found in MOSC include tannins, phytic acid, glucosinates, saponin, etc. [38]. Tannin content in control (unfermented seed cake) was found to be 5.10 mg/g of biomass. From the results as given in Fig. 6, it has been observed that there has been a significant decrease in the concentration of tannins which is almost 60% reduction by 4th day of incubation with A. niger, thus indicating that it is an efficient tannase producer, an enzyme that breaks down tannins. This is supported by the results shown by Knudson [39] which suggested that while A. niger is utilizing the organic compounds, it produces tannase and degrades tannic acid. This may also lead to a consequent accumulation of gallic acid.
However, A. oryzae was found to be an inefficient degrader of tannins. About 13.7% increase in the concentration of tannins was observed after the fourth day of fermentation. This is supported by the data provided by Sharath et al. [40] where it was suggested that the increase in the levels of tannins may be due to increase in free phenolic compounds after the fermentation process.
Even though both the strains were found to be efficient candidates for improving the property of MOSC, there are certain studies like amino acid profiling and toxicity analysis

Characterization of Amino Acids in Fermented MOSC by HPLC
The amino acid composition of protein isolates of MOSC after koji fermentation analyzed from HPLC chromatogram (Supplementary Appendix A-Fig. 2) has been Tabulated in Table 3, it is evident that the major essential amino acids namely histidine, threonine, methionine, valine, phenylalanine, isoleucine and leucine were found in the protein concentrates. In case of non-essential amino acids, arginine, glycine, alanine and tyrosine are found to have higher concentration (> 200 nmole/mL) in protein concentrates of fermented MOSC. The lower concentration of aspartic acid (57-63 nmole/mL) might be owing to its direct conversion to threonine and alanine by the enzymatic reaction [41,42]. However, essential amino acid content (3620 nmole/ mL) and non-essential amino acids (3278 nmole/mL) was found to be higher for sample treated with sulphosalicylic acid than the hydrolyzed one indicating that hydrolysis at higher temperature leads to the oxidation and loss of FAA owing to its undesirable changes in the physical and chemical properties [43]. The percentage of essential amino acid for the sulphosalicylic acid treated sample and HCl hydrolyzed sample was found to be 32.95% and 18.88% of the total FAA respectively. Since amino acids would serve as a nitrogen source for Aspergillus sp., the content varies based on the proteolytic (protease), biosynthetic enzymes (aminotransferase and aminopeptidase) activity and influence of process condition on amino acid structure and solubility [44,45]. A report by Sarker et al. [46] states that mustard cakes (black and yellow) are a source of essential and nonessential amino acids such as methionine, lysine, threonine, isoleucine, tryptophan, arginine and tyrosine were found in higher concentration. In general, lysine tends to react with other plant components leached during oil extraction thereby concentration seems to be low after oilseeds processing. It is expected to supplement essential amino acids such as threonine and methionine through animal feeds that contributes an improvement in cattle health. Enhancing the amino acid content through koji fermentation is a way forward costeffective strategy in MOSC based diets rather than fortification [47].

Conclusion
This study has screened the parameters for maximum benefit and enrichment of the nutrient level in unutilized inferior MOSC from cold press processing units. The optimization of these process parameters through statistical techniques is underway. Through the proof of this study, it can be inferred that adoption of koji fermentation in MOSC could serve as an alternate way to utilize the oilseed cake, which is an agricultural waste, thus beneficial for the industrialists. Farmers and animal breeders may  Serine  305  255  Glycine  339  548  Arginine  1023  535  Alanine  490  217  Tyrosine  687  309  Total non-essential amino acids  3278  2013  Total FAA  6898  4087 also be benefitted due to the low cost and easy availability of the seed cakes, as well as providing sufficient nutrition to the cattle. Thus, our results suggest that SSF using koji strains can serve as an effective way to enhance the nutrition and adapting this in large scale could have a positive impact on the agrarian society as well as industries. Similar strategies can be adopted for other industrial wastes such as cold pressed sunflower oilseed cakes, groundnut oil seed cakes, etc., as well as mixed cultures for further nutrient enhancement.