3.1 Characterization of MOSC
MOSC are typically yellow to brown in color. They have a pleasant smell and characteristic odor as reported in earlier studies [19]. The intensity of this odor varies in different type of mustard. It was observed that the carbohydrate, reducing sugars and free amino acids 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 [20]. In general, ruminants 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 un-degraded in the rumen. In spite of these routes, dependence of production of microbial protein in rumen solely is insufficient to supply required quantity of amino acids for optimal metabolism [21]. Therefore, the cattle feed should adequately constituted with these free amino acids thereby the limiting amino acids shall be supplied through consumption patterns. The objective of this study is to enrich such free amino acids through adoption of the Koji fermentation.
3.2Verification of Koji fermentation on the hot pressed and cold pressed MOSC
From Fig. 1(a), 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 reducing sugars was observed after fermentation. A.oryzae showed a significant increase in reducing sugar 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. 1 (c)). This can be attributed to the fact that A.oryzae MTCC 3107 is an efficient producer of amylase [22] and therefore breakdown the starch into simpler sugars, in addition to breaking down lignin and cellulose. A.niger is a well-known ligninase and cellulase producer and thereby degrades the lignin to make the cellulose accessible to the enzymes [23]. From Fig. 1 (b), 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 free amino acids was observed (Fig. 1 (d)) after fermentation. Being an efficient protease producer, A.oryzae showed a significant increase in free amino acids concentration (2.88 mg/g of MOSC) as compared to A.niger (1.634 mg/g of MOSC) after fermentation of the oilseed cake [22]. Briquetting of the cold pressed MOSC was done using hand-held extruder as shown in the Appendix A as (Fig. A1). 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 was observed as the 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, ease in feeding, 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 cm2.
A significant increase in the concentration of reducing sugar and free amino acids deciphers the breakdown of polymers into monomers or simpler units by the action of enzymes released by Aspergillus sp., [24]. Even 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 [9,10]. Hence, free amino acids and reducing sugar were chosen as the dependent factors for the selection of process parameters using two different Aspergillus sp., as these two plays a vital role in cattle feed quality of MOSC.
3.3 Selection of process parameters for solid state fermentation
Four process parameters (Solid to liquid ratio; incubation time; pH; inoculum volume) that affect fungal growth and fermentation were chosen and 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. 2 (a-h).
3.3.1 Effect of Solid: Liquid
Solid: liquid which indicates the moisture content plays a critical role in the growth of any microorganism. Alteration in the moisture content can lead to changes in the end-product. Higher moisture content would lead to increased humidity whereas lower moisture content would result in dry conditions inadequate for fungal growth [25]. Fig. 2 (a) 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 free amino acids (5.759 mg/g of MOSC) and reducing sugar (4.583 mg/g of MOSC) were observed with 2.5:1 solid: liquid which were found to be 8.22 and 2.29 fold increase as compared to control respectively. Similarly for A.niger, the maximum production of reducing sugar (6.6 mg/g of MOSC) and free amino acids (1.928 mg/g of MOSC) were observed with 2.5:1 solid: liquid with 3.3 and 2.82 fold higher than the unfermented MOSC. Thus, 2.5:1 solid: liquid ratio was chosen as the optimum for both the organisms. The comparison also elucidated the potential of A.oryzae and A.niger efficient in proteolytic and saccharolytic properties respectively.
3.3.2 Effect of pH
Understanding of pH is important as it affects the growth and sporulation of the microorganism. From Figs. 2 (c) and (d), it can be clearly deciphered that there is a gradual increase in free amino acid from initial pH of 6 to 7 (Fig. 2 (c)) 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. 2 (d)), the maximum production of reducing sugar (5.44 mg/g of MOSC) was observed at pH 7. Similarly for A.niger, the maximum production of reducing sugar (5.45 mg/g of MOSC) wasobserved 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. [26].
3.3.3 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 2-5 days [27], which forms the basis for choosing the range in the present investigation. Figs. 2 (e) 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 free amino acids (13.35 mg/g of MOSC) and reducing sugar (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 reducing sugar (6.042 mg/g of MOSC, 2.72 fold increase) and free amino acids (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 inter-reaction between the reducing sugar and amino acid in an aqueous condition like Milliard reaction [9] that might be the plausible reason for decrease in the sugar and free amino acid when incubated for 5 days.
3.3.4 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. Fig. 2 (g) 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 free amino acids (14.52 mg/g of MOSC) and reducing sugar (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 reducing sugar (8.159 mg/g of MOSC, 4.07 fold increase) and free amino acids (4.336 mg/g of MOSC, 6.19 fold increase) was observed with 2.5 mL of inoculum. Thus, 3mL for A.oryzae and 2.5mL 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.
3.4 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.
3.4.1 Bulk density of fermented MOSC
The bulk density of powder sample influence the texture, and the amount and strength of packaging material required for its distribution [28]. 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 food material [29].
3.4.2 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 Control<A.niger<A.oryzae. 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. In this 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.
3.4.3 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 [18].The foaming property is decreased from 11.26% (control) to 5% and6.70% with A. oryzae and A.niger respectively, because of the protein content also decrease after the fermentation of sample as indicated from the SSF potential studies.
3.4.4 Emulsifying capacity and stability
Emulsifying capacity and stability were found to be increased in the fermented MOSC, thus indicating improved digestibility of fats. Emulsifying capacity signifies the maximum quantity of oil that can be emulsified through dispersion, whereas emulsion stability 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 [30]. High HLB surfactant are generally water-soluble whereas low HLB surfactant is oil soluble, enzymatic hydrolysis during fermentation process generally results in improving emulsifying activity by producing lower molecular weight peptide that easily migrates into the oil-water interface.
3.4.5 Morphology characterization of fermented MOSC by SEM
The unfermented and fermented briquettes were analyzed by SEM to understand the fungal coverage morphology better and observe the growth of the mycelium inside the briquetted oilseed cake. Figs. 3 (a-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.
3.5 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. Fig. 4 (a-c) represents the EDS spectra for the unfermented cold pressed MOSC. The composition of elements present in different MOSC has been tabulated in Appendix (Table A1). From the obtained results, it has been observed that C-51.61 wt%, O-45.27 wt%, K-1.19 wt% and Ca-Mo traces were found to be the major elements in the unfermented cold pressed MOSC. Whereas with the briquettes fermented with A.oryzae, C-57.43 wt%, O-37.21 wt%, S-0.96 wt% and traces of Ni-P-Al were found to be the major elements of the fermented oilseed cake. In case of A.niger, C-42.54 wt%, O-47.73 wt%, K-1.57 wt%, Ca-1.81 wt%, Mg- 1.66 wt%, P- 1.49 wt% and traces of S-Al-Na were found to be the major elements of the fermented oilseed cake. In cattle diets, calcium, phosphorus and sodium are the major limiting elements. From 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. Cattles 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 in-turn 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 [31]. Nickel in cattle feed supplement improves feed efficiency and ruminal urease activity in ruminants [32]. Aluminium is known to alter the metabolism of other minerals as well as reduction of toxicosis in ruminants [33]. 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.
3.6 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 5 (a-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. [34] which state 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 micro-organisms. 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., (2015), 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 free amino acids. 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 [35]. 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. [30].
3.7 Analysis of ANFs (Tannins) in Cold Pressed MOSC
Anti-nutritional factors (ANFs) are compounds that are produced in human and animal feed by normal metabolic processes that interfere with other nutrients uptake. This leads to a decreased metabolic performance in animals. The primary ANFs found in MOSC include tannins, phytic acid, glucosinates, saponin, etc [36]. 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 [37] 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. [38] 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 are to be done before employing it for the cattles. The research is being directed towards it for complete applicability.