Metabolomics analysis showed that the overall metabolite profile of okara changed after B. subtilis WX-17 fermentation (Figs. 1 and 2). Most of the carbohydrate metabolites decreased after fermentation. This indicated that it was being consumed by B. subtilis WX-17 which could utilize the carbohydrates through glycolysis, to produce increased amounts of the energy molecule acetyl-CoA. This precursor could then enter the amino acids and fatty acid pathway. This suggested that the microorganism was able to utilise the carbon source present within okara and use it for metabolism to produce other components. It had been suggested that glucose is the preferred carbon source for B. subtilis (Singh et al. 2008; Tian et al. 2015). In this study, analysis of the various sugar pathways suggested that most other forms of carbohydrate were converted into glucose before being used for other processes.
The increase in amino acids were confirmed as shown in Table 1. This could be due to B. subtilis producing extracellular proteases which would breakdown the proteins in okara into amino acids thereby contributing to their increase after fermentation. The increase in amino acids is important as they can provide as a rich source of nitrogen that are essential to living organisms. For example, yeast such as Rhodosporidium toruloides and Saccharomyces cerevisiae requires nitrogen for the synthesis of amino acids, proteins, DNA and RNA (Cruz et al. 2002; Evans et al. 1984).
Fatty acid level also increased after okara fermentation (Table 2). Particularly, the increase in both linoleic acid and oleic acid levels, were desirable as studies have reported various health benefits when these fatty acids were consumed. Linoleic acid is a polyunsaturated fatty acid that contains numerous purported health benefits such as anti-obesity, anti-carcinogenesis, anti-atherosclerosis, anti-diabetic, osteosynthetic and immunomodulation effects (Benjamin and Spener 2009; Nagao and Yanagita 2005). Likewise, oleic acid is a monounsaturated fatty acid that had been linked to a reduction in coronary heart disease due to its ability to reduce LDL-cholesterol, thrombogenicity, LDL-oxidative susceptibility as well as insulin sensitivity factors (Lopez-Huertas 2010). The increase in linoleic acid and oleic acid were expected as B. subtilis are known to produce lipases that catalyses the hydrolysis of fatty acids (Ma et al. 2006; Sánchez et al. 2002).
Endogenous metabolic processes or exogenous chemicals in food system may generate free radicals which may cause oxidative damages by oxidizing biomolecules resulting in tissue damage or even cell death. Numerous traditional fermented food such as miso, tempeh, sufu and douche have free radical scavenging ability (Zhu et al. 2008). Therefore, it is of interest to analyse if B. subtilis WX-17 fermented okara displayed the same ability. In this regard, analysis showed that antioxidant activity increased by 6.4 times which strengthened the case of fermented okara as a functional food for animals.
To better understand the metabolic flux during fermentation, a metabolic pathway analysis was performed that would allow a hypothetical insight into the various metabolic pathways which were up regulated or down regulated during fermentation (Fig. 6). The pathway analyses were performed with reference to the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database (Kanehisa et al. 2017; Kanehisa and Goto 2000; Kanehisa et al. 2016; Ogata et al. 1999).
The precursor for glycolysis is glucose. It is converted into pyruvate through the intermediate glucose 6-phosphate (G6P) and phosphoenolpyruvate (PEP). The analysis showed that apart from ribose, all other forms of carbohydrates can be converted into glucose. Mannose can be converted into fructose through the enzyme mannose isomerase in a reversible reaction. Fructose is in turn converted into glucose via xylose isomerase. Both mannose and fructose were also produced through glycolysis through the intermediates mannan and d-fructose-2-phosphate respectively. The overall decrease in both mannose and fructose suggested that the rate of consumption is greater than the rate of production.
Glycolysis also produced sucrose through the intermediate UDP-glucose and sucrose synthase. Sucrose can then be converted into fructose and glucose through sucrose alpha-glucosidase. UDP-glucose can also be converted to maltose via maltodextrin which were then converted to glucose through maltose phosphorylase. Galactose can be converted into UDP-glucose via UDP glucose pyrophosphorylase.
The KEGG database suggested no direct pathway for conversion of ribose to glucose. This suggested that the reduction in ribose after fermentation is due to direct consumption of ribose by B. subtilis WX-17. This was supported by studies which showed that B. subtilis exhibited increased sporulation when provided with a mixture of glucose and ribose as carbon source compared to a medium with glucose as the sole carbon source (Warriner and Waites 1999).
One of the main intermediates of glycolysis, G6P can be converted into myo-inositol, a carboxylic sugar through myo-inositol 1-phosphatase (inositol phosphate metabolism pathway). During fermentation, B. subtilis WX-17 releases phytase which hydrolyses phytic acid, an antinutrient present in okara to produce myo-inositol which might explain the increased amount of myo-inositol detected after fermentation (Chen et al. 2015; Kerovuo et al. 1998). Myo-inositol can then be converted into acetyl-coA through malonate-semialdehyde dehydrogenase which can then enter the TCA cycle (inositol phosphate metabolism pathway).
Another intermediate in glycolysis, phosphoenolpyruvate (PEP) was involved in the shikimate pathway that produce tryptophan through a reversible reaction with (3-indole)-glycerol phosphate. The increased level of tryptophan detected suggested that the forward reaction (3-indole-glycerol phosphate to tryptophan) occurred at a higher rate than the backward reaction. Both phenylalanine and tyrosine are involved in the shikimate pathway as well and can be interconverted through prephenate which is an intermediate in the shikimate pathway. Their increased levels after fermentation suggested that reactions along the pathway are skewed towards phenylalanine and tyrosine metabolism rather than quinate.
Both valine and leucine which are essential amino acids were produced from the glycolysis intermediate, pyruvate (valine, leucine, isoleucine biosynthesis pathway). Valine is produced from the intermediate, 2-oxoisovalerate which can also be irreversibly converted into leucine. In addition, pyruvate can also be converted into leucine through pyruvate metabolism. Increased levels of valine and leucine suggested that their rate of synthesis is greater than their rate of degradation as leucine can be broken down into acetyl-coA. This implied that the bulk of the acetyl-coA were converted from pyruvate rather than leucine.
TCA cycle is a chain of reactions that are used by aerobic organisms to release stored energy in acetyl-coA through oxidation into adenosine triphosphate (ATP) and carbon dioxide. 4 of the key components in the TCA cycle were detected, of these both isocitrate and malate increased while fumarate and succinate decreased. Results also showed that isocitrate increased the most (3 times) after fermentation. This is in line with studies that had shown that B. subtilis produced the enzyme aconitate hydratase which catalysed the stereo-specific isomerisation of citrate to isocitrate (Cox and Hanson 1968; Dingman et al. 1987). The large amount of isocitrate produced likely drove reactions forward to produce succinate and fumarate which are consumed to produce malate.
The intermediates of the TCA cycle are involved in numerous reactions that produced important compounds. Isocitrate are broken down by the action of isocitrate dehydrogenase into 2-oxoglutarate which can then be converted into the non-essential amino acid, glutamate. Glutamate are in turn converted into ornithine via acetylornithine deacetylase which is part of the urea cycle. Critically, ornithine cyclodeaminase catalyses the conversion of ornithine to proline which is an essential amino acid (arginine and proline metabolism pathway). Glutamate, ornithine and proline were all upregulated after fermentation which strengthens the hypothesis that isocitrate were produced in excess. 2-oxoglutarate can also be converted into lysine through the lysine biosynthesis pathway.
From the pathway analysis, aspartate played a vital role in the synthesis of numerous important compounds. Firstly, in the lysine biosynthesis pathway, aspartate is converted into lysine through catalysis by diaminopimelate decarboxylase. Aspartate can also be directly converted into the TCA cycle intermediate, fumarate by aspartase (alanine, aspartate and glutamate metabolism pathway). Next, aspartate is also directly converted into asparagine, a non-essential amino acid by asparagine synthetase (alanine, aspartate and glutamate metabolism pathway). Aspartate is also involved in the synthesis of the essential amino acid, methionine which had been linked with optimising the immune function of the human intestines (cysteine and methionine metabolism pathway)(Ruth and Field 2013). Lastly, aspartate can be converted to threonine through threonine synthase (glycine, threonine and serine metabolism pathway). Threonine is also produced from the reversible reaction of glycine through catalysis by threonine aldolase (glycine, serine, threonine metabolism pathway). Glycine, another essential amino acid is in turn produced from the reversible reaction of serine through catalysis by glycine hydroxymethyltransferase (glycine, serine, threonine metabolism pathway). In addition, serine can also be converted to cysteine through cysteine synthase (cysteine and methionine metabolism pathway).
It was also observed that all the amino acids detected were glucogenic amino acids which means that they can be converted into glucose through gluconeogenesis. This could explain why although all the amino acids were up regulated after fermentation, the overall amount detected after fermentation were much lesser compared to fatty acids.
The metabolic pathways for the biosynthesis of fatty acids involved the conversion of acetyl-coA to malonyl-coA through acetyl-coA carboxylase which were then converted to the saturated fatty acids, palmitate and stearate through the fatty acid biosynthesis pathway. Palmitate and stearate then underwent elongation and unsaturation process to yield oleate and linoleate (biosynthesis of unsaturated fatty acids pathway). Both palmitate and stearate were down regulated while oleate and linoleate were up regulated. This suggested that a larger proportion of the saturated fatty acids were converted into unsaturated fatty acids. The reduction in saturated fatty acids are desirable as it is well known that consumption of high amount of saturated fatty acids are associated with increased risk of coronary heart diseases (Zong et al. 2016).
Overall, from the metabolic pathway analysis (Fig. 6), it can be summarized that the amount of various carbohydrates in okara decreased. This suggested that carbohydrates were consumed and utilised by B. subtilis WX-17 through glycolysis, to produce energy for its metabolism and cellular process. Subsequently, this led to the increased antioxidant amount, amino acids and fatty acids in fermented okara.
In conclusion, with the world’s population predicted to reach 9 billion by 2050, food security is becoming a rising global issue (Godfray et al. 2010). One strategy to combat these issues is the biovalorisation of food waste. With the growing popularity of soy-based products, the amount of okara produced are increasing rapidly, approximately 14 million tonnes globally every year. This work showed the possibility of reintroducing okara, a food waste back into the food chain through biovalorisation by fermentation. As the microorganism used is food grade, this study also presents the potential application of fermented okara into human diets in various forms. The study revealed that fermentation of okara using food grade B. subtilis WX-17 enhanced its nutrient profile. GC–MS and metabolic pathway analysis showed that both amino acids and fatty acids production increased after fermentation due to the release of hydrolases by B. subtilis WX-17 to break down complex macromolecules into simpler molecules which are easier to digest. Furthermore, antioxidant analysis also showed that post fermentation, fermented okara displayed higher antioxidant activities which indicated a higher phenolic content. Future work will include characterising the enzymes produced by B. subtilis WX-17, such as nattokinase, as well as other important components such as the valuable vitamin K2 MK7. In addition, downstream processing to extract the bioactive compounds from fermented okara will be carried out.