(Okara) after Probiotic Solid-State Fermentation - American Chemical

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Analysis of Improved Nutritional Composition of Potential Functional Food (Okara) after Probiotic Solid-State Fermentation Sulagna Gupta,†,‡ Jaslyn J. L. Lee,§ and Wei Ning Chen*,§ †

Interdisciplinary Graduate School, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore Residues and Resource Reclamation Centre, Nanyang Environment & Water Research Institute, Nanyang Technological University, 1 CleanTech Loop, CleanTech One, No. 06-08, Singapore 637141, Singapore § School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore ‡

ABSTRACT: Okara is a major agro-waste, generated as a byproduct from the soymilk and tofu industry. Since okara has a high nutritive value, reusing it as a substrate for solid state biofermentation is an economical and environmental friendly option. Rhizopus oligosporus and Lactobacillus plantarum were the probiotic FDA-approved food-grade cultures used in this study. The study revealed that biofermenting okara improves its nutritional composition. It was found that the metabolomic composition (by GC-MS analysis) and antioxidant activity (by DPPH test) improved after the microbial fermentations. Of the two, okara fermented with R. oligosporus showed better results. Further, the metabolites were traced back to their respective biosynthesis pathways, in order to understand the biochemical reactions being triggered during the fermentation processes. The findings of this entire work open up the possibility of employing fermented okara as a potential functional food for animal feed. KEYWORDS: okara, Rhizopus oligosporus, Lactobacillus plantarum, fermentation, metabolites

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released during SSF, adding to the nutritional value of the final product. These changes make fermented okara easier to digest than raw okara, which in turn makes it a potential contender to be employed as livestock feed. In the recent years, SSF has been chiefly exploited for the production of value-added products such as biologically active primary and secondary metabolites from a waste byproduct. Several industries are based on the commercialization of SSFderived products such as hydrolytic enzymes (e.g., cellulases, proteases, lipases, amylases), bioactive compounds (e.g., aflatoxin, penicillin, gibberellic acid, cyclosporin A), organic acids (e.g., citric acid, fumaric acid, lactic acid, oxalic acid), and other miscellaneous compounds (e.g., pigments, carotenoids, vitamins, biopesticides).12 Industrial SSF processes usually employ pure cultures in order to control substrate utilization and end product formation. If the fermented product is to be enlisted for consumption, it is imperative that the microbes used in the SSF process are of GRAS (Generally Recognized As Safe) status. Rhizopus and Lactobacillus are the most prominent fungi and bacteria genera, respectively, used by food industries for fermentation. These microaerophilic microbes can sustain relatively high temperatures of 30−40 °C and have been used as starter cultures since centuries. Although a lot of fermentation based work has been carried out on cooked soybean substrates (e.g., tempeh), a gap exists in utilization of raw soybean wastes (e.g., okara). This paper has detailed the investigation of improved nutritional composition

INTRODUCTION Okara, also known as soybean residue, is the insoluble waste byproduct from the manufacturing of soybean foods such as soymilk and tofu. Fresh okara is high in moisture content and thus putrefies very fast. Conventionally, it is either dumped into landfills or incinerated. The present awareness and demand for environmentally friendly disposal practices has promoted the recovery and utilization of agricultural wastes. Okara is one of the main agro-wastes generated in East and Southeast Asia. Raw okara possesses a high nutritive value (Table 1), which makes it potentially suitable for production of functional foods. However, since most of the nutrients are present in an insoluble form, pretreatment is required before employing it for feed purposes. Solid-state fermentation (SSF) is an economically viable option to biotransform crop residues for nutritional enrichment. Several advantages such as high reproducibility and product titers, minimal waste output, and absence of foam formation make SSF a convenient system to manage agricultural residues. Crop residues that have been successfully used in SSF so far include corn grits, soybean waste, wheat bran, cassava, sugar beet pulp, bagasse, etc. Soybean residue is one of the leading agro-wastes that has been engaged in studying SSF and its benefits. Enzymes secreted during the fermentation cause several desirable changes, leading to improved nutrition, texture, flavor, and aroma of the final product. Complex macromolecules (fats, proteins, carbohydrates) are hydrolyzed to smaller nutrients and antinutritional compounds (e.g., trypsin inhibitors, lectins, tannins) are considerably decreased after SSF.6,7 Fermentation releases phytases8 that act upon phytic acid (an antinutrient) and increase bioavailable iron,9 zinc,10 calcium, and inorganic phosphorus11 contents. Unbound forms of antioxidants are also © XXXX American Chemical Society

Received: February 22, 2018 Revised: April 16, 2018 Accepted: April 16, 2018

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DOI: 10.1021/acs.jafc.8b00971 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. Nutritional Composition of Okara S. No.

Composition

Quantity (per 100 g dry matter)

Reference

1.

Protein Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Cysteine + Methionine Valine Isoleucine Leucine Tyrosine + Phenylalanine Lysine Histidine Arginine Proline Fat Dietary fibers Insoluble dietary fiber Soluble dietary fiber Ash Carbohydrates Rhamnose Fucose Arabinose Xylose Mannose Galactose Glucose Uronic acids Stachyose Raffinose Sucrose Starch Phytic acid Minerals Potassium (K) Sodium (Na) Calcium (Ca) Magnesium (Mg) Iron (Fe) Copper (Cu) Manganese (Mn) Zinc (Zn) Phosphorus (P) Vitamins Thiamine Riboflavin Nicotinic acid Isoflavones Diadzin Glycitin Genistin Diadzein Glycitein Genistein Energy value

25.4−28.4 g 117 (mg/g protein) 41 (mg/g protein) 50 (mg/g protein) 195 (mg/g protein) 46 (mg/g protein) 46 (mg/g protein) 26 (mg/g protein) 51 (mg/g protein) 51 (mg/g protein) 81 (mg/g protein) 95 (mg/g protein) 65 (mg/g protein) 28 (mg/g protein) 75 (mg/g protein) 36 (mg/g protein) 9.3−10.9 g 52.8−58.1 g 40.2−43.6 g 12.6−14.6 g 3.0−3.7 g 3.8−5.3 g 0.85 g 0.45 g 6.35 g 5.14 g 1.26 g 10.83 g 15 g 5.03 g 140 g 30 g 230 g 59 g 50 g

1

2. 3.

4. 5.

6. 7.

8.

9.

10.

1046−1233 mg 16.2−19.1 mg 260−428 mg 158−165 mg 6.2−8.2 mg 1.1−1.2 mg 2.3−3.1 mg 3.5−6.4 mg 396−444 mg 0.48−0.59 mg 0.03−0.04 mg 0.82−1.04 mg 0.14 g 76 μM 16 μM 81 μM 88 μM 18 μM 60 μM 300 kcal

of okara after probiotic biofermentation with Rhizopus oligosporus (R. oligosporus) and Lactobacillus plantarum (L.

2

1

3

4

5

1

5

plantarum), thereby proposing its potential use as a functional food. B


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Journal of Agricultural and Food Chemistry

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solution (0.6 mM in ethanol) and 4 mL of ethanol was added and the mixture incubated in the dark at room temperature for 30 min. 1 mL of this solution was then measured for absorbance at 515 nm using a Nanodrop 2000c spectrophotometer (Thermo Scientific). Ethanol was used as blank, 4.5 mL of ethanol with 500 μL of DPPH was used as control and quercetin (0.1 mg/mL in ethanol) was used as a positive control. The antioxidant activity was calculated according to the formula:

MATERIALS AND METHODS

Materials and Microorganisms. Fresh okara was sourced from Unicurd Food Company Pte Ltd. (Singapore) and stored in airtight plastic bags at −80 °C, until used. Sodium chloride (NaCl), methoxyamine hydrochloride, pyridine, N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), trimethylchlorosilane (TMCS), 1,1diphenyl-2-picrylhydrazyl (DPPH), quercetin, ethanol, and methanol were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Dehydrated MRS and PDA media were bought from Becton, Dickinson and Company (BD Difco, USA) and reconstituted as per supplied instructions. Fungal strain Rhizopus oligosporus (DSM 1964), obtained from the Leibniz Institute DSMZ German Collection of Microorganism and Cell Cultures, was grown on Potato Dextrose Agar (PDA) plates at 30 °C for 48 h. Bacterial strain Lactobacillus plantarum (DSM 12028), obtained from the Leibniz Institute DSMZ German Collection of Microorganism and Cell Cultures, was grown on De Man, Rogosa and Sharpe (MRS) agar plates at 37 °C for 48 h. Fermentation. For fungal fermentation, 10 g of fresh okara was inoculated with a culture inoculum of 105 CFU Rhizopus oligosporus and incubated at 30 °C for 48 h. For bacterial fermentation, 10 g of fresh okara was inoculated with a culture inoculum of 107 CFU Lactobacillus plantarum and incubated at 30 °C for 48 h. Extracellular Metabolite Analysis. Sample Preparation. Extracellular metabolite extraction was carried out using the method followed by Sasidharan et al.13 with minor modifications. First, 5 mL of chilled saline (0.9% NaCl) solution was added to 10 g of fermented sample to quench the microbial cells. 30 mL of ice-cold methanol was then added to the samples as the extraction solvent for extracellular metabolites. The mixture was spiked with 600 μL of ribitol (2 mg/mL in water; internal standard) and centrifuged at 1400 rpm for 20 min at 4 °C. This was followed by centrifugation at 16 100g for 5 min at 4 °C. The supernatant was used as crude extract for further steps. The residual debris was discarded. 1 mL of crude extract was kept at 30 °C on a heat block until completely dry. Sample preparation for GC-MS (gas chromatography-mass spectrometry) was done using the method followed by Chen and Chen.14 First, 50 μL of 20 mg/mL methoxyamine hydrochloride in pyridine was added to the dried sample for carbonyls protection. The mixture was then vortexed for 1 min and subsequently incubated at 37 °C for 1 h. Following this, 100 μL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) was added to the sample for silylation. Samples were incubated at 70 °C for 30 min and finally centrifuged at 14 000 rpm for 1 h at room temperature before being transferred to glass vials for GC-MS analysis. GC-MS Conditions and Analysis. All samples for GC-MS were analyzed within 12 h of preparation. Chromatography was performed using 5975C Inert MSD with Triple-axis Detector from Agilent Technologies. The capillary column was 0.25 μm thick and had dimensions of 30 m × 0.250 mm. Hexane was used as the stationary phase. 1 μL of sample was injected in splitless mode; carrier gas helium was maintained at a purge flow of 50 mL per minute. The inlet was sustained at an isothermal temperature of 230 °C. The GC oven was programmed to initiate at 75 °C (4 min hold) and ramped to 280 °C at 4 °C per minute, with a final hold time of 2 min at 280 °C. The injection needle was cleaned with hexane thrice before starting sample run and twice after every measurement. Data was acquired in full scan from 30 to 800 m/z. Metabolites were identified using NIST08 mass spectral library. The chromatographic peaks were normalized according to the internal standard (ribitol) before being subjected to statistical analyses. Antioxidant Activity. The antioxidant activity of the samples was measured as a test of their free-radical scavenging capacity. 1,1Diphenyl-2-picrylhydrazyl (DPPH) was used for assessment, following the method used by Wan et al.15 with minor modifications. First, 0.1 g of sample was taken in an Eppendorf tube to which a small quantity of glass beads was added. The volume was made to 1.5 mL with ethanol and the tube was then spun on FastPrep-24 (MP Biomedicals) for 5 cycles of 20 s each following which centrifugation was carried out at 21 000g for 5 min. To 500 μL of supernatant, 500 μL of DPPH

DPPH scavenging activity (%) = [A C − A S]/A C × 100 where AC is the absorbance value of the control and AS is the absorbance value of the test samples Statistical Analyses. All data have been represented as an average of three replicates. Multivariate analysis was performed using OriginPro 2017. Clustering heatmap was generated using Groupaverage clustering and Euclidean distance calculation. Principal component analysis was carried out and represented as a twodimensional biplot to confirm the findings acquired by the cluster dendogram.

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RESULTS AND DISCUSSION Extracellular Metabolite Analysis Using GC-MS. The GC-MS metabolomic analysis performed on unfermented and fermented okara samples were examined to observe noticeable variations and similarities. Figure 1 displays the spectra

Figure 1. GC-MS chromatogram spectra of unfermented (control) and fermented okara samples. Key: Unfermented okara control (C); okara fermented with R. oligosporus (RO); okara fermented with L. plantarum (LP).

obtained after the (extracellular) metabolomic analysis of unfermented okara (control), okara fermented with R. oligosporus and okara fermented with L. plantarum. A comparative study of the metabolites detected in okara before and after fermentation was carried out. A total of 48 metabolites (excluding internal standard ribitol) were identified. The metabolites identified via GC-MS could be grouped into seven parent categories (Table 2). The parent categories include organic acids, amino acids, inorganic acids, fatty acids, carbohydrates, alcohols, and a miscellaneous group. As shown in Table 2, our results indicated that microbial fermentation of okara by both R. oligosporus and L. plantarum yielded greater number of metabolites. This is expected due to the action of microbial hydrolytic enzymes that break down complex molecules in okara to their constituent simpler units. Statistical Analyses. It is difficult to gauge how similar or unsimilar the fermented samples are to the control based on a C


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Table 2. Metabolites Detected via GC-MS Analysis of Unfermented and Fermented Okara Samples with Their Relative Abundancesa Metabolite Organic acids Lactic acid Propanedioic acid Butanedioic acid 2-Butenedioic acid Malic acid Propanoic acid Pentanedioic acid alpha-Aminoadipic acid 1-Propene-1,2,3tricarboxylic acid Ribonic acid Tartaric acid Galactonic acid Benzoic acid Gluconic acid Galactaric acid

Control (unfermented okara)

Okara feremented with R. oligosporus

N.D. 49178619.53 47319077.89 N.D. 218874578.4 183908980.5 N.D. N.D.

N.D. 47747086.23 1168762387 432057340.5 8138073830 139187140.3 76852921.1 40061997.42

2226350943 29160063.16 105777614.1 58278763.07 257104040.9 111897761.6 226444303.9 N.D.

N.D.

30273995.99

N.D.

28561671.19 28473751.19 22432674.37 N.D. 19813682.17 12928772.79

N.D. N.D. N.D. 70718886.84 39879817.93 N.D.

N.D. N.D. 211343057.3 N.D. 31770477.72 N.D.

Amino acids Valine Glycine Serine Threonine Alanine Proline Glutamine Asparagine Tyrosine

N.D. 51449134.35 N.D. N.D. 17196571.71 95264102.06 47565830.93 N.D 29793618.77

56497069.16 257095272.7 103765197.7 167135762.8 48575326.37 587747884.6 591453314.7 140334266.5 N.D.

33773725.94 24440822.09 N.D. N.D. 21814581.5 180307033.3 216215082.8 N.D. 254563179.1

Inorganic acids Phosphoric acid

33628651.21

516060826.3

34642488.11

499917693.4 117583950.4

309054417.3 791863674.2

564939870.8 88854174.89

Fatty acids Hexadecanoic acid 9,12Octadecadienoic acid

Control (unfermented okara)

Okara feremented with R. oligosporus

Okara fermented with L. plantarum

66987959.3

1012331836

67550136.8

192150287.6 18758145.81 44715610.1

318967929.2 N.D. N.D.

294716339.6 18918645.93 N.D.

N.D. 281993582.6 174027310.3 166752240

N.D. 75844360.79 N.D. N.D.

29703570.9 848342454.8 N.D. 153267558.5

140222925.4 50788938.89 905230558.9

N.D. 15579891.81 170337897.7

41874438.52 N.D. 130147920.9

233947793.8 586528356.2 549775864.5 N.D. 170885001.6

5136853386 70029415.51 63275035.18 54526863.11 N.D.

N.D. 639650164.6 593211327.1 N.D. 194296036.5

Alcohols Glycerol Inositol Glucitol Mannitol Oleyl alcohol 2-Monopalmitin

12719639.62 952742948.6 N.D. 256546418.8 202008498.9 14638032.6

N.D. 1477459369 46034227.91 14188906.93 N.D. 42404595.68

N.D. 1097722686 43990873.95 N.D. 147604065.8 52575702.53

Others Urea Pyrimidine

N.D. N.D.

94790100.22 102178230.9

130047746.8 N.D.

Okara fermented with L. plantarum

Metabolite trans-13Octadecenoic acid Octadecanoic acid Palmitic acid Decanoic acid Carbohydrates Erythro-pentose Galactose Mannose Glucopyranuronic acid Heptulose Turanose alpha-DGlucopyranoside Xylose Maltose Talose Xylo-hexos-5-ulose alpha-DXylopyranose

a

N.D.: Not detected.

Between Euclidean distances 2.50 × 109 and 8.37 × 109, it can be concluded that okara fermented with R. oligosporus and the cluster (composed of control and L. plantarum fermented okara) were significantly different from each other, with respect to their extracellular metabolic composition. Additionally, a PCA biplot was developed (Figure 3) to highlight the variance in distribution of the three groupsunfermented okara, okara fermented with R. oligosporus and okara fermented with L. plantarum, across the metabolites, with respect to the principal components. The GC-MS data was analyzed using a correlation matrix with listwise exclusion. The first two principal components (PC), PC1 and PC2, attributed to 50.63% and 22.74% of variance, respectively, and thus cumulatively accounted for 73.37% of the total variance. According to PC1, it can be clearly seen that L. plantarum fermented okara samples and control (unfermented) samples are similarly distributed, which differed from the distribution of R. oligosporus fermented okara. On the other hand, as seen in Figure 3, the control samples and okara fermented with R. oligosporus exhibited similar patterns of distribution across PC2,

visual scrutiny of Figure 1. Hence, statistical analysis tools were employed to make sense of the data generated by GC-MS. To validate the metabolic changes before and after fermentation, a hierarchical agglomerative clustering heatmap was generated (Figure 2). The heatmap indicated the abundance levels of the metabolites that were detected in unfermented and fermented okara samples. As evidenced by Figure 2, the cluster dendogram uncovered that L. plantarum fermented okara was in closer proximation to control (unfermented okara) than to R. oligosporus fermented okara, in terms of extracellular metabolite composition. The minimum and maximum values of the Euclidean distance used in our calculations were 0 and 10 × 109, respectively. The columns denoting okara fermented with L. plantarum and control (unfermented okara) converged at a Euclidean distance of 2.50 × 109. This cluster then converged with the column representing R. oligosporus fermented okara at a Euclidean distance of 8.37 × 109. Hence, we can reason that at a Euclidean distance below 2.50 × 109, all three columns are significantly unsimilar in their metabolite pattern distribution. D


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Figure 2. Heatmap dendogram of metabolites detected via GC-MS analysis in unfermented (control) and fermented okara samples. The scale indicates the relative abundance of each metabolite. Key: Unfermented okara control (C); okara fermented with R. oligosporus (RO); okara fermented with L. plantarum (LP).

Figure 3. PCA biplot derived from GC-MS data for unfermented (control) and fermented okara samples. Key: Unfermented okara control (C); okara fermented with R. oligosporus (RO); okara fermented with L. plantarum (LP).

which differed from that of okara fermented with L. plantarum. PC1 being the larger principal component, it can be interpreted that L. plantarum fermented okara and control (unfermented okara) are relatively similar, with respect to extracellular metabolite composition. The distribution across PC2, the smaller component, validates that a smaller degree of similarity exists between the control (unfermented okara) and okara fermented with R. oligosporus. The result interpreted from the PCA biplot study was in accordance with the prior implication made by the cluster dendogram.

The results established by this GC-MS metabolomic analysis and multivariate statistical studies can be used for an in-depth investigation of the metabolic mechanism of microbial fermentation. Tracing detected metabolites to their biosynthesis pathways. By analysis of the extracellular metabolites released before and after fungal (R. oligosporus) and bacterial (L. plantarum) biofermentation, a hypothesis can be drawn to understand the various metabolic pathways occurring during the process. Figures 4 and 5 display the link between the E


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Figure 4. Interlinkage of metabolic pathways occurring during biofermentation of okara by R. oligosporus.

Figure 5. Interlinkage of metabolic pathways occurring during biofermentation of okara by L. plantarum.

explain the increase in the levels of saturated medium-chain fatty acids (Table 2), such as hexadecenoic acid (palmitic acid), trans-13-octadadecenoic acid, 9,12-octadecanoic acid (linolenic acid), octadecanoic acid (stearic acid), etc. after fermentation.

various biochemical pathways that were believed to be triggered during the microbial fermentation of okara. It has been well established that both fungi and bacteria release lipases during fermentation.16,17 Lipid hydrolysis could F


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Benzoic acid is synthesized over multiple catalyzed steps in the shikimate pathway by fungi (Figure 4). Phenylalanine is the precursor for the compound. Amino acids lysine and tryptophan may account for production of glutaric acid, thus leading to elevated levels of glutaric acid in both R. oligosporus and L. plantarum fermented okara (Figures 4 and 5). As was expected, the levels of the aforementioned detected metabolites were higher in microbial fermented samples than the control (Table 2). The levels of tartaric acid dropped after fermentation. It may be reasoned that tartaric acid was converted to oxaloacetate via a two-way reaction and subsequently entered the TCA cycle. Aminoadipic acid was detected in okara sample fermented by R. oligosporus. Aminoadipic acid is an intermediate in the lysine metabolism via the α-aminoadipate pathway. The α-aminoadipate pathway is unique to fungi; this is evidenced by our results. Malonic acid levels increase after fermentation by both R. oligosporus and L. plantarum, which may be attributed to pyrimidine metabolism by the microbes. Propanoic acid (propionic acid) is a short-chain saturated triglyceride that is majorly formed as a product of amino acid (cysteine, isoleucine, and methionine) metabolism, odd-chain fatty acid metabolism, and from malonate after pyrimidine metabolism (Figures 4 and 5). Decreased levels of propionic acid after fermentation (Table 2) was in accordance with the expectation that microbes convert propanoic acid to propionylCoA and utilize it for carboxylic acid (organic acid) metabolism. Gluconic acid, glucuronic acid (a precursor of ascorbic acid), galactonic acid, and galactaric acid are sugar acids derived from glucose. The levels of gluconic acid and galactonic acid increase after fermentation, signifying oxidation of glucose and activation of the pentose phosphate pathway and pentose and glucuronate interconversion pathway, respectively. On the other hand, the levels of glucuronic acid and galactaric acid declined after fermentation. This decrease may be attributed to glucuronic acid being utilized for amino sugar and nucleotide sugar metabolism and galactaric acid being converted to pyruvate and entering the TCA cycle (Figures 4 and 5). The TCA cycle, being the chief energy generating source of aerobic microbes, is greatly activated during the fermentation process. Many of the metabolites detected from the samples are the byproducts of the TCA cycle (Figures 4 and 5). This accounts for the increased abundance of metabolites in the fermented samples. Butanedioic acid (succinic acid), 2butenedioic acid (fumaric acid), and malic acid are key intermediates in the cycle. Malic acid also enters the TCA cycle through its aspartate-arginosuccinate shunt. This can explain the remarkably elevated levels of malic acid in the fermented samples. Prop-1-ene-1,2,3-tricarboxylic acid (aconitic acid) is a minor intermediate in the conversion of citrate to isocitrate. It was expected that lactic acid be detected in okara fermented with L. plantarum (Table 2). Plenty of studies conducted earlier vouch that Lactobacillus species produce lactic acid during fermentation.26,27 Lactic acid is synthesized from the catabolism of pyruvate (Figure 5). Pyruvate is produced chiefly from the degradation of carbohydrates and amino acids and is primarily converted to acetyl CoA before entering the TCA cycle for yielding energy. Hence, lactic acid is one of the prominently abundant metabolites detected in okara fermented by L. plantarum. Since Rhizopus species are not

Cleavage of bonds in long-chain lipids also led to the increase in the levels of fatty acid derivatives such as 2-monopalmitin. On the other hand, derivatives like oleyl alcohol decreased as they entered lipid metabolism. Levels of decanoic acid (capric acid), a saturated medium-chain triglyceride, decreased after fermentation as well; this may be due to the lipolytic enzymes cleaving the saturated bonds in the compound. Glucitol (sorbitol), mannitol, myo-inositol, and glycerol were the alcohols detected during our metabolomics analysis (Figures 4 and 5). Sorbitol and mannitol are sugar alcohols derived from sugars such as glucose and fructose by reversible reactions; this may explain the increase in sorbitol levels and decrease in mannitol levels after fermentation. Mannitol then enters glycolysis, which further explains its decreased level. Glycerophospholipid hydrolysis generates glycerol and fatty acids; the former is converted to dihydroxyacetone phosphate (DHAP) before entering glycolysis (which may account for its decreased levels after fermentation), and the latter undergoes fatty acid hydrolysis to finally enter the TCA cycle as acetyl CoA. Further, both R. oligosporus and L. plantarum release microbial phytases during fermentation.18,19 Phytases are enzymes that hydrolyze phytic acid, an antinutrient present in soy. The hydrolysis yields myo-inositol and phosphoric acid (Figures 4 and 5). This can explain the increased levels of phosphoric acid and myo-inositol after fermentation. Microbial proteases released by both bacteria and fungi during fermentation20,21 aid in breakdown of complex proteins into their constituent amino acids. Serine, glycine, and threonine are synthesized via glycolysis (Figure 4) over a series of catalyzing reactions (serine, glycine, and threonine metabolism pathway). Reversible degradation of alanine yields pyruvate and glutamine; the former is involved in the synthesis of valine, an essential amino acid (Figures 4 and 5). Proline is produced from glutamine (alanine, aspartate, and glutamate metabolism pathway) and arginine (arginine and proline metabolism pathway). This pathway is also responsible for the catalysis of asparagine from aspartate (Figure 4). Alanine is synthesized from malonate after pyrimidine metabolism (Figures 4 and 5). However, tyrosine levels decreased after fermentation, indicating a triggering of the tyrosine metabolism pathway in which it was utilized to finally enter the TCA cycle. An interesting observation is that all the amino acids detected during the analysis belonged to the glucogenic category, i.e., they are capable of being converted into glucose via gluconeogenesis. This may justify their overall low abundance levels. Arginine either proceeds to undergo multistep reactions to be converted to proline or is enzymatically converted to urea (Figures 4 and 5). This would account for the increase in urea content in the fermented samples as compared to the control (unfermented sample). As seen in Table 2, several sugars such as xylose, galactose, mannose, turanose, maltose, etc. were detected by GC-MS analysis. It was noticed that the majority of the monomeric sugars decreased after fermentation; this could be explained by hypothesizing that they may have been interconverted to glucose and entered the glycolysis pathway or may have been converted to sugar alcohols such as sorbitol and mannitol. The other half of the detected carbohydrates increased after fermentation, supporting the fact that microbial enzymes such as amylases,22,23 cellulases,24 pectinases,25 etc. were released during the bioprocess, thereby breaking down long-chain polymeric carbohydrates to more simpler forms. G


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Interdisciplinary Graduate School (IGS), Nanyang Technological University, Singapore, for the award of research scholarship to Sulagna Gupta and the support for this research.

known for their lactic acid producing capabilities, it is in tandem with the obtained results. Thus, it may be summarized that bioactive compounds in fermented okara samples showed an overall positive trend, when compared to the levels in raw okara. DPPH Radical Scavenging Activity. The scavenging activity of samples to reduce the DPPH free radical was calculated (Table 3) from the DPPH assay. The results

Notes

The authors declare no competing financial interest. This article does not contain any studies with human participants or animals conducted by any of the authors.

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Table 3. Antioxidant Activity of Extracted Samples Sample

% RSA

Control (unfermented okara) Okara feremented with R. oligosporus Okara fermented with L. plantarum Quercetin (0.1 mg/mL)

27.44 52.81 29.10 94.33

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indicated that okara fermented with R. oligosporus had the greatest radical scavenging activity (% RSA) of 52.81%. Okara fermented with L. plantarum had a slightly better RSA of 29.10% than the unfermented control (27.44%). Quercetin, the positive control, displayed an RSA of 94.33%. It has been documented that the main isoflavones present in crude okara are daidzein, glycetein and genistein (aglycones), diadzin, glycitin and genistin (β-glucosides) and malonyldiadzin, malonyl-glycitin and malonyl-genistin (malonyl-glucosides).28 The increase in antioxidant activity in okara after fermentation may be attributed to an increase in phenolics content. In conclusion, it can be summarized that this work holds substantial potential. With the growth in popularity of soybased products, the quantity of okara being produced worldwide is on the rise. This work attempted to reuse okara, a food waste, as a potential functional food. The study revealed that biofermenting okara with food-grade probiotic microbes R. oligosporus and L. plantarum enhanced its nutrient profile. The GC-MS extracellular metabolite analysis showed that the fermentation processes released microbial hydrolases that degraded the complex macromolecules in okara into simple sugars, amino acids, and short-chain fatty acids, which are easier to digest. The levels of beneficial organic acids also increased after biofermentation, while the abundance of phytic acid, an antinutrient present in raw okara, decreased. Further, the antioxidant activity increased post fermentation, indicating an improvement in phenolics content. Our future target plan involves investigating the microbial enzymes released during biofermentation as well as examining the phenolics of fermented okara.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (+65)6316 2870. ORCID

Wei Ning Chen: 0000-0003-1111-5076 Author Contributions

W.N.C. designed the research. S.G. carried out the experiments and wrote the manuscript. J.J.L.L. commented on the experiments and manuscript. W.N.C. edited the manuscript. All authors read and approved the final manuscript. Funding

The authors would like to thank the Nanyang Environment and Water Research Institute (NEWRI), Singapore, and the H


Article

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(Okara) after Probiotic Solid-State Fermentation - American Chemical

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