Metabolomic analysis reveals the positive effects of Rhizopus oryzae fermentation on the nutritional and functional constituents of adlay millet seeds

doi:10.21203/rs.3.rs-4097492/v1

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Metabolomic analysis reveals the positive effects of Rhizopus oryzae fermentation on the nutritional and functional constituents of adlay millet seeds

https://doi.org/10.21203/rs.3.rs-4097492/v1

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published 29 Jul, 2024

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Adlay millet seeds are well known for excellent health benefits. However, using fungal fermentation to improve their nutritional and functional constituents and the underlying mechanisms has not been thoroughly investigated. Herein, we used Rhizopus oryzae as starter and applied metabolomics combining with quantitative verification to understand the changes of the nutritional and functional profiles of adlay millet seeds. Results showed that a total of 718 metabolites from 18 compound classes were identified. The fermentation with R. oryzae varied 203 differential metabolites, of which 184 became more abundant and 19 got less abundant, and many components such as amino acids, nucleotides, vitamins, flavonoids, terpenoids, and phenols significantly increased after the fermentation process. Interestingly, we found that R. oryzae synthesized high levels of two important beneficial compounds, S-adenosylmethionine (SAMe) and β-Nicotinamide mononucleotide (β-NMN), with their contents increased from 0.56 to 370.26 μg/g and 0.55 to 8.32 μg/g, respectively. KEGG analysis of enriched metabolites revealed the amino acid metabolic pathways were important for conversion of the primary and secondary metabolites. Specifically, aspartate can up-regulate the biosynthesis of SAMe and β-NMN. These findings improved our understanding into the effects of R. oryzae fermentation on enhancing the nutritional and functional values of cereal foods.

Adlay millet seed

Rhizopus oryzae fermentation

Nutritional and functional constituents

Metabolomics

S-adenosylmethionine (SAMe)

β-Nicotinamide mononucleotide (β-NMN)

Adlay millet (Coix lachryma-jobi L.) is being widely cultivated in East and Southeast Asia for centuries, including China, India, Japan, Korea, and Malaysia¹. Adlay millet seeds use both as a traditional Chinese medicine and food that is not only nutrient-rich, but also has beneficial physiological and pharmacological effects on human health². Adlay millet seed contains about 70% starch, 12.2-16.7% protein, 5.1-9.4% lipid, and 1.3-3% dietary fiber³. Since the Ming dynasty (14^th-17^th centuries CE) in China, adlay millet seed has been used in traditional Chinese medicine for various purposes, including inducing diuresis, stimulating lung and spleen function, treating heat-related ailments, reducing inflammation, and treatment of cardiovascular diseases and hypertension⁴. Modern studies found that adlay millet seed extract has functions such as antioxidant, anti-inflammatory, anti-obesity, immunomodulatory activity, lipid regulation, skin protection, and gastroprotection. These effects are thought to be related to the bioactive substances naturally present in adlay millet seed, such as polysaccharides, flavonoids, coixenolide (a fatty acid ester), vitamins, and phenols³.

Microbial fermentation is an effective and economical approach to improve the nutritional value, flavor, texture, shelf-life, digestibility, and functional components of cereal grains^5,6. The abundant enzymes (cellulases, amylases, proteases, and phytases) produced by microbial metabolic activities modify the chemical composition of cereal grains⁶. Many food-grade microorganisms have been widely reported to contribute to desirable modifications of nutritional quality traits of adlay millet seed. Fermentation of dehulled adlay by Monascus sp. produced a higher total phenol content than unfermented adlay, and greatly enhanced its antioxidant capacity⁷. Yeast, Bacillus subtilis and Lactobacillus plantarum fermentation of adlay millet seed increased the content of gama-aminobutyric acid (GABA), free amino acids, free fatty acids, soluble dietary, triterpenes, phenolics, flavonoids, and coixenolide, and increased its antioxidant capacity⁸^–10.

The members of fungus Rhizopus (R. microsporus, R. oligosporus, and R. oryzae), has been used for thousands of years as major components of starter cultures for mixed-culture, solid state rice fermentation of Chinese sweet rice wine (‘daqu’)⁶. The complex mixture of microbial species produces numerous enzymes, alcohol, amino acids, and many other flavor and bioactive compounds, which improve the nutritional value, sensory properties, and functional qualities of rice¹¹. Recent studies have shown that R. oryzae fermentation of rice bran increased the total contents of phenolics, flavonoids, carotenoids, anthocyanins, and enhanced the antioxidant capacity¹². Defatted adlay bran fermented with R. oryzae also increased the FRAP and ABTS⁺ radical scavenging capacity¹³. The R. oryzae is recognized as safe (GRAS) by the USA FDA¹⁴. In previous studies, R. oryzae was one of the typical strains used for lactic acid¹⁵, fumaric acid¹⁶, lipase¹⁷, amylase, protease biosynthesis¹⁸, and dicarboxylic acids¹⁹. In addition, as mentioned above, the fermentation of R. oryzae can improve the content changes of a particular compound or an overall compound class in adlay millet seed or defatted adlay bran^8,10,13. However, to the best of our knowledge, there has been no report of a comprehensive, i.e., metabolomic analysis of the detailed nutritional and functional compositional changes, especially the identification of bio-functional compounds resulting from microbial fermentation of adlay millet seed. Specially, the biosynthesis of functional substances such as S-adenosylmethionine (SAMe) and β-Nicotinamide mononucleotide (β-NMN) by R. oryzae fermentation has not been illustrated elsewhere.

Metabolomics is an analytical approach that provides comprehensive analysis of the hundreds of metabolites involved in complex biological processes, such as microbial fermentation²⁰, and allows holistic exploration of the key functional molecules and their possible complex effects on biological systems²¹. In this study, metabolomics based on ultra-high performance liquid chromatography-QTrap mass spectrometry (UHPLC-MS) was performed to explore global changes in the metabolites of adlay millet seed after fermentation with R. oryzae. Differential metabolites (DMs) analysis elucidated improvements in the nutritional and functional constituents of adlay millet seeds after R. oryzae fermentation, which was further confirmed by the quantitative verifications of amino acids closely related to the metabolic pathways of secondary metabolites and two bioactive metabolites with significant increase in abundance, i.e. S-adenosylmethionine (SAMe) and β-Nicotinamide mononucleotide (β-NMN). Our findings provide systematic insights into the effect of R. oryzae fermentation on boosting the comprehensive qualities of adlay millet seed and serve as a potential route for cereal processing into functional foods with high biological activity.

Features of R. oryzae and macroscopic appearance of adlay millet grits before and after fermentation

The colony of R. oryzae was loose on PDA medium and formed sporangia in later stage (Fig. 1a). The sporangia was black brown and spherical with erect sporangiophores (Fig. 1b). There were obvious stolons and rhizoids without diaphragm in the mycelium (Fig. 1c). The sporangiospores were oval and the funicular of sporangia was wedge-shaped (Fig. 1d). The adlay millet grits before fermentation was compact and well defined with each other (Fig. 1e,h). After 24 h of fermentation, there was clear liquid seeped out with pleasant sweet scent and some bubbles, and the adlay millet grits seemed relatively intact (Fig. 1f,i). After 54 h of fermentation, there was more glutinous liquid seeped out with clear wine aromas and more bubbles, and a membrane was formed by R. oryzae mycelium on the surface of the loosed adlay millet grits (Fig. 1g,j).

Metabolomic analysis of CAG and FAG samples

To investigate the global variations of metabolites in adlay millet seeds before and after R. oryzae fermentation, the metabolomic analysis was conducted for their accurate identification and annotation. Differences in metabolomics data from the CAG, FAG, and QC samples were visualized by PCA (Supplementary Fig. 1a). PC1 and PC2 explained 60.5% and 10.6% (total 71.1%) of the variation between CAG and FAG samples, respectively; all samples were within the 95% confidence interval, i.e., there were no outliers. The CAG samples were more closely grouped than the FAG samples, but the two groups were widely separated from each other and the QC samples, indicating a significant difference in metabolite profiles resulting from the R. oryzae fermentation.

In the OPLS-DA model, the FAG samples were more closely grouped, and the groups were still well-separated (Supplementary Fig. 1b). Permutation testing of the OPLS-DA model was performed (Supplementary Fig. 2); the abscissa represented the permutation retention, the point where the permutation retention equals 1 corresponds to the R2Y (goodness of fit) and Q2 (goodness of prediction) values of the original model, which were both close to 1, validating the authenticity, robustness and stability of the OPLS-DA model. Specifically, 718 metabolites were annotated in CAG and FAG samples from 18 compound classes (Fig. 2), of which the top 10 most abundant metabolites were flavonoids (113 metabolites), terpenoids (101 metabolites), alkaloids (78 metabolites), phenols (63 metabolites), amino acids and derivatives (53 metabolites), lipids and lipid-like molecules (44 metabolites), coumarins and lignans (37 metabolites), steroids and derivatives (28 metabolites), organic acids and derivatives (23 metabolites), and carbohydrates and conjugates (23 metabolites).

Identification and classification of differential metabolites

To analyze metabolic profile differences between FAG and CAG samples at the individual compound level, a hierarchical cluster analysis of the DMs was performed based on a Euclidean distance matrix and the complete linkage method (Supplementary Fig. 3). The abundance of most DMs was much higher in FAG than in CAG samples (Supplementary Fig. 3), indicating that the microbial fermentation had a great influence on the metabolic profile of adlay millet seed. A total of 203 differential metabolites were identified, of which 184 were more abundant and 19 were less abundant in FAG (Fig. 3a). The top ten DM compound classes were amino acids and derivatives (17.24%), flavonoids (11.82%), terpenoids (11.82%), alkaloids (8.37%), carbohydrates and conjugates (6.40%), lipids and lipid-like molecules (6.40%), organic acids and derivatives (5.42%), phenols (6.40%), nucleotides and derivatives (2.96%), and steroids (2.46%) (Fig. 3b). The abundances of amino acids and derivatives, nucleosides, nucleotides and derivatives, and terpenoids all increased (Fig. 3c), of which the first two classes included the three metabolites with the largest increases in abundance, namely, S-adenosylmethionine (SAMe), uridine diphosphate glucose (UDP-glucose), and β-Nicotinamide mononucleotide (β-NMN) (Fig. 3d). SAMe has health-beneficial biological activities, including prevention and treatment of arthritis, liver disease, depression, and jaundice²². β-NMN can ameliorate health conditions associated with nicotinamide

adenine dinucleotide (NAD⁺) deficiency, such as aging, cardiac, and cerebral ischemia, Alzheimer’s disease and obesity²³. In addition, most compounds in the classes of flavonoids, phenols, organic acids, lipids, and lipid-like molecules were more abundant.

Metabolism of nutritional components

Amino acids and derivatives

Adlay millet seed has been identified as a good source of dietary protein because of its relatively high protein/carbohydrate ratio compared with other cereals¹. In this study, the abundance of amino acids and derivatives notably increased (Fig. 3c, Supplementary Table 1), indicating that the R. oryzae fermentation induced positive effect on amino acids contents. In addition, the abundance of SAMe increased the most markedly (11,285-fold) of all the DMs (Table 1, Fig. 3d). SAMe is a key pleiotropic molecule involved in three types of cellular reactions: transmethylation, transsulfuration, and aminopropylation. It has been demonstrated to increase the antiviral effect of interferon, and

SAMe supplementation could restore hepatic glutathione (GSH) deposits and attenuate liver injury, treat arthritis, and depression, and reduce jaundice²². Gama-Aminobutyric acid (GABA) increased by 16.72-fold in FAG and was considered to be a potent bioactive compound because of its many physiological functions, including lowering blood pressure, enhancing memory, anti-inflammation, obesity prevention, enhancing immunity, and reducing insomnia²⁴. Similarly, fermentation of coix/adlay bran by B. subtilis resulted in an almost 14-fold increase in GABA⁸ and yeast fermentation increased the GABA content 6-fold⁹. In general, the R. oryzae fermentation improved the trophism and abundance of important amino acids.

Table 1

Representative DMs detected in samples of FAG compared with CAG.
Compounds					Type	Compounds		Type
Amino acids and derivatives						Nucleosides, nucleotides and derivatives
S-adenosyl methionine (SAMe)		11285.42		up		Uridine 5'-diphospho-D- glucose (UDP-glucose)	5789.74	up
L-alanine		24.80		up		β-Nicotinamide mononucleotide (β-NMN)	1189.04	up
γ-aminobutyric acid (GABA)		16.72		up		Vitamins
L-Glutamate		10.17		up		Nicotinic acid	10.39	up
L-Aspartate		7.98		up		Flavonoids
Carbohydrates						Gossypetin	8.46	up
β-Cyclodextrin	210.97			up		Liquiritin	8.05	up
Kojibiose	100.31			up		(-)-Epiafzelechin	6.28	up
β-D-Fructose-2-phosphate	52.14			up		Terpenoids
α-D-Glucose	12.40			up		Valechlorine	79.65	up
Sucrose	0.37			down		Germanicol	13.38	up
Lactulose	0.28			down		Simiarenol	10.46	up
Stachyose	0.25		down			Trans-caryophyllene	6.96	up
Lipids and lipid-like molecules						Alkaloids
Docosahexaenoic acid (DHA)	37.60				up	Rhynchophylline	21.10	up
1, 5-pentane dicarboxylic acid (pimelic acid)	17.80				up	Lupanine	6.81	up
Organic acids and derivatives						Phenols
(S)-2-Acetolactate	37.18				up	Maltol	16.06	up
cis-Aconitic acid	21.59				up	5-Heptadecylresorci–nol	12.21	up
2-Isopropyl-3-oxosuccinate	18.43				up	10-Gingerol	5.80	up

Carbohydrates and carbohydrate conjugates

Carbohydrate metabolism is one of the most important cellular metabolic processes and provides material and energy for growth and proliferation²⁵. A total of 12 carbohydrate and carbohydrate conjugate DMs were identified (Fig. 3c, Supplementary Table 1). Stachyose decreased significantly (Fig. 3d), followed by lactulose and sucrose (Table 1), whereas the Kojibiose, a good prebiotic, was increased by 100-fold. Kojibiose was found in sake and koji extracts, and was reported to strongly inhibit α-glucosidase I activity and showed antitoxic competence²⁶.

Lipids and lipid-like molecules

Adlay millet seed is richer in lipids than most cereal grains³. 44 lipids and lipid-like molecules were annotated in FAG and CAG, including various unsaturated fatty acids, such as Docosahexaenoic acid (DHA), 1, 5-pentane dicarboxylic acid (Pimelic acid), and conjugated linoleic acid (Bovinic acid) (Supplementary Table 1). Among them, 14 kinds of lipids were found as DMs (Fig. 3b, c). The abundance of DHA was 38-fold (the biggest increase of the lipids) more abundant in FAG than CAG (Table 1). DHA has been linked to many positive health effects, e.g. promoting the growth of nerve cells, and alleviating cardiovascular and inflammatory diseases²⁷. The abundance of pimelic acid increased 18-fold after R. oryzae fermentation. Pimelic acid is the initial precursor for biotin synthesis, suggesting that co-culture of R. oryzae with other biotin-producing microorganisms, such as B. subtilis, would increase the biotin yield from fermentation²⁸.

Nucleosides, nucleotides and derivatives

Nucleosides and nucleotides are not only components of genetic material, but also key components of cellular metabolic pathways. 12 compounds in this class were annotated in FAG and CAG, 6 of which were DMs, UDP-glucose and β-NMN were increased most (Table 1). UDP-glucose increased more than 5000-fold in FAG compared with CAG (Fig. 3d, Table 1). As one of the most important glycosyl donors for biosynthesis of glycosides, oligosaccharides, polysaccharides, and glycoproteins, UDP-glucose was closely associated with the biosynthesis of nucleotide sugars, galactose metabolism, amino sugar, and nucleotide sugar metabolism. It was notably the β-NMN was upregulated about 1200-fold in FAG samples (Fig. 3d, Table 1). It is an endogenous bioactive nucleotide and a key precursor for NAD⁺ biosynthesis²⁹. Beta-MNM has beneficial pharmacological effects on health problems arising from NAD⁺ deficiency, such as aging, cardiac, and cerebral ischemia, Alzheimer’s disease and obesity²³, so NMN-rich fermented adlay millet seed merits further study to evaluate its potential health benefits.

Vitamins

Vitamins are important nutritional components of adlay millet seed; vitamin E and β-carotene have been previously identified in adlay millet seed². In FAG and CAG, 10 vitamins were annotated, including vitamin E, β-carotene, ascorbic acid, nicotinic acid, vitamin A, pyridoxine, and thiamine, the last four of which were DMs (Table 1). The abundance of nicotinic acid increased 10.4-fold in FAG; it is a component of coenzyme I (NAD⁺) and coenzyme II (NADP⁺). At pharmacological level, its supplementation was beneficial for human health to combat cardiovascular disease, neurological problems, diabetes, and other skin disease³⁰.

Metabolism of functional components

Flavonoids

Flavonoids form a large group of bioactive plant secondary metabolites that is abundant in fruits and seeds, with human health benefits, such as anti-inflammatory, anti-aging, cardio-protective and immunomodulatory³¹. A total of 24 flavonoid compounds was annotated as DMs in FAG compared with CAG, namely 16 flavones (abyssinone V, gossypetin, liquiritin, (-)-epiafzelechin, kaempferol 3-O-rhamnoside, cyanidin, homoferreirin, naringenin chalcone, methyl hesperidin, quercitrin, 3-O-acetylpinobanksin, sciadopitysin, baicalein, morin, hinokiflavone, and 3,8'-biapigenin), 6 flavanones (spinosin, neohesperidin, diosmin, hesperidin, luteolin, and tangeretin), and 2 isoflavones (cajanol and 5,7,3',4'-tetrahydroxyisoflavone). Of these, 21 were more abundant in FAG and three were less abundant (Fig. 3c, Supplementary Table 1). Gossypetin (8-fold more abundant in FAG) is a hydroxylated flavone with strong antioxidant, antimicrobial, anti-inflammatory and anti-atherosclerotic activities³². Liquiritin and Spinosin were benifical for treatment of Alzheimer’s disease³³. Other flavonoids, such as Cyanidin, Hesperidin, and Quercetrin have anti-inflammatory and anti-cancer bioactivity³¹.

Terpenoids

Terpenoids constitute one of the largest classes of natural compounds present in many plants. They have many biological activities, such as anti-bacterial, anti-parasitic, anti-inflammatory, anti-tumor, and skin protection³⁴. In FAG, 24 terpenoid compounds were assigned as DMs, i.e., 3 monoterpenoids, 2 iridoids, 6 sesquiterpenes, 1 diterpene, 11 triterpenes, and 1 tetraterpenoid (Fig. 3b, Supplementary Table 1). All the terpenoid DMs were more abundant in FAG (Fig. 3c); the biggest increase was for Valechlorine (79-fold increase), the eighth biggest increase of all the DMs (Fig. 3d). Valechlorine alleviates hepatic steatosis by enhancing autophagy, and reduces oleic acid-induced lipid accumulation³². The triterpene germanicol (13-fold increase) has anti-fungal activity³⁵. R. oryzae fermentation effectively increased the contents of terpenoids in adlay millet seed, which should improve its biological functions and potential health value.

Alkaloids

Alkaloids have been isolated from many medicinal herbs and represent a rich source of bioactive molecules³⁶. A total of 17 alkaloid DMs were identified in FAG samples, of which 13 were more abundant (Supplementary Table 1). The abundance of rhynchophylline, (21-fold increase) increased the most after fermentation; this compound is a well-established treatment for central nervous system diseases³⁷. Lupanine is a positive modulator of insulin release, which helps to maintain glucose homeostasis³⁸.

Phenols

Phenols are one of the most widely distributed classes of plant secondary metabolites and have been widely applied in the food and pharmaceutical industries as flavorings, antioxidants and antibacterial agents³⁹. A total of 63 phenol compounds were annotated in FAG and CAG; of these, 13 were DMs and 12 were more abundant in FAG (Fig. 3b, c). The abundance of Maltol increased 16-fold, the largest increase of the phenolic compounds (Table 1). Maltol has various biological activities, such as neuroprotection, liver protection against acute carbon tetrachloride toxicity, reduction of drug-induced hepatotoxicity, and inhibition of osteoarthritis progression⁴⁰. The abundance of 5-heptadecylresorcinol increased 12-fold (Table 1); it is a member of the 5-n-alkylresorcinol (AR) family, found in many cereals. 5-Heptadecylresorcinol has a stronger inhibitory effect on cancer-cell proliferation than most other ARs⁴¹. The compounds of 10-Gingerol and curcumin, which have antitumor, antioxidant and anti-inflammatory effects⁴², were both more abundant in FAG. The results were similar to previous studies observed for increased phenolics from defatted rice bran by R. oryzae fermentation¹². Phenolic compounds in the cereals are predominantly bound to cell wall components, but the R. oryzae fermentation liberates them into the free form, increasing the apparent content and bioaccessibilities¹².

KEGG annotation, related pathway enrichment and network analysis of differential metabolites

The metabolism of various substances in the biological body is not carried out in isolation, but through the common intermediate substances to connect and transform to each other, forming a complete metabolic network to regulate the metabolism and perform cellular biological functions. To aid understanding of the metabolic activities of R. oryzae fermentation on adlay millet seed, the 203 DMs were mapped to KEGG pathways. A total of 120 DMs was mapped to the KEGG data sets and they were found to be enriched in 58 metabolic pathways (Fig. 4); most DMs were related to metabolic pathways and biosynthesis of secondary metabolites. The pathways of amino acids biosynthesis and D-amino acid metabolism showed significant enrichment of DMs (Fig. 4). In detail, the alanine, aspartate, and glutamate metabolism pathway enriched abundant DMs and had high enrichment factor (Fig. 4), indicating it played important role in the fermentation of adlay millet seed by R. oryzae.

By analyzing the several specific amino acid metabolic pathways in which the DMs were mainly enriched, the metabolic network among them were shown in Fig. 5. All DMs in the network were more abundant in the FAG samples compared with CAG samples (Supplementary Table 1). The pathways of alanine, aspartate and glutamate metabolism (Fig. 5, purple box), cysteine and methionine metabolism (Fig. 5, blue box), and glycine, serine and threonine metabolism (Fig. 5, green box) were interconnected with each other by a common DM, i.e., aspartate. It was obvious that the aspartate was transformed from the intermediate metabolite oxaloacetate in the tricarboxylic acid cycle (TCA) cycle by the pathway of alanine, aspartate and glutamate metabolism (Fig. 5, purple box). The aspartate could be transformed to homoserine and finally to generate SAMe through the cysteine and methionine metabolism pathway (Fig. 5, blue box). Besides, abundant aspartate could also be participated in the nicotinate and nicotinamide metabolism pathway (Fig. 5, Supplementary Fig. 4), leading to increased biosynthesis of

nicotinic acid (Table 1). The nicotinic acid could increase NAD⁺ content through Preiss-Handler pathway and should decrease the β-NMN consumption, so resulting in β-NMN accumulation⁴¹ (Supplementary Fig. 4, Table 1). Aspartate was also enriched in the glycine, serine and threonine metabolism pathway (Fig. 5, green box), the more abundant glycine, serine and threonine should be closely related to generation of terpenoids and steroids through methylglutarate pathway⁴⁴. Other intermediate metabolite, i.e. 2-oxoglutarate in the TCA cycle, could be converted to glutanate, and

then to GABA and glutamine ghrough alanine, aspartate and glutamate metabolism pathway (Fig. 5, purple box). Glutamate was also enriched in the pathways of arginine and proline metabolism and arginine biosynthesis. The increased content of arginine and proline can be used as precursors to synthesize alkaloids⁴⁵. Overall, amino acid metabolism played a crucial role in the transformation of primary metabolites into secondary metabolites by R. oryzae fermentation. Specifically, L-aspartate appeared to play a vital role in the metabolic network for biosynthesis of SAMe, β-NMN and other functional compounds.

Quantitative verification of typical DMs and important amino acids

Three metabolites with the most significant increase in abundance, i.e. SAMe, UDP-glucose, and β-NMN, and the important amino acids related to their metabolism were further quantitative verified. As shown in Fig. 6, SAMe, UDP-glucose, and β-NMN all exhibited extremely significant increase in FAG compared with CAG. The content of SAMe showed a sharp increase from 0.56 to 370.26 µg/g after R. oryzae fermentation; and the content of UDP-glucose and β-NMN increased significantly from 0.64 µg/g to 16.14 µg/g and 0.55 µg/g to 8.32 µg/g, respectively. The aspartate which was closely related to both SAMe and β-NMN biosynthesis showed a significant increase in content from 0.35–0.52%. Other correlative amino acids were also obviously increased after R. oryzae fermentation, including glutamate (from 1.31–1.70%), proline (from 0.48–0.62%) and arginine (from 0.24–0.31%), glycine (from 0.13–0.19%), serine (from 0.23–0.32%), and threonine (0.16–0.25%). Previous studies have reported R. oryzae fermentation effectively increased the content of free amino acids in defatted adlay bran and other cereals^13,46. The quantitative results were consistent with that of the metabolomics, confirming that the amino acid metabolism were important for R. oryzae fermentation of adlay millet seed to produce functional compounds.

In this study, the effects of R. oryzae fermentation on the nutritional and chemical composition of adlay millet seed were systematically investigated using UHPLC-MS-based metabolomics, indicating that R. oryzae fermentation is a promising way to improve the multiple qualities of adlay millet seed. After fermentation, the classes of amino acids and derivatives, lipids and lipid-like molecules, nucleotides and derivatives, and vitamins were significantly more abundant than in unfermented adlay millet seed. To the best of our knowledge, this is the first report of R. oryzae fermentation markedly increasing the abundance of two important bioactive compounds, i.e., SAMe and β-NMN. SAMe has health-beneficial biological activities, including prevention and treatment of arthritis, liver disease, depression, and jaundice. β-NMN can ameliorate health conditions associated with NAD⁺ deficiency, such as aging, cardiac, and cerebral ischemia, Alzheimer’s disease and obesity. In addition, R. oryzae fermentation increased the abundance of terpenoids, flavonoids, steroids, and phenols. KEGG annotation and pathway enrichment revealed that the pathways of alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism, as well as cysteine and methionine metabolism were important links between primary and secondary metabolism in R. oryzae fermenting adlay millet seeds. Specifically, aspartate appeared to play a vital role in the metabolic network of SAMe and β-NMN biosynthesis by R. oryzae. Future studies should combine multi-omics techniques, including genomics, transcriptomics, and metabolomics, to elucidate the functional genome bases and metabolic regulation mechanism of functional substances conversion by R. oryzae, as well as evaluation of the potential health promotion effects of fermented adlay millet seed products.

Chemicals and materials

Chromatographic grade formic acid and β-Nicotinamide mononucleotide standard were purchased from Sigma Aldrich (St. Louis, MO). Chromatographic grade methanol and acetonitrile were purchased from Shanghai Anpu Experimental Technology (Shanghai, China). S-adenosylmethionine and Uridine diphosphate glucose standard were purchased from Beijing spectrum analysis standard Technology Co., LTD (Beijing, China). Purified water was obtained using Merck Millipore water purifier (Ming-Che D 24^UV, Merck Millipore Corp., Shanghai, China). Adlay millet seeds were purchased from local market, and the R. oryzae starter was obtained from Angel Yeast Co., LTD (Hubei, China). Before experiments, the R. oryzae was cultured on Potato Dextrose Agar (PDA) medium for 7–8 days, and then the mycelium was selected and observed with a microscopy (Nikon Ti-E, Nikon, Japan).

Adlay millet pretreatment and fermentation

Adlay millet seeds were washed 2–3 times, and then soaked in water (1:3, w/v) overnight at room temperature. After draining the water, the soaked adlay millet was crumbled for 3–5 seconds at 50 Hz to granules with diameter of 2-2.5 mm using a domestic blender (JYL-C020E, Joyoung, China), then the adlay millet grits were steamed in a steamer for 30 minutes. After cooling on a clean bench for about 10 min, the adlay millet grits were stirred with distilled water (12% w/w), then were divided into six equal parts for further experiments. These three parts of the adlay millet grits were inoculated with R. oryzae starter culture (0.6% w/w, 10⁸-10⁹ spores/g), then incubated for 54 h at 30°C (ZSH-150 incubator, Shanghai Gemtop Scientific Instruments, Shanghai, China). These samples were designated as fermented adlay millet grits (FAG). The other three unfermented parts of the adlay millet grits were designated as control adlay millet grits (CAG). The FAG and CAG samples were stored at -80°C until needed for analysis.

Metabolite extraction

The FAG and CAG samples were freeze-dried, then crushed to a powder with the mixer mill for 60 s at 50 Hz. After that, aliquots (50 mg) were precisely weighed and transferred to Eppendorf tubes of extraction solvent (700 µL, methanol/water 3:1 v/v, containing the internal standard 2-chlorophenylalanine and precooled at -40°C). After vortex mixing for 30 s, the samples were homogenized at 35 Hz for 4 min and sonicated for 5 min in ice-water bath for 3 times. Then the samples were extracted overnight at 4°C on a shaker and centrifuged at 12000 rpm (RCF = 13800 (×g), R = 8.6 cm) for 15 min at 4°C). The supernatant was filtered through a 0.22 µm microporous membrane, diluted 10 times with extraction solvent, and vortexed for 30 s and transferred to 2 mL glass vials. A mixture of 60 µL aliquots from each sample was analyzed as a QC sample and stored at -80°C until needed for the UHPLC-MS analysis.

Widely targeted-metabolomic analysis

UHPLC-MS was carried out using an ExionLC AD System (SCIEX, Framingham, MA, USA), fitted with a Waters Acquity UPLC HSS T3 C18 (1.8 µm, 2.1 mm × 100 mm) chromatographic column, with a 400 µL/min flow rate and 40 ^oC column oven temperature. The mobile phases were water/0.1% formic acid (A) and acetonitrile (B). The auto-sampler temperature was 4°C and the injection volume 2 µL. The mobile phase linear gradient was: 0 min, 2% B; 10.0 min, 50% B; 11.0 min, 95% B; 13.0 min, 95% B; 13.1 min, 2% B; and 15.0 min, 2% B. A SCIEX QTrap 6500 + MS system was used for detection and identification of eluted compounds. Typical ion source parameters were as follows: Ion spray voltage, + 5500 V/-4500 V; curtain gas: 35 psi, temperature: 400°C; ion source gas I, 60 psi; gas II: 60 psi; declustering potential (DP), ± 100 V. Further, the standard curve method was used for the quantitative determination of SAMe, Uridine diphosphate (UDP)-glucose and β-NMN in CAG and FAG samples. All tests were carried out in triplicate.

Quantitative verification of amino acids

The content of amino acids in CAG and FAG samples was quantitatively analyzed by Hitachi L-8800 amino acid analyzer according to GB5009.124-2016 (National Food Safety Standard for the Determination of Amino Acids in Food, CN). The determination conditions were as follows: The separation column temperature was 57°C, and the reaction column temperature was 135°C. The sample size was 20 µL. The flow rate of buffer for elution was 0.40 mL/min and that of ninhydrin solution was 0.35 mL/min. The detection wavelengths were 440 and 570 nm, respectively. All the analyses were carried out in triplicate.

Statistical analysis

Total ion chromatograms (TICs) of multiple QC sample and internal standard were used for data quality control. The reproducibility of peak retention times and peak areas between runs was very good, and the RT and peak area of the internal standard 2-chlorophenylalanine was very stable (Supplementary Fig. 5), indicating the data acquisition was both repeatable and stable. The data preprocessing and annotation of untargeted-metabolomic analysis was as follows: the SCIEX Analyst Work Station Software (Version 1.6.3) was employed for multiple reaction monitoring (MRM), data acquisition, and processing. Multivariate statistical analysis was performed using SIMCA software (V16.0.2, Sartorius Stedim Data Analytics AB, Umea, Sweden). Data were scaled and logarithmically transformed to minimize the impact of both noise and high data variance. Unsupervised principal component analysis (PCA) with a 95% confidence interval was carried out to visualize the distribution and grouping of the samples. To visualize group separation and find significant differential metabolites (DMs), supervised orthogonal projections to latent structures-discriminate analysis (OPLS-DA) was applied. Analytical methods including seven-fold cross validation, cross validation, and permutation testing with 200 repetitions, were performed to estimate the stability, reliability, and validity of the OPLS-DA model. Thresholds including a p-value <0.05 from Student's T-tests and a variable importance in projection (VIP) >1 were applied to the first principal component of the OPLS-DA model to identify the DMs between FAG and CAG group samples. Metabolic pathway enrichment analysis of DMs was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and MetaboAnalyst databases.

For quantitative data, the result was provided as the mean and SD (n = 3). GraphPad Prism 8.0 software (GraphPad Software Inc.) was used to process image and analyze quantitative data. Multiple t-test was used for comparison of typical functional DMs and amino acids between FAG and CAG groups with the significant difference at the 5% level.

Research involving plants statement

This study was developed with commercial adlay millet seeds obtained from local supermarket in China, therefore nonexotic or at risk of extinction. The experimental research complied with relevant institutional, national, and international guidelines and legislation for cultivated plants.

Data Availability

All data generated and/or analyzed during this study are available from the corresponding author on reasonable request.

Author contributions

C.L.: Conceptualization, Methodology, Writing—Original Draft Preparation. J.W.: Visualization. M.S.: Data Curation, Methodology. X.H.: Writing-Review and Editing. Z.W.: Resources. Q.L.: Investigation. T.L.: Writing-Review and Editing, Supervision. Z.Z.: Supervision, Project Administration and Funding Acquisition.

Funding

This work is supported by the Guangxi Science and Technology Base and Talents Special Project (GuikeAD21220059); the Foundation for the Scientific Research Base and Distinguished Talents in Guangxi (GuikeAC22080006); the Guangxi First-class Disciplines (Agricultural Resources and Environment); and the National Natural Science Foundation of China (32101367). We also thank the instrument supports from and Guangxi Key Laboratory of Biology for Mango.

Additional information

Competing interests

The authors declare no competing interests.

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Metabolomic analysis reveals the positive effects of Rhizopus oryzae fermentation on the nutritional and functional constituents of adlay millet seeds

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Journal Publication

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Abstract

Figures

Introduction

Results and Discussion

Metabolomic analysis of CAG and FAG samples

Identification and classification of differential metabolites

Metabolism of nutritional components

Amino acids and derivatives

Carbohydrates and carbohydrate conjugates

Lipids and lipid-like molecules

Nucleosides, nucleotides and derivatives

Vitamins

Metabolism of functional components

Flavonoids

Terpenoids

Alkaloids

Phenols

KEGG annotation, related pathway enrichment and network analysis of differential metabolites

Quantitative verification of typical DMs and important amino acids

Conclusions

Materials and methods

Chemicals and materials

Adlay millet pretreatment and fermentation

Metabolite extraction

Widely targeted-metabolomic analysis

Quantitative verification of amino acids

Statistical analysis

Research involving plants statement

Declarations

References

Additional Declarations

Supplementary Files

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Journal Publication

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