REN Chuan-ying ,LU Shu-wen ,GUAN Li-jun ,HONG Bin ,ZHANG Ying-lei ,HUANG Wen-gong,LI Bo,LIU Wei,LU Wei-hong

1 School of Chemistry and Chemical Engineering,Harbin Institute of Technology,Harbin 150000,P.R.China

2 Food Processing Research Institute,Heilongjiang Academy of Agricultural Sciences,Harbin 150086,P.R.China

3 Safety and Quality Institute of Agricultural Products,Heilongjiang Academy of Agricultural Sciences,Harbin 150086,P.R.China

4 The Second Affiliated Hospital,Harbin Medical University,Harbin 150000,P.R.China

Abstract Germination and processing are always accompanied by significant changes in the metabolic compositions of rice.In this study,polished rice (rice),brown rice,wet germinated brown rice (WGBR),high temperature and pressure-treated WGBR (WGBR-HTP),and low temperature-treated WGBR (WGBR-T18) were enrolled.An untargeted metabolomics assay isolated 6 122 positive ions and 4 224 negative ions (multiple difference ≥1.2 or ≤0.8333,P＜0.05,and VIP≥1)by liquid chromatography-mass spectrum.These identified ions were mainly classified into three categories,including the compounds with biological roles,lipids,and phytochemical compounds.In addition to WGBR-T18 vs. WGBR,massive differential positive and negative ions were revealed between rice of different forms.Flavonoids,fatty acids,carboxylic acids,and organoxygen compounds were the dominant differential metabolites.Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database,there 7 metabolic pathways (phenylalanine/tyrosine/tryptophan biosynthesis,histidine metabolism,betalain biosynthesis,C5-branched dibasic acid metabolism,purine metabolism,zeatin biosynthesis,and carbon metabolism) were determined between brown rice and rice.Germination changed the metabolic pathways of porphyrin and chlorophyll,pyrimidine,and purine metabolisms in brown rice.In addition,phosphonate and phosphinate metabolism,and arachidonic acid metabolism were differential metabolic pathways between WGBR-HTP and WGBR-T18.To sum up,there were obvious variations in metabolic compositions of rice,brown rice,WGBR,and WGBR-HTP.The changes of specific metabolites,such as flavonoids contributed to the antioxidant,anti-inflammatory,anti-cancer,and immunomodulatory effects of GBR.HTP may further improve the nutrition and storage of GBR through influencing specific metabolites,such as flavonoids and fatty acids.

Keywords:brown rice,germination,metabolomics,metabolic pathway,high temperature and pressure

1.Introduction

Rice (Oryza sativaL.) is one of the main food crops in the world,accounting for more than half of the world’s population,especially in Asia.Usually,the embryo and bran layer of the staple rice are completely removed and only the endosperm is retained,providing starch and little protein as the main nutrients for human body.However,the bioactive components of rice mainly exist in the embryo and bran layer.Brown rice is an un-milled whole grain of rice containing the embryo and bran layer.It is recommended in routine diet due to the enrichment of nutritional components,such as fibre,iron,calcium,vitamins,and minerals (Tomioet al.2002;Matsuoet al.2012).The consumption of brown rice exhibits valuable health benefits for many diseases,such as coronary heart disease,hypertension,diabetes,metabolic syndrome,and colon cancer (Slavin 2004;Charoenthaikijet al.2010;Ravichanthiranet al.2018).However,the inferior sensory quality greatly limited the appealing of brown rice to consumers (Hunget al.2007).Evidence has proved that the germination is an effective way to solve this problem.Germination can not only improve the sensory quality,but also increase the production of physiologically active substances (Patil and Khan 2011).Therefore,the germinated brown rice (GBR) is considered as an attractive functional food with good taste and nutritional value (Xuet al.2011).

Significant changes in chemical compositions occur during the germination process of brown rice.Previous studies have proved that a large number of nutrients are increased in GBR,including potassium,zinc,magnesium,vitamin E/B1/B6,lysine,gamma-aminobutyric acid (GABA),and γ-oryzanol (Patil and Khan 2011;Cho and Lim 2016).In addition to these nutrients,there also many other differential chemical components are determined in GBR based on liquid chromatography (LC) and/or mass spectrum (MS).Tianet al.(2004) have shown that 6′-O-(E)-feruloylsucrose and 6′-O-(E)-sinapoylsucrose,two major soluble phenolic compounds are decreased in brown rice during germination,whereas free phenolic acids are increased.Tiet al.(2014) have found that the phenolic compounds of ferulic,coumaric,syringic,and caffeic acid are increased in GBR.Notably,a metabolomic analysis identified 25 metabolites (including acidic compounds,amino acids,sugars,lipid metabolites,and secondary metabolites) in GBR during germination,and these identified metabolites were enriched in the metabolomic pathways of carbohydrate metabolism,citric acid cycle,and lipid metabolism (Kimet al.2020).The above studies confirm the composition variation in GBR,while there still massive differential metabolites have not been revealed.

Because the moisture content of GBR is as high as 35–38%,fresh GBR needs to be treated to prolong the shelf life.Drying can reduce the moisture of GBR to 14–15%,but the energy consumption is extremely high,the moisture of the products is uneven,and the shelf life is still unsatisfactory (only 6 months).In order to have a long shelf life,high nutrition,and high taste value,a better treatment method for GBR is urgently needed.Until now,diverse controlled strategies have been discovered to further increase the contents of bioactive components,such as soaking (Thitinunsomboonet al.2013),high hydrostatic pressure (Xiaet al.2018),autoclaving (Renet al.2020),low-pressure plasma(Chenet al.2016),cold plasma (Yodpitaket al.2019),and ultrasound (Xiaet al.2020).In addition to low temperature,high temperature and pressure (HTP) is also an important processing method to facilitate the storage of GBR.It is noteworthy that HTP can also improve the taste value and increase the GABA and ferulic acid levels according to our previous research(Renet al.2020).However,the changes of metabolic compositions in HTP-treated GBR are still unclear.In this study,an untargeted metabolomics assay was performed in polished rice (rice),brown rice,wet GBR (WGBR),and processed WGBR (HTP and low temperature).The differential metabolites and involved metabolic pathways were isolated and determined.Our findings may reveal the metabolic characteristics and differences of rice in different forms,laying the foundation for the evaluation of specific nutritional value.

2.Materials and methods

2.1.Rice samples

Suijing 18,ajaponicacultivar,was provided by the Suihua Branch of Heilongjiang Academy of Agricultural Sciences(Harbin,China).Paddy rice was husked and processed into brown rice (without the husk),and brown rice was further milled and processed into rice (without bran layer and gern).After washed with clear water for three times,brown rice was germinated in an incubator at 30°C and 95% humidity for 40 h to produce WGBR.The generated WGBR has a moisture content of 35–38% (wet basis,w.b.).WGBR were further treated with 20 min of 115°C in pressure sterilizing pot (WGBR-HTP group),or 24 h of–18°C (WGBR-T18 group).

2.2.Metabolomic data acquisition and processing

Metabolomic data of the rice samples (rice,brown rice,WGBR,WGBR-HTP,WGBR-T18) were acquired by LC-MS.LC separation was performed on 2777C UPLC (Waters,USA) using ACQUITY UPLC HSS T3 column (100 mm×2.1 mm,1.8 μm,Waters,USA),and ion acquisition was performed on Xevo G2-XS QTOF(Waters,USA).The raw data were imported into Progenesis QI Software (ver.2.2,hereinafter referred to as QI) for peak picking,and getting the information of metabolites such as mass over charge,retention time,and ion area.Data preprocessing was performed using metaX.Low quality ions,including the ions in quality control (QC) samples containing over 50% missing value,and ions in actual samples containing over 80%missing value were filtered out.The ions in all QC samples with RSD ＞30% were further filtered out.Batch effect correction was performed using quality controlbased robust locally weighted scatterplot smoothing(LOESS) signal correction method.Principal component analysis (PCA) was performed using Soft Independent Modeling of Class Analogy (SIMCA) Software (ver.14.1).

2.3.Differential ions screening

Multivariate analysis of the variable importance in projection (VIP) values of the first two principal components of the PLS-DA model,combined with univariate analysis of fold-change andq-values were used to screen differential metabolites.The screening conditions included:VIP≥1,fold change ≥1.2 or ≤0.8333,andq-value＜0.05.The differential ions were identified using Progenesis QI (ver.2.2).

2.4.Functional annotation and metabolite pathway assay

The identified metabolites were functional annotated based on the Kyoto Encyclopedia of Genes and Genomes(KEGG) and Human Metabolome Database (HMDB)databases to reveal the classification and functional characteristics.In addition,the differential metabolic pathways were enriched based on the KEGG database to determine the major biochemical metabolic pathways involved by differential metabolites.

3.Results

3.1.The identification of positive and negative ions

An untargeted metabolomics assay was performed in rice,brown rice,WGBR,WGBR-HTP,and WGBR-T18 samples.A total of 7 795 positive ions and 5 719 negative ions were determined.Among these ions,6 122 positive ions and 4 224 negative ions with RSD≤30%were isolated for subsequent statistical analysis.The following primary identification revealed 4 481 positive ions and 2997 negative ions,and secondary identification revealed 2997 positive ions and 1 623 negative ions.

3.2.Classification and functional annotation of metabolites

The identified ions were classified and functional annotated based on the KEGG and HMDB database.As shown in Fig.1-A,the identified metabolites were mainly classified into three categories,including the compounds with biological roles,lipids,and phytochemical compounds.The amino acids/pepitdes/analogues,fatty acyls,and terpenoids were the dominant metabolites that enriched by positive ions in each category,respectively.The negative ions were dominant in the carbohydrates,polyketides,and flavonoids in each category,respectively.For the KEGG pathway enriched by positive ions,there 13,6,and 553 metabolites were annotated into the environmental information processing,genetic information processing,and metabolism,respectively.In negative ion mode,21 and 466 metabolites were annotated into the environmental information processing,and metabolism,respectively.It is noteworthy that the identified metabolites were involved in the metabolism of a variety of substances,including the amino acid,lipdis,terpenoids and polyketides,nucleotide,and carbohydrate(Fig.1-B).

Fig.1 Classification and functional annotation of identified metabolites based on the Kyoto Encyclopedia of Genes and Genomes(KEGG) and Human Metabolome Database (HMDB) database.A,classification of metabolites based on KEGG and HMDB database.B,functional annotation of metabolites based on KEGG database.

3.3.Differential ions in rice of different forms

The discreteness among different rice samples were firstly evaluated by PCA assay.As shown in Fig.2,the rice,brown rice,WGBR,and WGBR-HTP were separated in the PCA models of both positive and negative ions.However,the WGBR was relatively close to WGBR-T18 in both positive and negative ion modes.According to the secondary identification result,307 differential positive ions (256 up and 51 down) and 270 differential negative ions (213 up and 57 down) were revealed between brown rice and rice.Flavonoids,fatty acids,carboxylic acids,and organooxygen compounds were the dominant differential metabolites in both positive and negative ion modes.There 24 positive ions (19 up and 5 down) and 76 negative ions (44 up and 32 down) were determined to be different between WGBR and brown rice.These differential metabolites mainly included carboxylic acids,prenol lipids,flavonoids,and phenols in positive ion mode,as well as flavonoids,organooxygen compounds,fatty acids,and carboxylic acids in negative ion mode.In addition,HTP up-regulated 18 positive ions and 41 negative ions,and down-regulated 63 positive ions and 83 negative ions in WGBR.Between WGBRHTP and WGBR,the differential positive ions were dominated by prenol lipids,fatty acids,and flavonoids,and the differential negative ions were dominated by flavonoids,fatty acids,organooxygen compounds,and carboxylic acids (Fig.3).However,both the positive and negative ions in WGBR were not changed by T18.Besides,there 19 positive ions (15 up and 4 down) and 75 negative ions (48 up and 27 down) were identified between WGBR-T18 and WGBR-HTP.The detail number of differential ions between rice of different forms was listed in Table 1.

Fig.2 PCA assay of rice,brown rice,wet germinated brown rice (WGBR),high temperature and pressure-treated WGBR (WGBRHTP),and low temperature-treated WGBR (WGBR-T18) samples.Each dot in the figure represents a sample,and the different colors represent different groups.

Fig.3 The top 7 classifications enriched by differential metabolites in positive (pos) and negative (neg) ion modes.

3.4.Differential metabolic pathways in rice of different forms

Based on the KEGG database,the major biochemical metabolic pathways involved in the identified differential metabolites were enriched.A total of 9 and 14 differential pathways were determined between different samples in positive and negative ion modes,respectively.There 2 metabolic pathways (phenylalanine/tyrosine/tryptophan biosynthesis,and histidine metabolism) involving 4 positive ions,and 5 pathways (betalain biosynthesis,C5-branched dibasic acid metabolism,purine metabolism,zeatin biosynthesis,and carbon metabolism) involving 11 negative ions were determined between brown rice and rice.After germination,3,2,and 2 differential negative ions were determined to be enriched in the metabolic pathways of porphyrin and chlorophyll metabolism,pyrimidine metabolism,and purine metabolism,respectively.However,no differential metabolic pathways involving either positive or negative ions were revealed between WGBR and WGBR-HTP/WGBR-T18.Between WGBR-HTP and WGBR-T18,phosphpnate and phosphinate metabolism,and arachidonic acid metabolism were differential metabolite pathways in positive ion mode(Fig.4).The detail differential metabolites involving the above pathways were shown in Table 2.The differential metabolites and involved metabolic pathways in many other combinations were shown in Appendices A and B.

Table 2 The metabolites involved in differential metabolic pathways

Fig.4 The differential metabolic pathways in brown rice vs. rice,WGBR vs. brown rice,and WGBR-T18 vs. WGBR-HTP were enriched by identified differential positive (pos) and negative (neg) ions based on the KEGG database.The abscissa (rich factor)represents the number of differential metabolites annotated to the pathway divided by all annotated metabolites,and the dot size represents the number of differential metabolites.WGBR,wet germinated brown rice;WGBR-T18,low temperature-treated WGBR;WGBR-HTP,high temperature and pressure-treated WGBR.

Table 1 The number of differential ions in rice of different forms

4.Discussion

Nowadays,brown rice has increasingly been noticed due to high nutritional quality,which is recommended in diet throughout the worldwide.Compared with polished rice,brown rice is more enriched in fiber,iron,calcium,vitamins,and minerals (Patil and Khan 2011).The biochemical differences between brown and polished rice have been determined by many previous studies.

Vichaponget al.(2010) have found that the contents of phenolic compounds and total flavonoids are higher in brown rice than polished rice.Yanget al.(2016)have shown that the levels of tricin,tricin 7-O-β-Dglucopyranoside,tricin 7-O-rutinoside,and lycoperodine-1 are higher in brown rice than those in polished rice.In this study,an untargeted metabolomics assay based on LCMS was performed to discover the differential metabolites.PCA assay showed that rice and brown rice were separated in both the positive and negative ion models.Since PCA properly reflects the accumulation pattern and specificity of metabolites in corresponding samples,this result indicates the presence of metabolomics difference between rice and brown rice.Indeed,307 differential positive ions and 270 differential negative ions were determined between brown rice and rice.Among these differential metabolites,flavonoids and fatty acids were revealed to be the dominant classes.Flavonoids are well-known as the active components of medicinal plants and the ingredients of bioactive foods (Perez-Vizcaino and Fraga 2018).The consumption of flavonoid-rich foods is benefit for human health,and for the treatment of some human diseases,such as the cancer,stroke and coronary heart disease (Peluso and Palmery 2015).Fatty acids are substantial components of lipids,which exert important roles in structural function of cell membrane,material storage,and cell signaling in human(de Carvalho and Caramujo 2018).The consumption of unsaturated fatty acids is protective for human health,and some essential fatty acids (linoleic and alpha-linolenic)can only be taken from the diet (Tvrzickaet al.2011).Therefore,the metabolic changes of flavonoids and fatty acids may contribute to the high nutritional value of brown rice.Furthermore,the differential ions were annotated with the KEGG database,and total 7 differential metabolic pathways (4 positive ions and 11 negative ions)were determined.Among these metabolic pathways,tryptophan pathway is response to the defense responses of rice against pathogenic infection through producing serotonin (Ishiharaet al.2008).Phenylalanine ammonialyase is closely associated with brown planthopper resistance (Heet al.2020),and phosphate deficiency response (Ghoet al.2020).To combine with the physical difference between rice and brown rice (Patil and Khan 2011),the metabolites involved in the identified differential metabolic pathways may be mainly enriched in the bran layer.

For brown rice,germination is an effective way not only to solve the inferior sensory quality,but also to further improve the nutritional quality.It has been reported that GBR has higher nutrient and sweetness,as well as better digestion and absorption characteristics than non-germinated brown rice (Wuet al.2013).During germination,there are significant changes in the composition and chemical structure of many bioactive components,such as GABA,gamma oryzanols,and policosanols (Cho and Lim 2016).In this study,obvious differences on metabolites were observed betweenWGBR and brown rice,including 24 differential positive ions and 76 differential negative ions.Among differential metabolites,phenols,carboxylic acids,and prenol lipids were determined to be the main classes.These results are just similar with some previous findings.For examples,the germination increases the phenolic compounds in GBR,such as phenolic,ferulic,coumaric,syringic,and caffeic acids (Tianet al.2004;Tiet al.2014).There 25 metabolites are identified during the germination of brown rice by a metabolomic analysis,which mainly involved in the carbohydrate metabolism,tricarboxylic acid cycle,and lipid metabolism (Kimet al.2020).More importantly,flavonoids with beneficial effects were also changed following germination.These differential metabolites may be directly related with the improved nutritional quality of WGBR.Subsequently,the porphyrin and chlorophyll,pyrimidine,and purine metabolisms were identified as three differential metabolic pathways in a negative ion mode.It is known that photosynthetic activity is activated during germination,along with increased levels of lutein,b-carotene,and chlorophyll a (Leeet al.2013).The change of chlorophyll metabolism in WGBR may be attributed to the activation of germ.Because chlorophyll exerts chemoprotective and anti-carcinogenic roles in human (Hayes and Ferruzzi 2020),its increase may response for the high edible value of WGBR.In addition,pyrimidine and purine are building blocks for nucleic acid synthesis,which are urgently important for the germination,sprouting,organogenesis,storage organ development,and leaf senescence of plants (Stasollaet al.2003;Zrenneret al.2006).The metabolic changes of pyrimidine and purine conform to the physiological process of germination.

Low temperature is a common storage method for WGBR,which greatly blocks the overgrowth and the possibility of mildew.In this study,a relatively close relation between WGBR and WGBR-T18 was determined by PCA assay,and no differential metabolites were discovered.These results indicate that the low temperature cannot cause nutritional loss in WGBR.As another mean of storage,HTP is also a valuable processing method to improve the nutritive value of GBR.Our previous study has revealed that the HTP treatment improves the taste value and increases GABA and ferulic acid in GBR (Renet al.2020).In this study,81 and 124 differential metabolites were determined between WGBR and WGBR-HTP in positive and negative ion modes,respectively.These results confirm that HTP affects the metabolic compositions in WGBR.In addition to flavonoids and fatty acids,carboxylic acids were revealed as a dominant class for differential negative ions in WGBR-HTP.Since ferulic acid is an aromatic carboxylic acid,the metabolic changes of carboxylic acids may explain the increasing trend of ferulic acid in GBR.However,there no differential metabolic pathways involving either positive or negative ions were enriched.This phenomenon may be explained by the un-annotated functions of many isolated ions in the database.Since low temperature cannot affect the metabolites in WGBR,the metabolomic differences between WGBR-HTP and WGBR-T18 were further analyzed.There 19 and 75 metabolites were revealed in positive and negative ion modes,respectively.These differential metabolites may be attributed to the chemical structure changes and volatilization loss that induced by HTP.Furthermore,phosphonate and phosphinate metabolism,and arachidonic acid metabolism were determined as the differential metabolic pathways involving positive ions.A previous study based on high hydrostatic pressurestressed germination has shown that glycerol-3-phosphate is one of the differential metabolite signatures(Ruanet al.2021).Therefore,the high pressure may contribute to the change of phosphinate metabolism.For the arachidonic acid metabolism,its change may be caused by high temperature-induced oxidation.

5.Conclusion

The metabolic compositions were varied among rice,brown rice,WGBR,and WGBR-HTP.Flavonoids,fatty acids,carboxylic acids,and organooxygen compounds were the dominant differential metabolites.There 7,3,and 2 differential metabolic pathways were determined in brown ricevs.rice,WGBRvs.brown rice,and WGBRHTPvs.WGBR-T18,respectively.The metabolic characteristics of rice in specific forms may reflect the nutritional value to some degrees.However,the edible value and function of relevant metabolites are not revealed.Further researches on the validation of differential metabolites in specific rice forms,especially in WGBR-HTP are still needed.

Acknowledgements

This study was funded by the National Key Research and Development Program of China (2021YFD2100902),the Major Science and Technology Program of Heilongjiang,China (2019ZX08B02),the Research Funding for Scientific Research Institutes in Heilongjiang Province,China (CZKYF2021B001),the National Rice Industry Technology System,China (CARS-01-50),and the Heilongjiang Touyan Team,China (HITTY-20190034).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm