Germinated brown rice relieves hyperlipidemia by alleviating gut microbiota dysbiosis

REN Chuan-ying ,ZHANG Shan ,HONG Bin ,GUAN Li-jun ,HUANG Wen-gong,FENG Jun-ranSHA Di-xinYUAN DiLI BoJI Ni-na,LIU Wei,LU Shu-wen#

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

2 Heilongjiang Province Key Laboratory of Food Processing,Harbin 150086,P.R.China

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

4 Soybean Research Institute,Heilongjiang Academy of Agricultural Sciences,Harbin 150086,P.R.China

5 The Second Affiliated Hospital,Harbin Medical University,Harbin 150086,P.R.China

Abstract Hyperlipidemia is a frequent metabolic disorder that is closely associated with diet. It is believed that brown rice,containing the outer bran layer and germ,is beneficial for the remission of hyperlipidemia. This study established a rat model of hyperlipidemia by feeding a high-fat diet. The hypolipidemic potential of germinated brown rice (Gbrown) and germinated black rice (a germinated black-pigmented brown rice,Gblack) were explored in the model rats,mainly in the aspects of blood lipids,lipases,apolipoproteins,and inflammation. The gut microbiota in hyperlipidemic rats receiving diverse dietary interventions was determined by 16S rDNA sequencing. The results showed that the intervention of Gbrown/Gblack alleviated the hyperlipidemia in rats,evidenced by decreased TC,TG,LDL-C,and apolipoprotein B,and increased HDL-C,HL,LPL,LCAT,and apolipoprotein A1. Gbrown/Gblack also weakened the inflammation in hyperlipidemia rats,evidenced by decreased TNF-α,IL-6,and ET-1. In addition,16S rDNA sequencing revealed that the diet of Gbrown/Gblack elevated the abundance and diversity of gut microbiota in hyperlipidemia rats. At the phylum level,Gbrown/Gblack decreased Firmicutes,increased Bacteroidetes,and decreased the F/B ratio in hyperlipidemia rats. At the genus level,Gbrown/Gblack decreased Streptococcus and increased Ruminococcus and Allobaculum in hyperlipidemia rats. Some differential microbial genera relating to lipid metabolism were also determined,such as the Lachnospira and Ruminococcus in the Gblack group,and the Phascolarctobacterium,Dorea,Turicibacter,and Escherichia-Shigella in the Gbrown group. Notably,the beneficial effect of Gblack was stronger than Gbrown. To sum up,the dietary interventions of Gbrown/Gblack contributed to the remission of hyperlipidemia by alleviating the dysbiosis of gut microbiota.

Keywords: hyperlipidemia,germination,brown rice,black rice,gut microbiota

1.Introduction

Hyperlipidemia is a common disorder of lipid metabolism.It is characterized by the elevation of total cholesterol(TC),triglyceride (TG),and low-density lipoproteincholesterol (LDL-C) and the reduction of high-density lipoprotein-cholesterol (HDL-C) at circulating levels(El-Tantawy and Temraz 2019). In recent years,the incidence of hyperlipidemia is still increasing due to unhealthy lifestyles,particularly excess caloric intake and physical inactivity (He and Ye 2020;Donget al.2021).Since patients with hyperlipidemia are at high risk for cardiovascular disease with high mortality (Yaoet al.2020),the prevention and treatment of hyperlipidemia have become an urgent problem to be solved. Generally,diet is the cornerstone for the treatment of hyperlipidemia.Many dietary interventions are valuable in decreasing blood lipids,such as soluble fiber,soy protein,marinederived omega-3 fatty acids,and fortifying foods with plant stanols or sterols (Kelly 2010). The development of functional foods brings more possibilities for the prevention and treatment of hyperlipidemia.

Rice (OryzasativaL.) is one of the most important cereal crops worldwide (Songet al.2018). The endosperm of rice is usually consumed in daily life,while the removed bran layer and germ are rich in various nutritional and bioactive ingredients,such as fiber,iron,calcium,vitamins,and minerals (Patil and Khan 2011;Wuet al.2011). Emerging evidence has determined that brown rice containing the bran layer and germ presents a higher nutritional value,benefiting the remission of diverse human diseases,such as hypertension,coronary heart disease,diabetes,and metabolic syndrome (Slavin 2004;Ravichanthiranet al.2018). Since the sensory quality of brown rice is poor,germination has become an effective processing method to improve it. The germination can also improve the nutritional value of brown rice,evidenced by the increased production of bioactive components,such as γ-aminobutyric acid(GABA),ferulic acid,and γ-oryzanol (Patil and Khan 2011;Wuet al.2013;Cho and Lim 2016). Notably,germinated brown rice (Gbrown) exhibits obvious physiological effects on inhibiting hyperlipidemia (Roohinejadet al.2010;Esaet al.2013;Wuet al.2013;Shenet al.2016). In addition,black rice,a special pigmented brown rice,is also regarded as a healthy food widely consumed in China. Previous studies have determined that black rice enriched in anthocyaninis contributes to the alleviation of hyperlipidemia by reducing serum TG,TC,and LDL-C in animal models(Guoet al.2007;Yaoet al.2013;Songet al.2021).Consistently,germinated black rice (Gblack) also shows the potential to lower blood lipids (Hoet al.2012;Limet al.2016;Kanget al.2017). Therefore,Gbrown and Gblack are considered promising functional foods for preventing and treating hyperlipidemia.

Gut microbiota is a large and diverse group of microorganisms in the human digestive system,which plays an important role in hyperlipidemiaviaregulating host lipid metabolism (Gomaa 2020;Jiaet al.2021).Many previous studies have reported that the gut microbiota can affect lipid metabolism in the blood and tissues of both mice and human (Schoeler and Caesar 2019). The dietary interventions of prebiotics,probiotics,and traditional Chinese medicines have been determined to benefit the treatment of hyperlipidemia by targeting gut microbiota (Jiaet al.2021;Zhanget al.2021). In addition,some corps and related derivants also show therapeutic potential against hyperlipidemia through balancing gut microbiota,such as black rice (Songet al.2021),oats(Xuet al.2021),probiotics-fermented rice buckwheat(Yan Jet al.2022),red yeast rice (Yanget al.2020),and defatted rice bran (Yan Set al.2022). However,whether the function of Gbrown/Gblack on hyperlipidemia is related to gut microbiota is rarely reported yet.

In this study,the specific effects of Gbrown/Gblack on hyperlipidemia were identified in rats,mainly in aspects of blood lipids,lipases,apolipoproteins,and inflammation.The differences in gut microbiota in hyperlipidemia rats receiving diverse dietary interventions were further determined by 16S rDNA sequencing. Our study aimed to explore the hypolipidemic activity of Gbrown/Gblack and also the underlying mechanisms involving gut microbiota,providing guidance for dietary adjustment in patients with hyperlipidemia.

2.Materials and methods

2.1.Rice samples

Japonicacultivars,Suijing 18 and Heizhenzhu,were provided by the Suihua Branch of Heilongjiang Academy of Agricultural Sciences (Harbin,China). Brown rice was processed by removing the husk from paddy rice (Suijing 18),and then it was germinated in an incubator at 30°C and 95% humidity for 40 h to produce Gbrown. The black rice (Heizhenzhu) without the husk was germinated in the same condition to produce Gblack. Polished rice (Suijing 18) without the husk,bran layer and germ was used as a control check (R_CK group). In addition,the bran layer from Suijing 18 was also collected and defined as the Bran group.

2.2.Establishment of hyperlipidemia model in rats

Sprague Dawley (SD) rats (SPF grade,180-200 g,8 weeks) were purchased from Charles River (Beijing,China),and were maintained with free access to food and water in a stable environment at 22-25°C,65-70%humidity,and a 12 h light/dark cycle. Rats were randomly divided into six groups,including the blank,model,R_CK,Gbrown,Gblack,and Bran groups (n=6 for each group).After one week of adaptive feeding with a basic diet (4.24%fat),the hyperlipidemia model was induced by feeding a high-fat diet (18.90% fat;63.6% basic feed+15%lard+20% sucrose+1.2% cholesterol+0.2% sodium cholate) for 8 weeks. For dietary interventions,rats were fed with a high-fat diet combined with R_CK,Gbrown,or Gblack (38.2% R_CK/Gbrown/Gblack+25.4% basic feed+15% lard+20% sucrose+1.2% cholesterol+0.2%sodium cholate). In addition,rats in the Bran group were fed with 7.6% bran+22.3 basic feed+33.7% corn+15%lard+20% sucrose+1.2% cholesterol+0.2% sodium cholate. After 8 weeks of feeding,rats were weighed,and the fecal samples were collected. Subsequently,rats were anesthetized by an intraperitoneal injection of 50 mg kg-1pentobarbital sodium,and abdominal aortic blood was collected. Rats were finally sacrificed by cervical dislocation under anesthesia.

2.3.Detection of parameters involving lipid metabolism

The serum samples were separated from abdominal aortic blood by 20 min of centrifugation at 3 000 r min-1.The serum levels of TC,TG,LDL-C,and HDL-C were measured on an automatic biochemical analyzer (Gelite,Jinan,China) using corresponding kits (Jiancheng,Nanjing,China). In addition,the levels of hepatic lipase (HL),lipoprotein lipase (LPL),lecithin cholesterol acyltransferase (LCAT),apolipoprotein A1,apolipoprotein B,and inflammatory factors (TNF-α,IL-6,and ET-1) in the serum were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (Mlbio,Shanghai,China) following the manufacturer’s instructions.

2.4.16S rDNA sequencing

Total DNAs were isolated from fecal samples of rats by the CTAB method. The V3-V4 hypervariable regions of 16S rDNAs were amplified by PCR using a primer of 341F and 806R (341F: 5′-CCTACGGGNGGCWGCAG-3′;806R:5′-GACTACHVGGGTATCTAATCC-3′). The PCR program included an initial denaturation at 98°C for 30 s,32 cycles of 98°C for 10 s,54°C for 30 s,and 72°C for 45 s,and a final extension at 72°C for 10 min. The PCR products were separated by 2% agarose gel electrophoresis,purified using AMPure XT beads (Beckman Coulter Genomics,Danvers,MA,USA),and quantified on Qubit (Invitrogen,USA). The size and concentration of amplified libraries were assessed on Agilent 2100 Bioanalyzer (Agilent,USA)and using a Library Quantification Kit (Kapa Biosciences,Woburn,MA,USA). The libraries were finally sequenced on the NovaSeq PE250 platform.

2.5.Bioinformatic analyses

Paired-end reads were assigned to samples based on their unique barcode,truncated by cutting off the barcode and primer sequences,and merged using FLASH (v1.2.8).High-quality clean tags were obtained by quality filtering of raw reads using fqtrim (v0.94). Chimeric sequences were filtered out using Vsearch (v2.3.4). After dereplicated using DADA2,the sequences of amplicon sequence variants (ASVs) were obtained. Alpha diversity (Chao1,Observed species,Goods coverage,Shannon index,and Simpson index) and beta diversity (weighted_unifrac)were analyzed using QIIME2. In addition,the ASVs were annotated based on the SILVA (release 138) and NT-16S databases with a cut-off of 0.7.

2.6.Statistical analysis

Quantitative data generated from animal experiments were presented as mean±standard deviation. Statistical analyses were performed by GraphPad Prism (v7.0). The differences among multi-groups were determined by oneway ANOVA followed by Tukey’s text. AP-value＜0.05 was considered statistically significant.

3.Results

3.1.The lipid status in serum of hyperlipidemia rats receiving diverse dietary interventions

A hyperlipidemia model was established in rats with a high-fat diet. As shown in Fig.1-A,the weight gain was significantly higher in the model group than in the blank group (P＜0.05). Different dietary interventions all decreased the weight gain of the hyperlipidemia rats to varying degrees(P＜0.05). Blood lipid analysis revealed that the model group exhibited significantly higher TC,TG,and LDL-C levels and a lower HDL-C level compared with the blank group (P＜0.05).The above lipid changes in the hyperlipidemia rats were obviously weakened by the intervention of Gbrown or Gblack (P＜0.05). Bran exhibited similar results with Gbrown on lowering blood lipid in hyperlipidemia rats (P＜0.05),but its effects on decreasing TG and on increasing HDL-C were weaker than Gblack (P＜0.05). The intervention of R_CK only significantly decreased the TC in the hyperlipidemia rats (P＜0.05,Fig.1-B).

Three key lipases involved in lipid metabolism were also measured in rats of different groups. Compared with the blank group,the levels of HL,LPL,and LCAT were significantly lower in the model group (P＜0.05).Gbrown,Gblack,and Bran could all significantly elevate the levels of HL,LPL,and LCAT in the hyperlipidemia rats,and the effects were Gblack＞Gbrown＞Bran (P＜0.05).These lipases were not significantly influenced by the intervention of R_CK in the hyperlipidemia rats (Fig.1-C).

Apolipoprotein A1 and B,two important apolipoproteins in lipid transportation,were further analyzed. There significantly lower apolipoprotein A1 and higher apolipoprotein B were revealed in the model group compared with the blank group (P＜0.05). Both Gbrown and Gblack inhibited the abnormal changes of apolipoproteins in the hyperlipidemia rats,along with a stronger effect of Gblack than Gbrown (P＜0.05). Besides,R_CK did not influence the apolipoproteins,and Bran only significantly decreased the apolipoprotein A1 in the hyperlipidemia rats (P＜0.05,Fig.1-D).

Fig.1 The lipid status in serum of hyperlipidemia rats receiving different dietary interventions (n=6 each group). A,weight gain.B,the levels of blood lipids,including total cholesterol (TC),triglyceride (TG),low-density lipoprotein-cholesterol (LDL-C) and the reduction of high-density lipoprotein-cholesterol (HDL-C). C,the levels of lipases,including hepatic lipase (HL),lipoprotein lipase(LPL),and lecithin cholesterol acyltransferase (LCAT). D,the levels of apolipoprotein A1 and B. R_CK,rice_control check;Gbrown,germinated brown rice;Gblack,germinated black rice. Bars are SD. Different lowercase letters represent significant differences.

3.2.The inflammation in hyperlipidemia rats receiving diverse dietary interventions

Three inflammatory factors were detected to determine the effects of dietary interventions on inflammation in hyperlipidemia rats. The results showed that the levels of TNF-α,IL-6,and ET-1 in the model group were significantly higher than those in the blank group(P＜0.05). The enhanced inflammatory response in the hyperlipidemia rats was weakened by the intervention of Gbrown/Gblack/Bran,along with an anti-inflammatory degree of Gblack＞Gbrown＞Bran (P＜0.05). In addition,R_CK only decreased the IL-6 level in the hyperlipidemia rats (P＜0.05,Fig.2).

Fig.2 The levels of inflammatory factors,including tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6),and endothelin-1(ET-1) in hyperlipidemia rats receiving different dietary interventions (n=6 each group). R_CK,rice_control check;Gbrown,germinated brown rice;Gblack,germinated black rice.Bars are SD. Different lowercase letters represent significant differences.

3.3.Identification of gut microbiota in hyperlipidemia rats receiving diverse dietary interventions

The gut microbiota in rats was analyzedvia16S rDNA sequencing. A total of 1 543 ASVs were identified in the model group,which was lower than the blank group(4 569 ASVs). The dietary interventions all increased the numbers of ASVs in hyperlipidemia rats to some degree,including 2 590,2 404,5 365,and 2 075 ASVs in the R_CK,Gbrown,Gblack,and Bran,respectively. There were 540 ASVs shared between the blank and model groups,and similar amounts of coexisting ASVs were revealed between the model and diverse dietary intervention groups (496-647 ASVs) (Fig.3).

Fig.3 Venn diagrams of amplicon sequence variant (ASV) distributions in hyperlipidemia rats receiving different dietary interventions(n=6 each group). R_CK,rice_control check;Gbrown,germinated brown rice;Gblack,germinated black rice.

3.4.Diversity of gut microbiota in hyperlipidemia rats receiving diverse dietary interventions

The alpha diversity was analyzed to reflect the sequencing depth,abundance,and uniformity of gut microbiota (Quet al.2017). As shown in Fig.4-A,the goods_coverage index was close to 1 in all enrolled groups,indicating enough sequencing depth in the samples. The microbial abundance (chao1 and observed_utos indexes) was obviously lower in the model group than in the blank group. Different dietary interventions elevated the microbial abundance in the hyperlipidemia rats with the degree of Gblack＞R_CK＞Gbrown＞Bran. The microbial abundance in the Gblack group was even higher than in the blank group (Fig.4-B and C). Consistently,the Shannon (diversity),pielou_e(uniformity),and Simpson (both abundance and uniformity) indexes exhibited the same trends with the chao1 index in different groups (Fig.4-D-F). Besides,beta diversity was further analyzed to reveal microbial differences. In a weighting method,all the enrolled groups showed overlapping microbial compositions.Among them,the Gblack group had a relatively low coincidence with the Model group but had a high coincidence with the blank group (Fig.4-G).

Fig.4 The alpha and beta diversities of gut microbiota in hyperlipidemia rats receiving different dietary interventions (n=6 each group). A,goods_coverage (sequencing depth). B,chao1(abundance). C,observed_utos (abundance). D,Shannon(diversity). E,pielou_e (uniformity). F,Simpson (abundance and uniformity). G,PCoA plot in a weighting method. Points of the same color represent duplicate samples within a group. R_CK,rice_control check;Gbrown,germinated brown rice;Gblack,germinated black rice.

3.5.Classification of gut microbiota in hyperlipidemia rats receiving diverse dietary interventions

Gut microbiotas were annotated and classified to determine changes between different groups. Cluster analysis revealed that the gut microbiota in the Blank and Gblack,Gbrown and R_CK,and Bran and Model were clustered into a clade,respectively,at both the phylum and genus levels (Fig.5-A and C).

At the phylum level,Firmicutes was the dominant microbial phylum in the black group,accounting for 71.36%. The abundance of Firmicutes was obviously increased in the model rats (87.33%). Except for the Brain,the interventions of different rice all decreased Firmicutes abundance to some degree: Gblack (68.98%)＜R_CK (75.56%)＜Gbrown (78.60%). As another frequent microbial phylum,Bacteroidetes enriched in the blank group (21.63%) were reduced by a high-fat diet in the model group (2.92%). Different dietary interventions recovered the abundance of Bacteroidetes in the hyperlipidemia rats with the degree of Gblack (17.09%)＞Gbrown (11.82%)＞R_CK (9.91%)＞Bran (3.20%)(Fig.5-A). Because Firmicutes/Bacteroidetes (F/B)are critical in balancing the gut environment,the F/B ratio was compared among different groups. As shown in Fig.5-B,the F/B ratio was the highest in the model group,followed by the Bran group without significant differences. Gbrown,Gblack,and R_CK all significantly decreased the F/B ratio in the hyperlipidemia rats,and the effect was Gblack＜Gbrown＜R_CK (P＜0.05). The F/B ratio in the Gblack group was close to that in the blank group.

At the genus level,a relatively higher abundance ofStreptococcuswas observed in the model group (4.96%)than in the blank group (1.04%). On the contrary,the abundance ofRuminococcuswas decreased in the model group (0.51%) compared with the blank group(1.72%). Different dietary interventions decreasedStreptococcus(Gbrown＞Bran＞Gblack＞R_CK) and increasedRuminococcus(Gblack＞Gbrown＞R_CK＞Bran)in hyperlipidemia rats to some degree. In addition,dietary interventions also recovered the low abundance ofAllobaculumin hyperlipidemia rats (Fig.5-C).

Fig.5 Classification of gut microbiota in hyperlipidemia rats receiving different dietary interventions (n=6 each group). A,top gut microbiota at the phylum level. B,Firmicutes/Bacteroidetes (F/B) ratio. C,top gut microbiota at the genus level. R_CK,rice_control check;Gbrown,germinated brown rice;Gblack,germinated black rice.

3.6.ldentification of differential microbiota in rats receiving diverse dietary interventions

Linear discriminant analysis Effect Size (LEfSe) analysis was performed to determine the differential gut microbiota in different groups. The differential microbiota was the most abundant in the blank group (74),and the dominant genera included Muribaculaceae,Firmicutes,Clostridia_UCG_014,Quinella,and Bacteroidetes (Top 5). On the contrary,the model group had the lowest differential microbiota (13),mainly including the genera ofLactobacillus,HT002,Streptococcus,andEnhydrobacter.Dietary interventions increased the differential gut microbiota in the hyperlipidemia rats,and the number is Gblack (55)＞R_CK (37)＞Gbrown (26)＞Bran (19).Notably,some differential microbiota in the interventions groups were related to lipid metabolisms,such as theLachnospiraandRuminococcusin the Gblack group,Phascolarctobacterium,Dorea,Turicibacter,andEscherichia-Shigellain the Gbrown group (Fig.6).

4.Discussion

Hyperlipidemia is a prevalent disorder characterized by an excess of lipids in the bloodstream (Konget al.2021).In clinical,hyperlipidemia is usually accompanied by a high risk of cardiovascular diseases,making it a great threat to human health (Yaoet al.2020). Although many drugs are available to treat hyperlipidemia,the first-line therapy is still a healthy diet and lifestyle (Yaoet al.2013).Brown rice is an un-milled whole grain containing the outer bran layer and germ,exhibiting a higher nutritional value than polished rice (Tomioet al.2002;Matsuoet al.2012). Evidence has determined that the rice brain enriched in nutritional and bioactive components benefits the remission of hyperlipidemia. For example,rice bran inhibits the accumulation of body fat and hepatic lipids in obese rats by maintaining hepatic lipid homeostasis (Yanget al.2019). The insoluble dietary fiber from defatted rice bran ameliorates hyperlipidemia by improving lipid and glucose metabolisms and inhibiting inflammation and oxidative stress (Liuet al.2021). The hydrolyzed bound phenolic from rice bran ameliorates hyperlipidemia by inhibiting the uptake of cholesterol and fatty acid in the liver and gut (Zhaoet al.2022). In this study,the dietary intervention of rice bran decreased the TC,TG,LDL-C,TNF-α,and IL-6 and increased the HDL-C,HL,LPL,LCAT,and apolipoprotein A1 in hyperlipidemia rats. These findings are consistent with previous studies and illustrate that rice bran contributes to the remission of hyperlipidemia by balancing lipid metabolism and inhibiting inflammation.

However,the bran and brown rice are less acceptable to consumers due to inferior sensory quality (Hunget al.2007). Germination is an ideal choice to improve the sensory quality of brown rice,which can further improve its nutritional value (Patil and Khan 2011;Wuet al.2013;Cho and Lim 2016). Similar to rice bran,Gbrown also exhibits a high therapeutic potential against hyperlipidemia. Roohinejadet al.(2010) have shown that Gbrown decreases TC and LDL-C and increases HDL-C in the serum of hypercholesterolaemic rats(Roohinejadet al.2010). Shenet al.(2016) have found that Gbrown relieves hyperlipidemia in mice by mediating blood lipids and adipocytokines (Shenet al.2016). Esaet al.(2013) have revealed that Gbrown enhances antioxidant enzyme activity and inhibits lipid peroxidation in hypercholesterolaemic rabbit(Esaet al.2013). Consistently,we found that Gbrown exerted a hypolipidemic role in hyperlipidemia rats,and its effects on reducing HL and LPL and elevating apolipoprotein A1 were stronger than bran. Gbrown also presented a higher anti-inflammatory effect than bran in hyperlipidemia rats. Our findings indicate that Gbrown with a good taste is a promising functional food for preventing and treating hyperlipidemia. Balancing lipid synthesis and metabolism may be the dominant action mechanism of Gbrown in hyperlipidemia.Evidence has determined that fibers,GABA,ferulic acid,and γ-oryzanol are effective in lowering lipid profiles(Bhaskaragoudet al.2016;Derosaet al.2019;Nie and Luo 2021). These ingredients enriched in Gbrown may directly contribute to the amelioration of hyperlipidemia.On the other hand,black rice is a pigmented brown rice with beneficial effects on human health (Ito and Lacerda 2019). Similarly to Gbrown,Gblack also exerts a beneficial role in protecting against hyperlipidemia.Kanget al.(2017) have shown that Gblack decreases serum levels of TG,TC,LDL-C,aspartate transferase,and alanine transferase in diabetic rats. Limet al.(2016) have found that Gblack extracts inhibit adipocyte differentiation through down-regulating adipogenic enzymes (aP2,LPL,and FAS) and adipogenic transcriptional factors (C/EBP-α and -β,PPAR-γ,and SREBP-1c)invitro. Consistent with previous studies,an obvious hypolipidemic effect was determined in Gblack. Notably,among diverse interventions,Gblack exhibited the best effects on improving lipid metabolism and also on inhibiting inflammation. Therefore,Gblack is recommended as a more functional food for people with hyperlipidemia or at risk of hyperlipidemia. In addition to the same active ingredients in Gbrown,anthocyaninis is believed to be another important active ingredient in Gblack,contributing to the alleviation of hyperlipidemia (Guoet al.2007;Yaoet al.2013;Songet al.2021).

The gut microbiota plays an important role in host metabolism,such as the homeostasis of glucoses and lipids,and the production of energy and vitamins(Pascaleet al.2018). Notably,the gut microbiota is closely associated with the development of hyperlipidemia since it can mediate host lipid metabolismviainfluencing satiety,cholesterol metabolism in liver,lipid oxidation in muscle,energy storage in adipose tissue,and integrity of the intestinal barrier (Jiaet al.2021). Some microbiota-related metabolites,such as bile acids,lipopolysaccharide,and short-chain fatty acids,are considered effectors in regulating hyperlipidemia (Jiaet al.2021). Until now,targeted therapy based on gut microbiota has achieved exciting results in improving hyperlipidemia (Jiaet al.2021;Zhanget al.2021). Many kinds of crops also exhibit therapeutic potential against hyperlipidemiaviatargeting gut microbiota (Yanget al.2020;Songet al.2021;Xuet al.2021;Yan Jet al.2022;Yan Set al.2022). Here,the gut microbiota was further explored in different intervention groups by 16S rRNA sequencing,aiming to discover the underlying biological mechanisms. The results showed that hyperlipidemia rats exhibited a lower microbial abundance than normal rats,and the low microbial abundance was recovered by the intervention of Gblack/Gbrown. These findings indicate that Gblack/Gbrown restores the abundance and diversity of gut microbiota in hyperlipidemia rats.

After annotation and classification,the microbial differences in rats receiving diverse interventions were analyzed. At the phylum level,Firmicutes and Bacteroidetes were observed as the dominant microbial phylum in normal rats. The high-fat diet increased Firmicutes but decreased Bacteroidetes in the model rats,which are just consistent with previous studies (Round and Mazmanian 2009). Bacteroidetes and Firmicutes are known as two of the most abundant groups of prokaryotes in human. An increased F/B ratio may be induced by ethanol,a high-fat diet,and high fructose (Jandhyalaet al.2015;Ballway and Song 2021). Previous studies have reported that the decreased F/B ratio is associated with obesity and inflammatory bowel disease (Stojanovet al.2020). Notably,emerging evidence has determined that decreased F/B ratio also contributes to the hypolipidemic role of many bioactive substances. For example,Konget al.(2021) have shown that glycosaminoglycans improve gut microbiota imbalance by reducing the F/B ratio(Konget al.2021). Kanget al.(2022) have found thatLactobacillusacidophilusreverses high-fat diet-induced gut dysbiosis,evidenced by the decreased F/B ratio (Kanget al.2022). Heet al.(2020) have revealed that tomato seed oil effectively attenuates hyperlipidemia by reducing the F/B ratio. The highland barley whole grain (Denget al.2020),AgaricusblazeiMurrill polysaccharides (Liet al.2020),and gypenosides (Huanget al.2019),kynurenic acid (Liet al.2021) with the therapeutic potential against hyperlipidemia can also decrease the F/B ratio. Similar to previous studies,Gblack/Gbrown elevated the F/B ratio in hyperlipidemia rats,illustrating the improvement of gut microbiota dysbiosis. In addition,the elevating effect of Gblack on the F/B ratio was stronger than Gbrown,which is consistent with the findings on lipid metabolism and inflammation.

This study also isolated some important gut microbiota at the genus level.Streptococcusis a kind of Gram-positive bacteria responsible for a variety of inflammatory diseases in human,such as sepsis,meningitis,pneumonia,endocarditis,arthritis,and pharyngitis (Haenniet al.2018). Weiet al.(2021) have found thatStreptococcusis a hub genus in the fecal micro-ecosystem of high-fat diet mice (Weiet al.2021).Consistently,a high abundance ofStreptococcuswas also revealed in high-fat diet-induced hyperlipidemia rats in this study. Notably,the enrichment ofStreptococcusin hyperlipidemia rats was weakened by the intervention of either Gblack or Gbrown (Gbrown＞Gblack). A previous study has reported that the abundance ofStreptococcusis positively correlated with body weight,serum TC and LDL-C in high-fat diet-induced hypercholesterolemia mice (Dinget al.2022). Therefore,the decreasedStreptococcusmay contribute to the hypolipidemic and anti-inflammatory role of Gblack/Gbrown against hyperlipidemia.Ruminococcusis another kind of gram-positive bacteria involved in the digestion of resistant starch (Zeet al.2012).Ruminococcuscan be elevated by many bioactive substances with therapeutic potential against hyperlipidemia,such as FermentedRosaroxburghiiTratt juice (Jiet al.2022),LactobacillusparacaseiFZU103 (Lvet al.2021),and microencapsulatedLactobacillusplantarum(Songet al.2017). In this study,Gblack/Gbrown also reversed the low abundance ofRuminococcusin hyperlipidemia rats(Gblack＞Gbrown). SinceRuminococcusis negatively correlated with serum and hepatic lipid profiles (Li Let al.2019),the recovery ofRuminococcusmay benefit the remission of hyperlipidemia.Allobaculumis also a probiotic contributing to the effects of various bioactive substances in inhibiting hyperlipidemia,such as ginsenoside Rb1 (Jiaet al.2021),probiotic-fermented black tartary buckwheat (Renet al.2021),purple yam (Li Tet al.2019),andAgaricusblazeiMurrill polysaccharides(Liet al.2020). Similarly,the interventions of Gblack/Gbrown also increased the abundance ofAllobaculumin hyperlipidemia rats. Besides,some differential microbial genera related to lipid metabolism were also determined in this study,such asLachnospiraandRuminococcusin the Gblack group,Phascolarctobacterium,Dorea,Turicibacter,andEscherichia-Shigellain the Gbrown group. These microbial genera may participate in the function of Gblack/Gbrown on hyperlipidemia through mediating blood lipids.

However,this study is still limited to animal experiments. The function of Gbrown/Gblack in hyperlipidemia and the underlying mechanisms involving gut microbiota need to be verified in human. In addition,the hypolipidemic function of specific gut microbiota targeted by Gbrown/Gblack also needs to be explored.The underlying molecular mechanism of Gbrown/Gblack in hyperlipidemia is also a direction for future research.

5.Conclusion

The dietary intervention of Gbrown/Gblack decreases blood lipids and weakens the inflammatory response in hyperlipidemia rats. The therapeutic potential of Gbrown/Gblack in hyperlipidemia is associated with the balance of gut microbiota,mainly evidenced by the decreased F/B ratio at the phylum level,and the decreasedStreptococcusand increasedRuminococcusandAllobaculumat the genus level. Some microbial genera may also contribute to hypolipidemic outcomes,such asLachnospira,Ruminococcus,Phascolarctobacterium,Dorea,Turicibacter,andEscherichia-Shigella. Our findings prove that Gbrown and Gblack are promising foods to alleviate hyperlipidemia by balancing gut microbiota. Since the beneficial effect of Gblack is relatively stronger than Gbrown,Gblack is more recommended to be consumed.

Acknowledgements

This study was funded by the National Key Research and Development Program of China (2021YFD2100902),the Outstanding Youth Project of Provincial Agricultural Science and Technology Innovation and Leaping Project,China (2022JCQN005),the Research Funding for Scientific Research Institutes in Heilongjiang Province,China (CZKYF2022-1-B021),and the National Rice Industry Technology System,China.

Declaration of competing interest

The authors declare that they have no conflict of interest.