Understanding the nutritional functions of thermally-processed whole grain highland barley in vitro and in vivo

Journal Pre-proofs Understanding the nutritional functions of thermally-processed whole grain highland barley in vitro and in vivo Bo Zheng, Shaowen Zhong, Yukuo Tang, Ling Chen PII: DOI: Reference:

S0308-8146(19)32122-3 https://doi.org/10.1016/j.foodchem.2019.125979 FOCH 125979

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Food Chemistry

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19 August 2019 26 November 2019 28 November 2019

Please cite this article as: Zheng, B., Zhong, S., Tang, Y., Chen, L., Understanding the nutritional functions of thermally-processed whole grain highland barley in vitro and in vivo, Food Chemistry (2019), doi: https://doi.org/ 10.1016/j.foodchem.2019.125979

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Understanding the nutritional functions of thermally-processed whole grain highland barley in vitro and in vivo Bo Zheng a, Shaowen Zhong a, Yukuo Tang a, Ling Chen a*

a

Ministry of Education Engineering Research Center of Starch & Protein Processing, Guangdong

Province Key Laboratory for Green Processing of Natural Products and Product Safety, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China

*Corresponding author. Email addresses: [email protected]; Tel: +86 20 8711 3252

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Abstract: The objective of this study was to investigate the nutritional functions of highland barley subjected to heat-moisture treatment (HMT) and dry heat treatment (DHT) in vitro and in vivo. In vitro test indicated HMT and DHT play part in the reduced glycemic potency of highland barley. Meanwhile, in vivo results showed that thermally-processed highland barley (THB) supplementation significantly decreased the body weight and serum glucose, improved oxidation resistance and altered the composition of gut microbiota. Bifidobacteria, Fusicatenibacter and Desulfovibrio were identified as types of bacteria that might related to the relatively higher content of dietary fiber in THB. The Spearman’s correlation analysis revealed that Fusicatenibacter and Desulfovibrio were positively correlated with T-AOC levels. In addition, the putative metagenomes implicated that THB might regulate the metabolic pathways of gut microbiota. Overall, our findings provide important information for the rational design of highland barley-based health-promoting foods with nutritional functions.

Keywords: Highland barley; thermal process; glycemic index; nutritional functions; intestinal microflora

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Abbreviations GI

glycemic index

HMT

heat-moisture treatment

DHT

dry heat treatment

MDA

Malondialdehyde

SOD

Superoxide dismutase

T-AOC

Total antioxidant capacity

OTUs

Operational Taxonomic Units

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1. Introduction In recent years, the demand for nutritious and healthy food is becoming more and more urgent with the increasing improvement of living standards and the continuous evolution of the disease spectrum(Barratt, Lebrilla, Shapiro, & Gordon, 2017). Therefore, it has become a research focus in the field of staple food industry and research to develop characteristic food with personalized nutritional functions (de Toro-Martín, Arsenault, Després, & Vohl, 2017). Highland barley cultivates in the Qinghai Tibet Plateau, as the smallest staple food cereals and the largest coarse cereals in China, has attracted much attention because of its unique nutritional value(F. Wang, Yu, Zhang, Zhang, & Fan, 2015). Therefore, the design of highland barley food with good quality and healthy nutrition has important application significance and broad market prospects. Whole grains are definite as whole, ground or compressed caryopsis, including starch endosperm, germ and bran (Klerks, Bernal, Roman, Bodenstab, Gil, & Sanchez-Siles, 2019). Due to its high dietary fiber, low fat, low energy, simple processing and low cost, whole grains food becomes more and more popular in developed countries(Călinoiu & Vodnar, 2018; Karl, Meydani, Barnett, Vanegas, Goldin, Kane, et al., 2017). Increasing studies supported that whole-grain consumption was associated with a reduced risk of cardiovascular disease(Aune, Keum, Giovannucci, Fadnes, Boffetta, Greenwood, et al., 2016; Zong, Gao, Hu, & Sun, 2016), high blood pressure(Ndanuko, Tapsell, Charlton, Neale, & Batterham, 2016) and colonic inflammation(Vetrani, Costabile, Luongo, Naviglio, Rivellese, Riccardi, et al., 2016). A few nutritional studies showed whole grain highland barley could also reduce the risks of chronic health problems such as hypertension(Lin, Guo, Gong, Lu, Lu, Wang, et al., 2018), obesity(Y. Shen, Hu, Zhang, & Jiang, 4

2018; Xia, Li, Song, Zheng, & Kan, 2018) et al. In addition, highland barley beta-glucan appears to have a favorable impact on hyperglycemia and glucose homeostasis(Lin, et al., 2018; R. Liu, Zhao, Guo, Liu, Yu, Wang, et al., 2019). However, whole grains also have relatively high carbohydrate content, of which starch accounts for about 90%, which is likely to cause hyperglycemia in human body(K. Liu, Zhang, Chen, Li, & Zheng, 2019; R.-L. Shen, Zhang, & Dong, 2016). Therefore, it is of great significance to regulate the nutritional components of highland barley by food processing methods, thereby reducing the glycemic index (GI) of highland barley. Heat-moisture treatment (HMT) and dry heat treatment (DHT) have already played an important role in the green processing of food due to they only involve the use of thermal energy and moisture, which is efficient and safe(Gunaratne, 2018; Marston, Khouryieh, & Aramouni, 2016). Previous observations have collectively suggested that HMT and DHT can effectively change the multi-structure of starch. It was reported that HMT could simultaneously disorder and reassemble the starch molecules across multi-scale lengths and convert some fractions of rapidly-digestible starch into slowly-digestible starch and resistant starch while DHT could easily destroy starch granules, and some of the amylopectin chains could easily degrade into amylose, which lead to the formation of starch-lipid complex. These all contributed to reducing the digestibility of starch and thus improved the nutritional functions of starch (de Carvalho Teixeira, Queiroz, Rocha, Amorim, Soares, Monteiro, et al., 2016; K. Liu, Zhang, Chen, Li, & Zheng, 2019). However, limited work has been undertaken for evaluating the effect of HMT and DHT on the nutritional functions of whole grain highland barley in terms of glucose metabolism, oxidative stress and intestinal microflora. Therefore, the effects of thermal process on the regulation of glycemic index of whole grain highland barley was firstly 5

investigated. Furthermore, physiological and biochemical metabolic indexes such as blood glucose and oxidative stress and intestinal microbial diversity in rats were also studied, which would contributing to establish a relation between thermal process and nutritional functions in vitro and in vivo. In this study, 6 rats were exposed to chow diet, 6 rats were exposed to the high-fat diet and 18 high-fat-diet-fed rats were supplemented with highland barley (the HB group), HMT-treated highland barley (the MHB group) and DHT-treated highland barley (the DHB group) to understand the alteration of nutritional functions by thermal process. To the best of our knowledge, it is the first study comprehensively comparing these detailed nutritional function parameters between native highland barley and thermally-processed highland barley.

2. Material and methods 2.1 Sample preparation and Composition analysis Native highland barley powder was purchased from Qinghai Gaojian biotechnology co. Ltd (Xining, China). Based on our previous studies, two thermal processing methods were selected and the processing parameters were followed by the reference we reported previously (K. Liu, Zhang, Chen, Li, & Zheng, 2019). For the HMT method, highland barley was firstly equilibrated at 4 °C for 24 h until the moisture content reached 25%. Then, they were moved into a 1000 mL screwed stainless steel reactor with continuous rotation and heated at 110 °C for 2 h. During HMT, the highland barley powder were stirred by the paddle when the reactor was continuously rotated. As for the DHT of highland barley powder, the highland barley powder was firstly placed in an open 1000 6

mL screwed stainless steel reactor with continuous rotation and heated at 160 °C for 3 h. During DHT, the highland barley powder was stirred by the paddle when the reactor was continuously rotated. Finally, the DHT samples were dried at 35 °C for 12 h and ground. The moisture contents of HB, MHB and DHB were analyzed in accordance with reference to AOAC methods 934.01/4.1.03 (Dubat, 2010). Available Starch content was determined with thermostable α-amylase according to Åman et al (Åman, Westerlund, & Theander, 1994). Dietary fibre was analyzed according to the AOAC method 994.13 (Theander, Aman, Westerlund, Andersson, & Pettersson, 1995). Total carbohydrate content were determined according to BeMiller et al (BeMiller, 2017). 2.2 Animals Thirty healthy SPF male adult Sprague-Dawley (SD) rats (non-obese) weighing 180±20 g were purchased from the animal house, Guangdong Medical Laboratory Animal Center (China). These rats were fed at a clean-grade facility in a well-maintained and hygienic environment (temperature 27±1 °C; 60±10% humidity; and a 12h/12h light/dark cycle). The rats were given access to water and diet. After one week’s adaptive feeding with a basal diet, no difference in the body weight was observed. These rats were divided into five groups (six rats for each group) randomly, including two control groups and three treatment groups. In two control group, one group of rats were fed with a standard chow diet (NC group), the other group rats were fed with a high-fat diet (HFD group). In three treatment groups, the first group of rats were fed with a high-fat diet supplemented with normal highland barley powder, and the second group were fed with a high-fat diet supplemented with HMT-modified highland barley powder (MHB group), and the third group were supplemented with DHT-modified highland barley powder (DHB group). For each group, the intervention was 7

implemented for 8 weeks. After 8 weeks of intervention, all rats were sacrificed by anaesthesia (1 mg/kg pentobarbital sodium) and then dissected. Their body weights were monitored weekly. Feces were collected on the day of necropsy. All animal procedures were approved by the Ethical Committee for the Experimental Use of Animals, Guangdong Medical Laboratory Animal Center (Approval No: SCXK 2013-0002). 2.2 Experimental diets The experimental high-fat diet was prepared by Research Diets (Choi, Gwon, Ahn, Jung, & Ha, 2013). The composition of the diet is as follows: carbohydrate source 54.5%, casein 20%, lard oil 10%, corn oil 5%, cholesterol 0.5%, cellulose 5%, mineral mix (based on AIN76) 3.5%, vitamins (based on AIN76) 1%, methionine 0.3%, and choline bitartrate 0.2%. The three HB groups of rats were fed with the experimental high-fat diet where 44% carbohydrate source was replaced by HB, MHB and DHB respectively. 2.3 In Vitro GI analysis The GI of native highland barley and thermally-processed highland barley were analyzed with a modified procedure established by Goni et al. (Goñi, Garcia-Alonso, & Saura-Calixto, 1997). The kinetics of starch hydrolysis was calculated using the equation: [C = C∞ (1 − e−kt)], where C, t, C∞, and k denote the hydrolysis degree at each time, digestion time, the maximum hydrolysis extent, and the kinetic constant, respectively. Then, the hydrolysis index (HI) was calculated by dividing the area under the hydrolysis curve of each sample by the correspond ding area of a reference sample (fresh white bread). The GI value was calculated using the equation: pGI = 39.71 + 0.549HI. 8

2.4 Blood and tissue analysis After treatment, the rats were weighted and then fasted overnight before the blood glucose was recorded. Blood samples were taken from the neck of the rats for the determination of physiological and biochemical indexes. The liver tissue were quickly removed after sacrifice and then weighed. Body composition was determined using rodent MRI (EchoMRI 700). Three main indexes for characterizing oxidative stress including Malondialdehyde (MDA), Superoxide dismutase (SOD) and Total antioxidant capacity (T-AOC) were measured spectrophotometrically using their corresponding diagnostic reagent kits (Jian Cheng Biotechnology Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. 2.5 Microbial community analysis 2.5.1

DNA extraction, and 16S rRNA gene sequencing

Approximately 80 mg feces samples were collected in a sterile container and immediately stored at −80 °C until further processing. Total genome DNA from samples was extracted using the hexadecyltrimethylammonium bromide/sodium dodecyl sulfate (CTAB/SDS) method according to the reference we reported previously (Zheng, Wang, Shang, Xie, Li, Chen, et al., 2018). The DNA concentration and purity were monitored on 1% agarose gels. 16S rRNA genes of distinct regions in V4 were amplified using the specific primers of 515F‐806R, with barcode. All PCR reactions were performed with Phusion High Fidelity PCR Master Mix (Biolabs, New England). Samples with bright main bands between 400-450 bp were chosen for the following library preparation and

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sequencing. The library was sequenced on an IlluminaHiSeq2500 platform, generating 250 bp paired-end reads. 2.5.2

Intestinal microbiota analysis

After the removal of the low-quality sequences, pyrosequencing errors, and chimera, all the sequencing reads were denoised. Sequences of >97% similarity levels were assigned to the same Operational Taxonomic Units (OTUs). Representative sequences for each OTU were screened for further annotation. The rare fraction curves and alpha diversity indices were performed using MOTHUR software, and the beta diversity (among samples) was analyzed using principal component analysis (PCA). The function of intestinal microbiota was performed by online phylogenetic investigation of communities by reconstruction of an unobserved states program (PICRUSt, http://picrust.github.io/picrust/). 2.6 Statistical analysis For in vitro studies, all analyses were conducted in triplicate and statistical analyses were performed using the SPSS 22.0 software. Results were reported as mean ± s.e.m. For in vivo studies, two-tailed unpaired Student’s t-test was applied for comparison. ANOVA was used on comparisons that involved multiple groups. Here, different lowercase letters above the same column indicate a significant difference (P≤ 0.05). Nonparametric Spearman’s correlation coefficient was calculated to identify the correlations between metabolic biomarkers and bacterial abundances.

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3. Results and discussions 3.1 Body weight, liver and adipose tissue weights Effect of the supplementation of highland barley before and after thermal processing on body weight gain, liver coefficient and total fat coefficient was presented in Fig. 1. The initial body weights (week 1) between these five groups were not significantly different (P > 0.05), showing the rationality of the grouping method. After two weeks’ treatment, there were apparent increasing trends of the body weight (P > 0.05), and significant differences between the normal control group (NC) and other four groups, indicating weight growth in the rat was more rapid on high-fat diets (HFD) than on normal fat diets. At the end of the study, the three treatment groups exhibited a decrease to values that were significantly different from the HFD-fed rats (Fig. 1B). Additionally, we did not observe any body weight differences in the MHB, DHB and NC group rats (p<0.05), but the body weight gain in the DHB group rats were significantly lower than that in the HB group rats (p<0.05). The liver coefficient in the HFD group rats was significantly higher than that in the other four group rats at the end of the study (Fig. 1C). Three treatment groups significantly reduced this increase (p<0.05). Meanwhile, a significant difference was found in DHT group rats when compared to HB group. However, there is no significant difference between HB group and MHB group. The total fat coefficient in the HFD group rats was also significantly higher than that in the other four group rats at the end of the study (Fig. 1D). Interestingly, the fat coefficient in HB and MHB group rats were significantly decreased compared with the NC group (p<0.05).

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3.2 In vitro GI, Blood glucose, insulin and glucagon levels To evaluate whether the modification of thermal process in highland barley reduces their glycemic index, we firstly measured free glucose during in vitro digestion. Results showed that the in vitro GI value of native highland barley was 82.2 while the MHB and DHB were 70.3 and 61.0, respectively (p<0.05), indicating the GI value of highland barley decreased after thermal process (Table 1). Meanwhile, we measured the glucose level, insulin level and glucagon levels respectively, the results were also shown in Fig.2. It can be seen that the blood glucose, insulin level in the HFD group were significantly higher than those in NC group (p<0.05). When compared with HFD group, there showed a significant decrease in blood glucose, insulin level for the HB group. Interestingly, the administration of DHB significantly reduced blood glucose, insulin level compared with HB group (p<0.05). The glucagon level between these five groups showed no significant differences. The result indicated that DHB has a strong impact on the regulation of the blood glucose level. 3.3 Oxidative stress indices In this study, the changes in MDA, SOD and T-AOC levels in the serum of rats were measured to understand the influence of native HB and thermally-processed HB on these oxidative stress indices. The results (Figure 2) showed a statistically significant decrease in serum levels of MDA and SOD, as well as an increase in T-AOC level for the HB group compared with for the HFD group (P < 0.05). Besides, the administration of MHB and DHB significantly reduced MDA and increased T-AOC compared with HB group. Overall, the serum oxidative stress status of rats fed with DHB was more effectively improved than that of rats in the HB and MHB group.

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3.4 Intestinal microflora To further explore the effects of thermal-processed highland barley on gut microbiota composition, the 16S rRNA pyrosequencing based on V3-V4 region was performed. A total 31584 raw sequences were generated, and low quality sequences were removed. The remaining clean tags were clustered into 760 Operational Taxonomic Units (OTUs) based on 97% similarity. Fig. 4 shows the gut microbiota composition in rats after the 8-week intervention for the five groups. The observed species indices in the NC group was significantly higher than those in the other four groups (P < 0.05), which indicates a higher intestinal flora abundance of the NC group (shown in Fig. 3a). Meanwhile, the observed species indices in the HF group was significantly higher than those in three highland barley groups (P < 0.05), and there was no significant difference among three highland barley groups. In addition, ANOSIM analysis with an unweighted UniFrac revealed that there were significant differences among these five groups (P = 0.001). Then, we analyzed the statistical results of species percentage among different species classification levels. At the phylum level (shown in Fig. 3d), bacteria among these five groups were mainly composed of Bacteroidete, Firmicutes, and Proteobacteria. Compared with the NC group, the increased level of the phylum Bacteroidetes and decreased level of the phylum Firmicutes were found in HFD rats (P <0.05). At the same time, compared with HF group, the phylum Bacteroidetes level in MHB1 and DHB1 group increased while the phylum Firmicutes level dereased (P <0.05). Next, the relative abundances of the intestinal bacteria at genus level among these five groups were analyzed (shown in Fig. 3e). Compared with the NC group, the HF group was characterized by a higher amount of Bacteroides, Parasutterella, Parabacteroides, Barnesiella, Clostridium XI, 13

Butyricimonas and lower amount of Lactobacillus, Clostridium XlVa, Prevotella, Flavonifractor, Bifidobacterium and Alloprevotella, indicating a disruption of gut composition in the rats fed on high-fat diet. Besides, highland barley supplementation significantly decreased the abundance of the HFD-enriched genera Barnesiella, Clostridium XI and Blautia and significantly increased the abundance of Lactobacillus, Prevotella, Bifidobacterium, which had the same trend of the NC group. Meanwhile, as shown in Fig.3f, we focused on the enteric bacteria which were the main producers of short chain fatty acids including Bacteroides, Bifidobacterium, Prevotella, Ruminococcus, Blautia, Clostridium , Veillonella and Coprococcus. The results showed that all these bacteria increased by highland barley treatment. Interestingly, DHB can help the growth and reproduction of these intestinal bacteria more than native highland barley and MHB. Metagenomics analysis using the linear discriminant analysis effect size (LEfSe) method was performed to compare the microbial community composition and its abundance diversity in feces between four high-fat diet groups of rats from the genus levels, and to further select the dominant flora of microbial communities. It can be seen in Fig. 4 that there were large differences in each level of microbial communities between the four groups. According to the LDA score, the dominant bacteria (from genus level) in the HF group were Barnesiella, Butyricimonas, Parasutterella, Roseburia, and Gemella while Veillonella occupied important places in the HB group, Bifidobacterium occupied important places in the MHB group and Bifidobacterium, Fusicatenibacter and Desulfovibrio were the most important bacteria in the DHB group. We also assessed the functional diversity of the different putative metagenomes using PICRUSt software (Langille et al., 2013), which allows the prediction of metabolic pathways from the 16S 14

rRNA reads. The results showed that the pathways displayed a difference in the mean proportion between the high-fat diet group and three highland barley treated groups (Fig. 5a). The pathways including xenobiotics biodegration, transcription were over-represented in the high fat diet group, whereas the translation, replication and repair were over-represented in the HB group, metabolic disease was over-represented in the MHB group, metabolism of amino acids was over-represented in the DHB group. These results indicate that highland barley associated with thermal process may influence the functional diversity, especially predicted by putative metagenomes. The Spearman’s correlation coefficient was used to investigate the relationship among bacterial abundance, blood glucose and oxidative stress indexes, and the result was shown in Fig.5b. Among the bacteria at genus level, Fusicatenibacter, Desulfovibrio were negatively correlated with blood glucose and MDA index while positively correlated with T-AOC. In addition, Barnesiella, Gemella, Turicibacter, Allobaculum, Eubacterium and Ruminococcus2 were nagetively correlated with blood glucose and MDA indexes while positively correlated with T-AOC.

4. Discussion Highland barley, as the most important staple food in Qinghai and Tibet, has unique nutrient content and high nutritional value compared with other cereal crops and it occupies an increasingly important position in Chinese dietary structure. However, due to its high starch content and crude fiber content, highland barley also has disadvantages such as high digestibility, high blood sugar response and rough taste, which is inconsistent with the concept of modern nutrition and health. Therefore, controlling the digestive properties of highland barley through appropriate processing technologies not only improves the nutritional deficiency of highland barley, but also gives it higher 15

application value. In this study, for the first time, we demonstrated that thermal-processed whole grain highland barley improved the physiological and biochemical indices, which may be associated with modulating gut microbiota with obese rats. Nutrient composition analysis showed that highland barley presented a relatively similar content of carbohydrate and a lower content of dietary fibre compared with other whole grains, such as hard wheat, millet, rye and Sorghum (Mengesha, 1966). The higher content of starch is consistent with that of highland barley reported by Jane (Yangcheng, Gong, Zhang, & Jane, 2016). However, after thermal process, the carbohydrate, fibre, and starch contents were changed significantly (Table 1). This mainly due to the change of the aggregate structure and chain structure of the highland barley starch under heat energy and the migration of water molecules. Meanwhile, the starch molecules form complexes with lipids, proteins and some small molecule compounds such as polyphenol combining effects of aggregation, entanglement, static electricity and hydrogen bonding, which resulted in the increase of resistant starch (H. Wang, Liu, Chen, Li, Wang, & Xie, 2018). However, the carbohydrate and starch content in DHB were significantly lower than that of HMB, this mainly due to the high heat energy under DHT could destroy starch granules more easily, and part of starch was broken down into dextrin or reducing sugar (B. Zhang, Zhao, Li, Zhang, Li, Xie, et al., 2014). Previous studies showed that resistant starch can’t access to human digestive enzymes and are fermented in the colon producing short chain fatty acids, which is recognized as a type of dietary fiber. Therefore, this is one of the reasons for the increase in dietary fiber content. In addition, we also found that the protein content of highland barley increased after thermal process while fat ccontent decreased. This may be due to the fact that fat breaks down into fatty acids and 16

monoglycerides at high temperatures and forms complexes with starch and protein. GI value is an indicator of glucose elevation after eating, which has a guiding role in the dietary reference of diabetic patients. Previous studies showed that GI is mainly influenced by the hydrolization of carbohydrate during digestion, and glycemic response rose with an increasing amount of available rapidly digestible carbohydrate (Lehmann & Robin, 2007). Our in vitro results showed that the GI value decreased significantly after thermal process, which was consistent with the changes of carbohydrate, starch and dietary fibre content. Surprisingly, the DHT treated highland barley was classified as moderate GI food having GI value of 61. Overall, thermal process could reduce glycemic potency of highland barley. The effect of highland barley after thermal process on physiological and biochemical indices was investigated in vivo through 8-week administration on HFD rats. After 8 weeks of high-fat diet intervention, compared with the normal control group, the body weight of the HFD group increased significantly, and serum glucose level, oxidative stress index, liver coefficient and fat coefficient were significantly inferior (p <0.05), indicating that the high-fat diet produced a certain degree of damage to these physiological and biochemical indicators to the test rats. Consistent with our results, it has previously been reported that high-calories diet leads to hyperphagia and obesity together with obesity-related phenotypes such as insulin resistance, dyslipidemia, hepatic metabolic disorder and so on(Park, Pichiah, Yu, Oh, Daily, & Cha, 2012; Wan & Kwan, 2018). Interestingly, these physiological and biochemical indexes of mice fed with highland barley before and after thermal process were improved obviously. A growing number of studies have demonstrated that highland barley and highland barley-enriched products can significantly decrease the body weight(Y. Shen, 17

Zhang, Cheng, Wang, Qian, & Qi, 2016; Xia, Li, Song, Zheng, & Kan, 2018). Our results also provided supportive evidence for the function on controlling the rising trend of body weight and fat coefficient during high-fat diet especially feeding with DHB. Although, the mechanism of helping controlling weight has not been fully understood, at least parts of the contributions could be attributed to the property of dietary fiber. At the level of blood glucose changes, we found that highland barley could stabilize the blood glucose level and reduce the speed of change into a hyperglycemia condition. The result confirmed that DHB has a strong impact on the regulation of the blood glucose level. Nonetheless, the DHB group displayed a significantly lower serum insulin level (P < 0.05), indicating a lower insulin resistance of the DHB group than those of other two groups. Thus, the DHB intervention improved the biological effect of insulin as well as the sensitivity of the insulin receptor tissue to insulin. In addition, high-fat diet was reported could increase free radical metabolism and lipid peroxidation, which finally resulted in the metabolic diseases. As a biomarker of oxidative stress, Malondialdehyde (MDA) has been identified to reflect the presence of oxidative radicals and is responsible for the observed cellular damage. In our study, the increased MDA level was observed in HF group, suggesting the presence of oxidative radicals in rats under high-fat diet. After highland barley treatment, the MDA level was reduced significantly, which indicated highland barley could reduce the degree of lipid peroxidation in rats and improve the degree of damage to cells. Meanwhile, the protective effect is related mostly to the free-radical scavenging ability of antioxidant enzymes such as SOD and T-AOC. Zhang et al. reported that highland barley was found to have strong superoxide radical, hydroxyl radical and scavenging activity due to its high level of polyphenols. The current study also demonstrated that highland barley could elevate SOD activities 18

and T-AOC, indicative of protective mechanism to combat ROS damage. Compared with native highland barley, DHB could improve the lipid oxidation of rats remarkably. Previous studies have proven that diet drove gut microbiota composition and metabolism, marking microbes a relationship between diet and different physiological index through their capacity to generate microbial metabolites due to the dietary intake (Bultman, 2017). Our study showed that high-fat diet did significantly change the relative abundance of gut microbiota, including Bacteroides, Barnesiella, Butyricimonas, Parasutterella, Prevotella and Flavonifractor, which is similar to those reported by Zhang et al (X. Zhang, Zhao, Xu, Xue, Zhang, Pang, et al., 2015). We also noted that the abundance of Lactobacillus and Bifidobacterium were decreased remarkably in the HF group, reflecting high-fat diet inhibited the growth and reproduction of probiotics in the intestine of the rats. Short-chain fatty acids (SCFAs) such as acetate, propionate and butyrate are the main products of intestinal bacterial fermentation of dietary fiber and complex carbohydrates, playing an important role in the intestinal energy supply, maintaining intestinal mucosal barrier, regulating intestinal hypersensitivity and intestinal motility, immune regulation and anti-tumor effects (Koh, De Vadder, Kovatcheva-Datchary, & Bäckhed, 2016). Koh et al. reported that dietary fibers could escape digestion by host enzymes in the upper gut, and were metabolized by microbiota in the colon (Koh, De Vadder, Kovatcheva-Datchary, & Bäckhed, 2016). Cummings et al. reported that the major products of dietary fibers from the microbial fermentative activity in the gut were SCFAs (Shortt, Hasselwander, Meynier, Nauta, Fernández, Putz, et al., 2018). In our study, based on the significant difference in dietary fiber content of highland barley before and after thermal processing, we focused on the abundance of SCFAs-producing bacterial groups and found a greater abundance of 19

Bacteroides, Veillonella, Bifidobacterium in rats consuming highland barley when compared with HF group (shown in fig.4f). Interestingly, DHB groups also presented a higher relative abundance of Prevotella, Blautia, Coprococcus and Ruminococcus. Cosistent with our results, a positive correlation between SCFAs-producing microbiota and dietary fiber intake is commonly reported and seen in the current study as well. Besides, we selected the dominant microbial communities of each group by using LEfSe. Compared with HF group, we found Veillonella occupied the most important place in HB group while Bifidobacterium in MHB group and Bifidobacterium, Fusicatenibacter and Desulfovibrio in DHB group. Interestingly, these were all SCFAs-producing bacteria, in which Veillonella mainly produced acetate, Bifidobacterium, Fusicatenibacter and Desulfovibrio.mainly produced propionate and butyrate. In addition, it has been suggested that Veillonella may perform a protective role and aid in human immune system development and is negatively correlated with autism, asthma and bronchiolitis (Poppleton, Duchateau, Hourdel, Matondo, Flechsler, Klingl, et al., 2017). Bifidobacterium were found one of the probiotics associated with the glucose metabolism, and higher amount of bifidobacteria might contribute to normalizing the blood glucose levels, which is consistent with the glucose level in this paper (Zheng, et al., 2018). What’s more, Bifidobacterium were demonstrated in enhancing antitumor immunity in vivo (Sivan, Corrales, Hubert, Williams, Aquino-Michaels, Earley, et al., 2015). In agreement with the literatures and the 16S rRNA sequencing results, the Spearman’s correction result further confirmed that the genus Fusicatenibacter and Desulfovibrio were positively correlated T-AOC, negatively correlated with blood glucose and MDA while Barnesiella and Eubacterium were negatively correlated with T-AOC. 20

However, the related biochemical pathways and the connection between the function of Fusicatenibacter and Desulfovibrio and oxidation stress should be further explored. The shifts in microbiome structure of rats were consistent with imputed functional predictions. Many of the significantly altered imputed functions in DHB groups were related to metabolic diseases. Based on our findings, high-fat diet could cause a large proliferation of harmful bacteria while native highland barley and thermal-processed highland barley were favorable for the proliferation of probiotics producing SCFAs. Especially, DHB could significantly improve the oxidation stress by restoring the disorders of the intestinal microbiota. In conclusion, thermally-processed highland barley could decrease the blood glucose and reversed the insulin resistance and oxidation stress of rats under high-fat diet. Remarkably, the administration of thermally-processed highland barley could contribute to the proliferation and growth of probiotics, especially short-chain fatty acid producing bacteria. Moreover, many OTUs affiliated to the Fusicatenibacter and Desulfovibrio Genus were positively correlated to T-AOC level and thus inferred in metabolic pathways. These findings open an exciting avenue for providing potentially effective approaches for food interventions to regulate the metabolic diseases.

Potential conflict of interest statement The authors declare no competing financial interest.

Acknowledgements This research has been financially supported by the National Natural Science Foundation of China (NSFC)-Guangdong Joint Foundation Key Project (U1501214), YangFan Innovative and Entrepreneurial Research Team Project (2014YT02S029). 21

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Figure captions Figure 1. Changes in a) body weight, b) weight gains, c) liver coefficient and d) fat coefficient of rats during the 8-week intervention for the five groups. (NC: chow diet group; HF: HFD group; HB: highland barley group; MHB: MHT-treated highland barley group; DHB: DHT-treated highland barley group). Figure 2. Changes in a) blood glucose, b) serum insulin, c) serum glucagon, d) MDA, e) SOD and f) T-AOC level of rats during the 8-week intervention for the five groups. (NC: chow diet group; HF: HFD group; HB: highland barley group; MHB: MHT-treated highland barley group; DHB: DHT-treated highland barley group). Figure 3. Gut microbiota composition in mice after the 8-week intervention for the five groups. a): observed species; b: β diversity of intestinal microflora; c) analysis of weighted UniFrac distance matrix) (adjusted P value < 0.05); d-e: The relative abundance of bacterial community at the taxa level (d: Phylum; e: Genus) for the five groups; f) the relative abundance of short chain fatty acids-producing bacteria. (NC: chow diet group; HF: HFD group; HB: highland barley group; MHB: MHT-treated highland barley group; DHB: DHT-treated highland barley group). Figure 4. Significant analysis of intestinal microflora in rats for the a) five and b) four groups (NC: chow diet group; HF: HFD group; HB: highland barley group; MHB: MHT-treated highland barley group; DHB: DHT-treated highland barley group). Figure 5. a) Function in KEGG module prediction using 16S data with PICRUSt between the four groups; b) Correlations between specific genera, blood glucose and oxidative stress indices. (HF:

28

HFD group; HB: highland barley group; MHB: MHT-treated highland barley group; DHB: DHT-treated highland barley group).

29

NC NC

500

300

c

body weight gain (g)

body weight (g)

HFD HB

400

MHB DHB

300

a

HFD b

ab

a

200

HB MHB DHB

100

200

0 WEEK1

WEEK2

WEEK4

WEEK6

NC HFD HB MHB DHB

WEEK8

Time

a

d

4

HFD c

c

b

HB

HB MHB

a

DHB

2 1 0 NC

c

HFD

HB

HFD

6

fat coefficient (% )

liver coefficient (% )

NC

NC

5

3

Group

b

4

b

c a

b

a

MHB

DHB

2

0

MHB DHB

NC

Group

d

Figure 1

30

HFD

HB

Group

MHB DHB

80

HFD

8.0 6.0

NC

NC

c

HB b

b

MHB

b a

4.0

60

Insulin (uIU/mL)

Blood glucose (mmol/L)

10.0

DHB

2.0

HB

b

b

a

MHB

a

DHB

40 20

0.0 NC

HFD

HB

MHB

0

DHB

NC

Group

a

6

a

a

a

MHB DHB

2000

DHB

4

HFD

c

HB

MDA (nmol/mL)

a

MHB

NC

d

HFD

3000

HB

b

4000

a

HFD

Group

NC

glucagon (pg/mL)

HFD

c

b

a

HB

a

MHB DHB

2

1000

0

0 NC

HFD

HB

MHB

NC

DHB

Group

c

NC

a

6

MHB DHB

200

NC

b

b

4

b

HFD

HB

MHB

NC

DHB

Group

f

Figure 2

31

HFD

HB DHB

a

0 NC

HFD MHB

2

0

e

DHB

c

HB

T-AOC (U/mL)

SOD (U/mL)

400

b

b

MHB

8

HFD c

HB

Group

d

600

b

HFD

HB

Group

MHB

DHB

600

observed species

NC HFD HB

400

MHB DHB 200

0 NC

a

HFD

HB

Group

c

b

d

MHB

e

32

DHB

Bacteroides

Prevotella

8 6 4 2

10

Relative abundance

Relative abundance

Relative abundance

10

60 40 20 0

0 NC

f

HF

HB

MHB

HF

HB

6 4 2

2 1

3 2 1 0

NC

HF

HB

NC

MHB DHB

Bifidobacterium

Ruminococcus 6

Relative abundance

4 3 2 1 0

4

2

0 NC

HF

HB

HB MHB DHB

4

3

MHB DHB

HF

Coprococcus

0

0

Relative abundance

2

NC

Relative abundance

Relative abundance

8

HB

4

MHB DHB

4

HF

6

Blautia

10

NC

8

0

NC

DHB

Veillonella

Relative abundance

Clostridium

80

MHB DHB

NC

Figure 3

33

HF

HB

MHB DHB

HF

HB

MHB DHB

a

b

Figure 4

34

a

b

Figure 5

35

Table 1 characteristics and in vitro digestibility of highland barley powder before and after HMT and DHT (mean ± SEM). Native highland

HMT-highland

DHT-highland

barley

barley

barley

Carbohydrate (g/100g)

70.70±1.21b

71.10±2.62b

64.80±1.68a

Dietary fibre (g/100g)

3.77±0.06a

10.90±0.13c

7.32±0.66b

Moisture content (mg/100g)

12.80±1.03c

8.52±0.68b

6.43±0.38a

Starch (mg/100g)

61.32±0.90b

63.05±1.20b

58.90±1.30a

Protein (g/100g)

10.2±0.13a

11.5±0.20b

12.1±0.10b

Fat (g/100g)

1.92±0.03b

1.88±0.04b

1.79±0.03a

GI value

82.20±2.00c

70.30±1.60b

61.00±3.20a

Different lowercase letters above the same column indicate a significant difference (P ≤ 0.05).

36

1. HMT and DHT reduced glycemic potency of highland barley. 2. DHB had better performance in improving blood glucose metabolism and oxidation stress. 3. MHB and DHB contributed to the proliferation and growth of SCFAs producing bacteria. 4. Fusicatenibacter and Desulfovibrio were typical bacteria positively correlated with T-AOC levels. 5. HMT and DHT-modified highland barley helped modulate nutritional function pathways.

37