Rice, a staple food for over half the world's population, is rapidly digested, leading to high blood glucose levels and increased risk of obesity, type II diabetes, and cardiovascular disease. Reducing the rate of starch digestion in rice is crucial for improving public health. This can be achieved through in-plant breeding of high-resistant starch (RS) rice varieties or by processing strategies like lipid complexation. Complexation involves incorporating lipids into starch, creating a stable structure resistant to amylase digestion. This undigested starch-lipid complex reaches the colon, where it serves as a substrate for gut microbiota fermentation, increasing the production of short-chain fatty acids (SCFAs) like acetate, propionate, and butyrate, which have various health benefits. Previous studies demonstrated the impact of starch-lipid complexes on digestibility and SCFA production, but often lacked a realistic in vitro digestion step before fermentation. This study aimed to comprehensively investigate the in vitro physiological behavior of high-lipid rice (RS4), low-lipid rice (GZ93), and their palmitic acid-complexed counterparts to understand their interaction with gut microbiota.

Existing literature supports the concept of reducing starch digestibility to mitigate health risks associated with high glycemic index foods. In-plant breeding techniques like chemical and radiation mutagenesis, and genetic engineering, have been employed to create high-RS rice varieties. Alternatively, complexation with lipids, proteins, or polyphenols has shown promise in slowing down starch digestion. Lipids, particularly fatty acids, can embed into the starch structure, forming undigestible domains. Several studies have shown that exogenous lipid complexation decreases starch digestibility. The undigested starch-lipid complex (RS type 5, RSV) in the colon serves as a substrate for gut microbiota, influencing its composition and the production of beneficial SCFAs. However, previous research often examined starch-lipid complexes without considering the preceding digestion process in the small intestine, limiting the realism of the fermentation studies. This gap highlighted the need for a comprehensive investigation that integrates in vitro digestion and colonic fermentation.

Two rice varieties, RS4 (high amylose and lipid content) and GZ93 (low amylose and lipid content), were used. Rice flour and isolated starch were prepared from both varieties, and starch-palmitic acid complexes were created by mixing starch and palmitic acid and heating them to 100°C for 30 min. The composition (starch, protein, and lipid content) and thermal properties (differential scanning calorimetry, DSC) of all samples were measured. In vitro digestion was performed using the INFOGEST protocol, simulating oral, gastric, and intestinal phases. The percentage of digested starch was calculated, and the undigested fractions were used for in vitro colonic fermentation using fecal batch cultures from two healthy donors. Short-chain fatty acid (SCFA) concentrations (acetate, propionate, butyrate) were measured at different time points (4h, 24h, 48h) using gas chromatography. 16S rRNA gene sequencing was employed to analyze the gut microbiota composition and diversity after 48h fermentation. PICRUSt was used to predict microbial gene functions. MIMOSA2 analysis was performed to identify associations between SCFA concentrations and gut microbiota.

RS4 samples showed significantly higher lipid content in their flour, starch, and complexes than GZ93 samples. In vitro digestion revealed significantly lower digestibility in RS4 samples (RPA and RN) than GZ93 samples (GPA and GN), indicating increased resistant starch content (RSV) in the RS4 samples due to the higher endogenous and exogenous starch-lipid complexes. Colonic fermentation of the INFOGEST undigested fractions demonstrated significantly higher concentrations of acetate and propionate in the starch-lipid complexes (RPA and GPA) compared to the respective native flour and starch samples. Butyrate production did not show a significant difference. The starch-lipid complexes resulted in distinct microbial composition shifts, characterized by higher abundances of Bacteroidetes and Proteobacteria, and lower abundances of Firmicutes. Analysis revealed increased gene expression levels related to fructose, mannose, and pyruvate metabolism in starch-lipid complex samples. MIMOSA2 analysis confirmed a strong association between SCFA production and specific gut microbiota taxa. Enterobacter kobei was identified as a major contributor to acetate variation, while several taxa were implicated in propionate and butyrate variations. The results show that lipid complexation decreased the digestibility of rice starch in vitro, resulting in increased SCFA production during in vitro colonic fermentation.

The findings demonstrate the effectiveness of lipid complexation in reducing rice starch digestibility and promoting SCFA production. This effect is likely mediated by selective modulation of the gut microbiota, leading to alterations in metabolic pathways involved in SCFA biosynthesis. The observed increase in acetate and propionate, and the involvement of specific microbiota, highlight the potential of this approach to improve gut health. The integration of in vitro digestion with fermentation provided a more realistic assessment compared to previous studies that only considered the fermentation of un-digested complexes. The differences between RS4 and GZ93 varieties, both in the natural state and after palmitic acid complexation, illustrate the interplay between genetic predisposition and processing techniques in influencing the gut microbiota. Future research could investigate the long-term effects of consuming lipid-complexed rice and its impact on host health markers. In vivo studies are needed to confirm and extend these in vitro findings.

This study provides strong evidence that lipid complexation in rice starch decreases digestibility and enhances SCFA production via selective modulation of gut microbiota. The results indicate the potential of this approach for developing healthier rice-based products with improved gut health benefits. Future research should explore the optimal lipid types and concentrations for complexation and extend these findings through in vivo studies examining the impact on human health parameters.

The study utilized in vitro models, limiting the direct translation of findings to in vivo conditions. The number of human fecal samples used was limited, potentially restricting the generalizability of the microbiome findings. The study focused on palmitic acid, and further research is needed to assess the effects of other lipids. Also, the study primarily investigated short-term effects; the long-term consequences of consuming lipid-complexed rice need further study.