Prebiotics are a class of indigestible carbohydrates in foods that selectively stimulate the growth and metabolic activity of beneficial gut microorganisms, providing health benefits to the host. Currently, common prebiotics on the market include oligosaccharides, resistant starch, inulin, lactulose, and levans, which are often added to food matrices as the important health promoters. In addition to altering gut microbiota, prebiotics have been reported to improve intestinal integrity and function, promote the production of SCFAs, and reduce levels of nitrogenous metabolites in the gut [1]. Accordingly, prebiotics play an important role in promoting consumer health [2].

Dietary fiber (DF), often referred to as the “seventh nutrient”, is an important part of the human diet [3]. Studies have confirmed that DF, especially soluble dietary fiber (SDF), having the ability to reduce the risk of cancer, inflammatory bowel disease, obesity, and diabetes [4]. These health benefits are closely related to the modulatory effects of SDF on the gut microbiota of host [5]. Therefore, the prebiotic properties of SDF have received much attention from researchers. With the in-depth study of SDF, researchers have found that SDFs from different sources tend to exhibit different probiotic properties, which can be attributed to their different structural characteristics [6]. It has been shown that gut microbes showed preferential utilization of glucose [7], and polysaccharides with higher glucose content have better probiotic proliferation-promoting effects [8]. When the glucose residues of polysaccharides are linked to each other by β-1, 4 glycosidic bonds, they have better prebiotic activity than polysaccharides linked through β-1, 3, β-1, 6 and β-1, 1 glycosidic bonds [9]. In addition, the polysaccharides with lower molecular weight, polymerization degree, and crystallinity degree usually present a better prebiotic effect [10]. Although the effect of polysaccharide structure on its prebiotic activity has been preliminarily studied, the current study is not sufficient to clearly elucidate the correlation between the structural properties of polysaccharides and their prebiotic effect. Accordingly, the relationship between the structural properties of polysaccharides and their prebiotic activities urgently needs to be further investigated.

In recent years, the important functions of SDF have been continuously discovered, hence the market is flooded with more and more food products containing SDF. The search for new sources of natural SDF has become an emerging trend with the increase in health awareness and the booming application of natural bioactive in the food industry [11]. Grapefruit belongs to the genus Citrus in the family of Rutaceae, with a delicious flavor and high nutritional and medicinal value. According to the data from the Food and Agricultural Organization of the United Nations, grapefruit is widely grown in countries such as China and the United States [12]. Grapefruit is rich in flavonoids, carotenoids, coumarins and organic acids, which have a variety of health-promoting properties, such as anti-inflammatory, anti-cancer, and weight loss [13]. In addition to daily consumption, large quantities of grapefruit are reportedly utilized to produce grapefruit juice [14]. In daily consumption and industrial production, grapefruit peel is often buried or incinerated as waste, resulting in a waste of resources and increasing the environmental burden. Interestingly, recent studies have shown that these by-products are rich in SDF [15]. Many researchers have extracted SDF from grapefruit peel and extensively investigated its physicochemical properties [4]. Nowadays, some pomelo peel SDFs have been reported to present the prebiotic activity [16], while no studies have been conducted on the prebiotic property of SDF extracted from grapefruit peel sponge layer (GSLSDF). Our previous study found that GSLSDF had better prebiotic activity than other pomelo peel SDFs (Fig. S1). Nevertheless, the probiotic proliferation mechanism of GSLSDF is unclear, hindering its application. Therefore, in this study, the key prebiotic fraction of GSLSDF was identified. Then the relationship between structure and prebiotic activity of key prebiotic fraction was investigated. Finally, the modulatory effect of key prebiotic fraction on intestinal microorganisms was studied by in vitro fecal fermentation experiment.