Effect of resistant starch type 5 on gut health through modulating gut microbiota

Abstract

Resistant starch is a dietary fiber that escapes digestion in the small intestine and undergoes fermentation by gut microbiota in the colon, producing beneficial short-chain fatty acids (SCFAs). Among the various types of resistant starch, resistant starch type 5 (RS5) has gained significant attention due to its unique and stable structural and functional properties. RS5 is a self-assembled V-type inclusion complex formed when amylose helices encapsulate guest molecules. This formation occurs through non-covalent interactions after the native starch structure is disrupted, and a guest compound is introduced. This structure provides enhanced resistance to enzymatic digestion, slows fermentation, and facilitates targeted release of bioactive molecules, making it effective in modulating gut health. RS5 promotes the proliferation of beneficial gut microbiota while suppressing pathogenic species, leading to increased SCFAs production, mostly butyrate, acetate, and propionate, which maintain intestinal integrity, reduces inflammation, and supports metabolic regulation. RS5 also contributes to preventing and managing chronic diseases such as obesity, type 2 diabetes, and colorectal cancer. While prior research has focused on its preparation methods and physicochemical characteristics, the influence of RS5 on gut microbiota and host health remains inadequately explored. This review summarizes the formation, classification, and structural diversity of RS5 complexes and how these factors influence digestibility and fermentation kinetics. Furthermore, it explores how RS5 modulates the composition and metabolic activity of the gut microbiota, enhancing SCFAs production. By comparing RS5 with other RS types, this review highlights its superior prebiotic potential and supports RS5-based functional food development for improving gut and metabolic health, targeting gut microecology.

Graphical abstract

1. Introduction

Starch, a key plant-derived energy source, constitutes a significant part of human diets worldwide. It primarily comprises two polysaccharides: i.e., amylose, which contains few or no α-1,6 linkages, and amylopectin, which has 5–6% α-1,6 glycosidic bonds branching from its primarily α-1,4-linked backbone [1]. While humans possess enzymes such as pancreatic and salivary amylases, glucoamylase, and sucrase-isomaltase to break down starch into absorbable sugars, not all starches undergo complete digestion before reaching the colon. These undigested starches are termed resistant starch (RS) due to their resistance to human enzymatic digestion, making them available for fermentation by colonic gut microbiota [2]. Starch is classified into the rapidly digestible (RDS), slowly digestible (SDS), and RS based on the rate of digestibility. High intake of RDS is linked to a greater risk of chronic diseases, such as diabetes and metabolic disorders, encouraging research into increasing SDS and RS content in foods to improve health outcomes [3].

RS is categorized into five types based on structural and functional attributes. RS1 resists digestion due to its physical inaccessibility, as it is trapped within intact plant cell walls, protein matrices, or other structures [4]. RS2, found in native uncooked raw potatoes, unripe bananas, and high-amylose maize starches, resists digestion due to its tightly packed crystalline structure [5,6]. RS3 forms when gelatinized starch cools and the amylose and amylopectin chains recrystallize during retrogradation [7]. RS4 is referred to chemically modified starches produced through cross-linking or substitution processes [7]. RS5, the most recently recognized type, consists of starch molecules complexed with guest compounds to form stable V-type complexes, which offer distinct benefits compared to RS1-RS4 [8]. RS5 was first introduced as a concept by Jane and Robyt in 1984 [9]. Starch-lipid V-type complexes were originally classified as RS5. Later, in 2021, Gutiérrez and Tovar broadened this category to include other emerging starch complexes, such as those formed with glycerol, amino acids, proteins, lipids-proteins, polyphenols, and polysaccharides, highlighting the evolving classification system for RS5 structures [10]. Guest molecules can either occupy the helical cavity to form inclusion complexes, or interact via hydrogen bonding and other forces to create non-inclusion complexes [11]. These complexation phenomena have functional consequences. The formation of amylose-lipid inclusion complexes (ICs) reduces starch digestibility, enhances RS content, and may improve glycemic control and lipid metabolism [12]. Compared to the other RS types, RS5 exhibits enhanced texture, gelation, and thermal stability, and compatibility with bioactive compounds [11]. Its dual role as a fermentable fiber and delivery matrix enables targeted nutrient release and antioxidant retention, making RS5 a promising candidate for functional foods and potential chronic disease prevention [13]. However, the mechanisms of starch-ligand interactions and the best methods to optimize RS5 formation are still not fully understood.

At the microbiome interface, different RS sources exert distinct effects on the human gut microbiota due to variations in their amylose-to-amylopectin ratio, crystallinity, starch chain lengths, crystal type, and granule morphology [14]. In general, they support increased SCFA production, reduced gut pH, enhanced mucus secretion, and modulation of the gut microbial community [15]. Like other RS, RS5 acts as a prebiotic, selectively stimulating the growth of beneficial gut microbes, such as the RS primary degrader Ruminococcus bromii and the butyrate producer Faecalibacterium prausnitzii [16]. However, among the RS types, RS5 stands out due to its ease of production, broad array of starch-to-guest molecule combinations, and potential synergistic health effects as a controlled-release guest molecule delivery system [17]. Such attributes underscore the need for detailed assessments of RS5 properties to clarify how specific RS-guest molecule attributes influence gut microbiota and health outcomes.

The impact of RS5 on gut health is linked to improved gut barrier function, reduced inflammation, and enhanced lipid metabolism. RS5 reduces glucose absorption by resisting digestion in the small intestine. It is fermented in the colon by gut microbiota to produce SCFAs, including acetate, propionate, and butyrate. Acetate is a metabolic fuel for peripheral organs such as the brain, spleen, and heart. Once transported to the liver, propionate can suppress the production of cholesterol and fatty acids, which helps lower lipid levels in the blood [18]. Butyrate is most closely linked to colon health. It supports colonic cell health by serving as the preferred energy source of colonocytes and reducing inflammation [19]. It also regulates glucose and energy homeostasis and improves gut histology by decreasing permeability and enhancing mucin production [16,20]. Collectively, it offers protection against obesity, insulin resistance, intestinal inflammation, and reduces cholesterol absorption [14,21,22].

Although extensive research exists on resistant starch types RS1 through RS4 and their roles in human health, RS5 investigations remain limited. This review summarizes the formation, structure, and properties of starch-guest ICs, typical shifts in microbial composition and SCFA profiles, and the health-promoting effects of RS5. While each RS source exhibits unique properties and effects on gut microbiota and host health, this article explores RS5′s properties, impact on gut microbiota, and potential health benefits compared to other RS types.

2. Types of RS5

2.1. Starch-lipid inclusion complex

The nature of the IC in RS5 is critical to determining its properties. Starch-lipid ICs, the original IC used to define the RS5 type, form through non-covalent interactions such as hydrogen bonding, hydrophobic forces, and van der Waals forces, resulting in V-type crystalline structures formation [23,24]. Fig. 1 illustrates standard methods for forming starch-guest ICs, beginning with converting starch into random helices and culminating in the formation of V₆-type ICs with various lipids and polyphenols. Interestingly, these ICs can naturally form in the human digestive tract during the digestive process. The hydrophobic tail of the lipid inserts into the non-polar cavity of the amylose helix, resulting in the formation of left-handed single-helical structures [25]. The inner cavity is lined with methylene groups and glycosidic oxygen atoms to form a hydrophobic cavity that holds compatible ligands [26,27]. Meanwhile, the hydrophilic regions of the lipid, such as the carboxyl or glyceryl ester group, remain outside the helix due to spatial constraints and repulsive electrostatic forces [23,28,29]. The lipid-containing helices stack together to form crystalline layers, which eventually become starch-lipid ICs with a V-type crystal structure. The V-type structure is identified by X-ray diffraction (XRD), with the V_6h type showing characteristic peaks at 2θ ≈ 12.9°, 13.7°, 18.9°, and 20.2° [[30], [31], [32], [33], [34]]. These complexes usually have six glucose units in each turn, but there are also complexes with seven or eight units, designated as V₇ and V₈, respectively.

Fig. 1.

Formation of starch-lipid inclusion complex and starch-polyphenol inclusion complexes and non-inclusion complexes [[35], [36], [37]].

These ICs can be classified into two types based on the melting temperature of the crystalline components and a few structural models have been proposed (Fig. 2). According to Biliaderis and Galloway [40], an amorphous helical structure characterizes type I (VI) and exhibits a melting temperature (T_m) between 95-105°C, which consists of a partially ordered structure with no distinct crystalline regions. On the other hand, Type II (VII) is a semi-crystalline lamellar structure with higher thermal stability, with a T_m ranging from 115-130°C. Type II further subdivides into two subtypes: Type IIa, which has a T_m around 115°C, and type IIb, which has a T_m of approximately 121°C (Fig. 1a) [41]. Type IIb exhibits greater crystallinity and is more resistant to digestion, making it particularly useful for food applications where digestion resistance is desired [8,23,[42], [43], [44]]. Other structural models were proposed such as the one illustrating the thermal behavior of amylose-decanol ICs as revealed by DSC analysis (Fig. 2b). Form Ia represents non-crystalline complexes composed of randomly oriented amylose helices, each hosting a single decanol molecule per helical segment. Form Ib reflects crystalline complexes with V-type antiparallel packing, where guest molecules alternate the orientation of their functional groups. In contrast, Form II contains two decanol molecules packed tail-to-tail within each amylose helix, resulting in a more ordered structure. This tighter packing leads to thicker lamellae and larger crystals, resulting in higher thermal stability and dissociation temperature [39].

Fig. 2.

Proposed structures for Form I and II of the V-type amylose inclusion complexes by various researchers: (a) crystal-thickness vs. melting point and chain/lipid stoichiometry for Types I, IIa, IIb [38]; (b) DSC peaks linked to Forms Ia, Ib, II with their molecular packing [39]; (c) V6/V7/V8 helical variants and guest accommodation [37].

The formation and stability of these complexes are influenced by several factors such as starch botanical sources [45], lipid chain length and saturation [46], processing conditions (temperature, pH, moisture) [[47], [48], [49]], and interactions during gelatinization [50]. For instance, the conversion from the VI-type to the more stable VII-type complex typically occurs between 90–110 °C, and the VIIa-type can further transition into the even more stable VIIb-type under these same conditions [51]. Similarly, longer-chain saturated fatty acids tend to enhance crystallinity and thermal stability, while unsaturated fatty acids reduce the structural order, affecting the digestibility of the complexes [52]. Moreover, processing techniques such as ultrasound and microwave treatments can modify the crystallinity and digestibility of starch-lipid ICs, offering specific applications in food technology [53,54].

2.2. Starch-polyphenol complexes

Starch-polyphenol complexes, a type of RS5, form through non-covalent interactions, such as hydrogen bonding, Van der Waals forces, electrostatic interactions, hydrophobic interactions, and ionic interactions between polyphenol and starch [55,56]. Amylose can form helical cavities that encapsulate the hydrophobic regions of polyphenols. In contrast, hydrogen bonds form between the hydroxyl groups of starch and polyphenols, protecting both from degradation, thereby delaying their bioavailability to further downstream in host digestion [57,58]. These interactions cumulatively form complexes that resist enzymatic digestion and allow them to transit to the colon and exert their health benefits [58]. Starch can form complexes with a variety of polyphenols, including phenolic acids (caffeic acid, gallic acid, ferulic acid, trans-p-coumaric acid, and protocatechuic acid) [59], flavonoids (anthocyanidins, flavones, flavonols, flavanones) [60], tannins [61], and lignans [62]. The interaction between starch and polyphenols is influenced by several factors, such as polyphenol types (molecular size, chemical structure, solubility, structural configuration), sources, and structural characteristics of starch [36,63,64]. Polyphenols with multiple hydroxyl groups, such as epigallocatechin gallate (EGCG) and caffeic acid, form strong hydrogen bonds with starch [56]. Polyphenols with low molecular weight (M_w), such as salicylic acid, can fit into amylose helices to form stable ICs, while larger polyphenols, such as tannic acid, are hindered by steric effects and form non-ICs [65].

The amylose-to-amylopectin ratio in starch is a key determinant of starch-polyphenol interactions. Amylose facilitates the formation of ICs with polyphenols due to its linear helical structure [[66], [67], [68]], whereas amylopectin forms a weaker interaction and a less stable complex due to its branched structure [55,69]. Starch varieties with higher amylose to amylopectin ratio, such as high amylose maize starch, display increased polyphenol binding affinity and promote more structured crystalline arrangements in amylose-polyphenol complexes, which has been notably observed with compounds such as apigenin [57,70]. Starch granules consist of alternating crystalline and amorphous regions, with the crystalline structure playing a key role in polyphenol interactions and digestibility [71]. Starches from different botanical sources display variations in their crystalline structure (defined as A-, B-, or C-type) dependent on factors such as their amylopectin chain length or amylose content. In A-type starches, polyphenols primarily interact with amylopectin, altering crystallinity and promoting RS formation. B-type starches are found in both tuber sources, such as potato, which have low amylose and long amylopectin chains, and pea and high-amylose cereals, for example, maize and wheat, which display intermediate crystalline patterns and amylopectin chain length profiles [72]. While both show B-type crystallinity, high-amylose starches more effectively form amylose-polyphenol ICs via hydrogen bonding and enhancing ordered structures and RS formation [73]. C-type starches show a balanced interaction, with polyphenols engaging both amylose and amylopectin [57,74,75]. Additionally, increasing the concentration of polyphenols enhances hydrogen bonding with starch, promoting complex formation. For example, at low concentrations, polyphenols (gallic acid and tannic acid) disrupt the crystalline structure of starch and form less dense complexes [61,76]. In contrast, higher concentrations lead to form dense, stable networks by forming V-type crystalline structures that limit water penetration and swelling, resulting in more ordered complexes [55,61].

Starch-polyphenol complexes alter starch's physicochemical properties, including swelling power, solubility, pasting, and thermal properties, causing an enhancement or limiting water accessibility to starch molecules, depending on their specific molecular characteristics [77]. Phenolic acids can reduce the peak viscosity, final viscosity, elastic modulus, and viscous modulus of starch by disrupting the amylose-amylopectin network, causing weak gel structures [78]. They reduce starch digestion rate, aiding blood sugar management and providing a lower glycemic index. Additionally, these complexes alter nutrient and antioxidant bioavailability, improve starch granule microstructure, reduce viscosity and elasticity, and exhibit prebiotic properties that promote beneficial gut microbiota and improve gut health [58,61,78,79].

2.3. Other types of RS5

In addition to starch-lipid, starch-polyphenol V-type complexes, recent studies have explored other RS5 guests, including starch-glycerol in which glycerol acts as a plasticizer, disrupting hydrogen bonds in starch, lowering the glass transition temperature (Tg) and increasing chain mobility [80]. These structures can be formed through ultrasonication, enzymatic debranching, or high-pressure treatments. Similar to glycerol, glycerol esters, such as glycerin monostearate can be used to produce RS5 [10]. Certain starch-protein complexes could be another RS5 formed through several types of molecular interactions: (i) hydrogen bonds form between starch’s hydroxyl groups and the protein’s amino or carboxyl groups. (ii) hydrophobic interactions between the inside of the amylose helix and the nonpolar parts of the protein and (iii) electrostatic forces occur when starch and protein have opposite charges, depending on the pH. This interaction promotes the formation of a physical barrier around the starch matrix, thereby reducing enzymatic hydrolysis during digestion and developing a more stable molecular structure [81]. Starch-protein complexes are typically formed during hydrothermal treatments or enzymatic modifications, where proteins bind to exposed starch chains, enhancing structural integrity and stability [10]. The complexation is influenced by several key factors, including starch composition, protein characteristics, and processing conditions [23]. These complexes significantly influence the functional properties of starch, including its solubility, gelatinization, and retrogradation behavior, as well as its physicochemical properties, including digestibility and thermal stability [10,23]. Other guest combinations for RS5 include starch-lipid-protein complexes, which are advanced ternary structures that exhibit enhanced stability and functionality compared to binary complexes [82]. These inclusion complexes are more structurally stable and ordered, forming protective barriers that block enzymes, making them harder to digest and increasing the RS content [82]. Proteins provide additional structural order by forming electrostatic and hydrogen bonding interactions, which strengthen the crystalline network. This complex formation is influenced by several factors such as starch, lipid, and protein types, fatty acid chain length, and experimental conditions, such as temperature, pH, and moisture content [[83], [84], [85]]. For example, lipids with longer chains form stronger and more stable complexes unless excessive length reduces solubility. Similarly, fewer double bonds in fatty acids yield more ordered and stable complexes, whereas higher unsaturation weakens crystallinity [51,86].

3. General knowledge of RS on gut microbiota

Over 1,000 species of bacteria have been identified in the human gut, mostly falling within five phyla- Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria, and Verrucomicrobia. Firmicutes, followed by Bacteroidetes, comprise the majority [87,88]. Collectively, the gut microbiota possesses an array of enzymes that break down polysaccharides resistant to host enzymes, but only a select few members can efficiently degrade RS. While there are only two chemical bonds that need to be broken in starch, the variety of physical complexities that render RS resistant to digestion means that RS-degrading bacteria often need complex systems for RS degradation. This is especially true of the keystone RS degrader Ruminococcus bromii (Firmicutes), which utilizes a set of multi-enzyme complexes known as amylosomes to accomplish this degradation efficiently. R. bromii is frequently a member of the gut microbial community and plays a critical role in making RS accessible to other microbial members [16,89]. R. bromii can utilize most types of RS from a wide variety of sources, digesting them to maltose and maltooligosaccharides, which are fermented predominantly to acetate [89,90]. This acetate can be utilized by some bacteria in the production of butyrate. However, interestingly, many R. bromii strains are unable to utilize glucose directly for growth, likely due to the lack of a transporter, leaving it behind as a growth substrate for other members of the microbiota [91]. Other important RS degraders in the gut include Bifidobacterium adolescentis (Actinobacteria), Clostridium butyricum (Firmicutes), and the proposed Ruminococcoides bili strain FMB-CY1 (Firmicutes) [16,[92], [93], [94]].

With the exception of C. butyricum, the primary RS-degrading organisms do not directly produce butyrate. Instead, cross-feeding interactions with other members of the microbiota are required. This can include some organisms that serve as secondary starch degraders which have more limited enzymatic systems for starch digestion but can produce butyrate [95,96]. Additionally, several butyrate producers, such as E. rectale and Faecalibacterium prausnitzii, can utilize the acetate produced by R. bromii in the production of butyrate [97]. Furthermore, some organisms can utilize the lactate produced by B. adolescentis for butyrate production [98]. Most butyrate in the gut is produced from carbohydrate precursors through the microbial BUK (butyrate kinase) or BUT (butyryl-CoA: acetate CoA-transferase) pathway, while amino acid (glutamine and lysine) fermentation contributes only minimally [99]. RS generally elicits higher SCFA production compared to non-starch polysaccharides [100,101]. In contrast, non-starch polysaccharides may be in a recalcitrant form of glucose polysaccharide (i.e., cellulose with beta-glucose bonds) or composed of non-glucose sugar residues that stimulate different microbes and metabolic pathways than RS. Microscale localization, facilitated by specialized substrate-specific binding modules, also plays a role in RS rendering exceptional SCFA production, with B. adolescentis, R. bromii, and E. rectale typically occupying the surface of fecal excreted starch granules [102,103].

The microbiota is relatively stable over time in the absence of major disruptions (e.g., diet change, antibiotics), however, introducing RS to the microbiota may result in 'blooms' in specific groups of bacteria [104]. These particular members are generally considered beneficial to human health, and in this sense, RS is a prebiotic [17,105]. RS increases the proportion of RS degraders and butyrate producers, such as R. bromii, F. prausnitzii, Roseburia spp., and E. rectale [89,104]. In a human in vivo study, a high RS diet produced among the most significant changes in E. rectale (8% increase) and R. bromii (5% increase) community composition. In another in vivo human study, a high intake (48 grams of RS/day for two weeks) of RS upregulated genes encoding pathways for starch degradation and sugar uptake [89].

Interestingly, enrichment in the microbiota towards those associated with RS degradation and butyrate production does not guarantee a measurable alteration in SCFAs concentration at lower doses [106]. The particular types and sources of RS can induce unique changes even in the same microbiota, with different RS degraders and butyrate producers selected for by different starch sources [107]. Particularly within the RS2 type, it has been found that potato starch tends to select for B. adolescentis, while high amylose maize starches select for R. bromii [108]. However, although RS can be resistant to enzymatic degradation by the human gut microbiota, the classification of RS type (RS type 1-5) explains the mechanism of digestion resistance from a human enzymes perspective; it does not cover starch digestion resistance (or lack thereof) pertaining to enzymes of the colonic microbiota, nor dictation of their metabolite profiles and community shifts. In short, within and between different RS types and different gut microbial communities, alterations in the SCFA profile and effects on the gut microbial community composition can converge and diverge [103,[109], [110], [111], [112]]. Mapping the response of the microbiota to different types of starch sources requires consideration of the finer molecular structure of starches (i.e., branch chain lengths, type of crystal polymorph, extent of modifications and treatments, etc.) and the features of the gut microbiome involved, with an exception for RS5 [113]. RS5 is compositionally different from other RS due to the inclusion of a non-starch guest. In the case of RS5, consideration of the guest molecule properties and starch/guest interaction effects are also necessary to gain a full picture of where host health benefits are derived. However, the guest must first be released from the starch carrier and made available, emphasizing a potentially critical role of key RS degrading microbes in garnering guest-molecule associated health benefits of RS5.

4. RS5 and its role in gut health

RS5 is characterized by its unique V-type crystalline structure, which forms through complexation with guest molecules, such as lipids or phenolics. This association is responsible for enhanced resistance to enzymatic digestion, distinguishing RS5 from other RS types. It is a promising functional ingredient for the promotion of gut health by combining the benefits of RS and bioactive guest molecules. Studies have shown that the specific guest molecule used in RS5 formation influences its physicochemical properties, digestion profile, and effect on the gut microbiome. While health effects can overlap or diverge within and between all RS types, the potential for additional health benefits from a guest molecule is a feature explicit to type 5 RS (Fig. 3).

Fig. 3.

Mechanism of starch-guest inclusion complexes (ICSs) in enhancing gut health. The health benefits offered by guest molecules that are shuttled to the colon and made accessible over time by the gut microbiota augments the health benefits traditionally associated with resistant starch.

From the studies in Table 1, Table 2, RS 2-5 generally produce a superior butyrogenic response from the gut microbiota alongside a decrease in pH. RS5 is potentially more butyrogenic than RS 1-4 [118,119,121,125]. As with other types of RS, RS5 tends to decrease alpha diversity [114,115,[117], [118], [119],121,123], indicative of prebiotic effects in stimulating select members of the community. From the phylum level, all RS types tend to increase the relative abundance of Firmicutes, especially RS5 [114,115,117,119,121]. The morphology of RS5 is rough and irregular, in contrast to the smooth morphology associated with other types of RS (i.e., RS2) [116,117,123]. The relevance of this defining feature for gut microbiota, if any, has not been heavily explored. In the context of in vivo RS5 studies (Table 2), weight loss is associated with RS5 intake [122,123]. Compared to other RS types, the effect of RS5 is more pronounced in increasing high-density lipoprotein cholesterol while RS as a whole generally decreases triglycerides and total cholesterol [122,123]. In contrast to no intervention, RS supplementation generally improves liver histology, increases liver weight, and colon length in disease-induced states, with RS5 performing as well, or better than other types of RS [122,123,126].

Table 1.

In vitro studies on RS5 and its impact on gut health.

Substrate and microbe source	Parameters measured	Outcomes	Refs.
Debranched (DB) HAMS with (RS5) and without (RS3) lauric acid (12C), myristic acid (14C), palmitic acid (16C), or stearic acid (18C), HAMS (RS2). Not mock digested. 3 different adult human fecal donors.	Alpha diversity. Human fecal fermentation.	Chao1 and ACE: NSD between RS sources. Shannon: Significantly higher in RS5 made with shorter lipids compared to RS2 and RS3 Simpson: Significantly higher in RS5 compared to RS2 and RS3.	[114]
	Microbial community	All RS sources increased the relative abundance of Roseburia and Prevotella and decreased Megamonas relative abundance.
	RS content	RS5 sources had significantly higher RS content vs RS3 (RS5: 52.9-55.8%, RS3: 36.8%, RS2: 58.5%).
	SCFA	RS5 supplementation increased propionic, acetic, and total SCFAs more than RS2 and RS3.
Octenyl succinic anhydride (OSA)-modified potato starch + monolauryl glycerol (MLG) ((RS4+RS5) composite), potato starch-MLG (RS5), OSA-modified potato starch (RS4, two levels of OSA substitution), potato starch (RS3), potato starch (RS2). Not mock digested. 4 male and 4 female adult human fecal donors.	Alpha diversity. Human fecal fermentation.	Goods-coverage, Chao1, and Shannon: All RS sources decreased. Simpson: Decreased for all RS sources except the RS4 with higher substitution.	[115]
	Microbial community	(RS4 + RS5) composite: Increased Firmicutes and decreased Bacteroidota relative abundance. All other RS sources: Increased Bacteroidota and decreased Firmicutes relative abundance.
HAMS + stearic acid (RS5), Octenyl succinic anhydride (OSA)-modified HAMS (RS4), debranched HAMS (RS3), HAMS (RS2). Mock digested. 3 adult human fecal donors.	SCFAs	Total SCFA concentration: RS3<RS4<RS2<RS5 Butyrate concentration: RS4<RS3<RS2<RS5 Propionate concentration: RS2 and RS3 and RS4<RS5 Acetate concentration: RS3<RS2 and RS5<RS4	[116]
	Glucose liberation during mock digestion	Relative starch hydrolysis rates: RS5>RS3>RS2 and RS4
Rice starch + purple red rice anthocyanin extract (RS5), rice starch (RS3). Anthocyanin guest dosages of 0% for the RS3 and 5, 10, 20% for the RS5 based on dry starch weight. Mock digested. 3 adult male human fecal donors.	Alpha diversity. Human fecal fermentation.	All RS sources had decreases in the Chao1, Shannon, and Simpson value but no significant differences between each other.	[117]
	Microbial community	Higher anthocyanin concentrations correlated with increased relative abundance of Bacteroidetes and decreased relative abundance of Firmicutes.
	SCFAs	RS5 made with progressively higher anthocyanin concentrations had increasingly lower SCFA production.
Chestnut starch + proanthocyanidins (RS5), chestnut starch (RS3), chestnut starch (RS2). RS5 made with 0.5, 1.0, 2.0, or 4.0% proanthocyanidins (PR) on a chestnut starch dry basis. Mock digested. 5 healthy human fecal donors.	Alpha diversity. Human fecal fermentation.	RS5: lower Shannon, Simpson, Chao1 and Ace values vs RS3. Shannon and Simpson values increased with higher concentrations of PR.	[118]
	Microbial community	The relative abundance of Proteobacteria was lower and Actinobacteria higher in all RS5 fermentations compared to RS3. Firmicutes/Bacteroidetes ratio was lowest in RS5 with 4% PR.
	RS content	Rapidly digestible starch content significantly decreased with higher PR concentrations (83% decreased to 68%), while slowly digestible starch and RS content significantly increased (14% increased to 19% and 3% increased to 12%, respectively).
	SCFAs	Acetate, propionate, and butyrate levels were significantly higher in RS5 sources vs RS3 with concentrations positively correlating with PR concentration, RS content, α-amylase inhibitory activity, antioxidant activity and advanced glycation end product inhibition.
	α-amylase inhibition	Higher PR concentrations correlated with lower α-amylase activity.
	Antiglycation activity	RS5: significantly higher inhibition of advanced glycation end products and glycoxidation products (all PR concentrations) compared to RS3.
	Antioxidant activity	All RS sources had strong antioxidant potential (DPPH and ABTS assay), with higher PR concentration positively correlating with significantly higher antioxidant potential.
Debranched HAMS-palmitic acid complex (RS5), HAMS (RS3), HAMS (RS2). Not mock digested. 4 healthy fecal donors (2 males and 2 females, ages 25-33 with normal body mass index). RS5 prepared in sodium acetate buffer.	Alpha diversity. Human fecal fermentation.	Simpson, ACE, and Chao1: NSD between RS sources and blank at time 24 hours (B24). Shannon: B24 and RS2 significantly higher diversity than RS3 and RS5.	[119]
	Microbial community	All RS treatments decreased the relative abundance of Bacteroidetes and increased the relative abundance of Firmicutes.
	SCFAs	RS5 fermentation produced significantly higher butyric, acetic, and propionic acid concentrations in the first 12 hours of fermentation compared to RS2 and RS3.
Starch (pea, chestnut, corn, rice, or cassava) + epigallocatechin gallate (5% weight/weight starch) (RS5), RS3 pea, chestnut, corn, rice, or cassava starch. Not mock digested. 6 healthy adult fecal donors (3 males and 3 females, ages 22-26 with normal body mass index).	Alpha diversity. Human fecal fermentation.	ACE and Chao: Significantly decreased in RS5 treatments compared to the equivalent RS3 versions. Shannon: All RS3 and RS5 treatments significantly decreased, except RS3 and RS5 cassava starch, RS3 pea and chestnut starch. Simpson: Significantly increased in four of five RS5 sources, while one of five RS3 sources significantly increased.	[120]
	Microbial community	RS3 and RS5 sources both favored increases in the relative abundance of Firmicutes and Bacteroidota and decreased relative abundance of Proteobacteria or Actinobacteriota.
	SCFAs	All RS5 sources produced higher acetate and total SCFA concentrations than their RS3 counterpart.
Lotus seed starch-epigallocatechin gallate (1 part per 10 parts starch weight) (RS5), HAMS (RS2). Not mock digested. 12 eight-week-old ICR male mice fecal donors.	Alpha diversity. Mouse fecal fermentation.	Chao1 and Shannon indices significantly decreased in RS5 and RS2 treatment. Simpson index significantly decreased for RS2 treatment, but not RS5.	[121]
	Microbial community	The relative abundance of Firmicutes and Proteobacteria increased in both RS2 and RS5 fermentations, while Bacteroidota relative abundance decreased.
	SCFAs	RS2 treatments had significantly higher acetic and propionic final concentrations than RS5, while RS5 had significantly higher butyrate. The total SCFA production did not significantly differ between RS2 and RS5.

ABTS = 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); DPPH = 2,2-diphenyl-1-picrylhydrazyl; NSD = No significant difference; SCFAs = Short chain fatty acids.

Unless noted, production of RS5 was through a heated water-based method.

Table 2.

Studies on RS5 and its impact on gut health in rodent models.

Substrate and microbe source	Parameters measured	Outcomes	Refs.
Rice starch-oleic acid (RS5), rice starch (RS3), rice starch (RS2). 18 male non-obese SD rats.	Body weight	RS5 was associated with the lowest body weight.	[122]
	Hepatic enzymes and blood glucose	Aspartate transaminase: RS5<RS2<RS3; alanine aminotransferase: RS5<RS2<RS3; alkaline phosphatase: NSD. Relative levels of blood glucose: RS2<RS3 & RS5
	Microbial community	Relative abundance of Firmicutes: RS3<RS2<RS5; relative abundance of Bacteroidetes: RS5<RS2<RS3.
	Necropsy	Liver weight: RS5<RS3<RS2; Epididymal fat weight: NSD; Perirenal fat weight: RS3 & RS5<RS2
	Serum lipid profile	TG: RS3 & RS5<RS2. HDL: RS3<RS2<RS5. LDL: RS2 & RS5<RS3. TC: RS3 & RS5<RS2.
	Oxidative stress markers	Malondialdehyde: RS5<RS2<RS3; superoxide dismutase: RS2 & RS3<RS5; glutathione peroxidase: RS2<RS3 & RS5.
	Rapidly digestible, slowly digestible, and RS content, respectively	RS2: 35.2, 52.8, 12.0% RS3: 58.4, 25.7, 15.9% RS5: 42.7, 34.1, 23.2%
	Serum insulin	NSD
Canna edulis starch-lauric acid (RS5, two dosage levels used in mouse supplementation – high and low), Canna edulis starch (RS2), simvastatin (10 mg/kg mouse). 50 fifty-six-week-old hyperlipidemic male mice (control – 10 mice, high-fat diet (HFD) - 40 mice) *Mouse model assessed the RS5, not the RS2	Alpha diversity. In vivo fermentation.	Chao1 and ACE: Significantly decreased for the HFD alone and diet supplemented with a “high” dosage of RS5 vs control diet. Shannon: Significantly decreased for the HFD supplemented with a “high” dose of RS5 vs control diet. Simpson: Significantly increased for HFD supplemented with a “high” dosage of RS5 vs control diet. HFD alone vs control diet: NSD in Shannon and Simpson indices.	[123]
	Hepatic enzymes and blood glucose	Aspartate aminotransferase: NSD between RS5 supplemented HFD vs HFD alone. Alanine aminotransferase: Higher dosages of RS5 supplemented HFD significantly lowered vs HFD alone. RS5 supplementation significantly decreased blood glucose vs HFD alone.
	Microbial community	Compared to the control diet, the relative abundance of Firmicutes increased and Bacteroidota decreased in HFD without intervention. The opposite was true for RS5 and simvastatin treatment.
	Necropsy	Hematoxylin and eosin (HE) stained and Oil Red O-stained liver: HFD mice with no RS intervention had greater lipid content. RS5 intervention greatly decreased the number of lipid droplets and degree of hepatocyte deformation. HE stained epididymal fat: HFD mice with no intervention trended towards larger and more irregularly shaped fat cells. Simvastatin and RS5 greatly decreased adipocyte damage.
	RS content	RS2: 53.20% RS5: 56.12%
	Serum lipid profile	The HFD mouse group without RS supplementation had significantly higher serum TC, TG, and LDL levels than the control mice. RS5 (low and high) supplementation: Decreased serum TC and TG, while increasing HDL. The HDL level associated with the higher RS5 supplementation group was significantly higher than the control diet group. NSD was observed in LDL levels for both RS5 groups against the control.
	Body Weight, Lee’s Index, and eWAT Weight	RS5 (low and high dose) supplemented mouse groups on a high-fat diet had significantly lower body weight than mice on the high-fat diet alone. Lee’s index: Higher in HFD group, while lower in the simvastatin and RS5 (low and high dose) group. HFD group had significantly higher epididymal fat mass than all other mouse groups.
Debranched HAMS + lauric acid (RS5), HAMS (RS2). Male C57BL/6J mice, seven weeks old – eighteen with dextran sulfate sodium (DSS)-induced colitis and 6 without as a negative control. RS5 prepared in heated aqueous DMSO.	Alpha diversity. In vivo fermentation	Chao1: NSD between all treatments Simpson and Shannon: RS2 supplemented mice did not significantly differ from negative control mice, while the positive control and RS5 supplemented mice were significantly lower alpha diversity.	[124]
	Body Weight	RS5 supplemented mice lost significantly more weight than negative control mice. RS2 supplemented mice lost significantly more weight than RS5 supplemented mice. Positive control mice lost significantly more weight than RS2 supplemented mice.
	Microbial community	The relative abundance of Firmicutes was lower and Bacteroidota higher in negative control mice compared to positive control, RS2, and RS5 supplemented mice. The relative abundance of Proteobacteria was highest in positive control mice, with genus level indicating a sizable contributor came from Escherichia-Shigella. The relative abundance of Akkermansia was lowest in positive control mice, in tandem with lower mucus.
	Necropsy	Relative colon lengths: Negative control>RS5>RS2>positive control. The colons of RS5 supplemented mice were significantly longer than the colons of positive control mice. RS2 and RS5 supplemented mice maintained a significantly thicker mucus layer and more goblet cells per crypt than positive control mice.
	SCFAs	RS2 and RS5 supplemented mice produced significantly more acetate, propionate, and butyrate than positive control mice. Compared to RS2 supplementation, RS5 supplementation produced equal or significantly more acetate, propionate, and butyrate.
	Pro and anti-inflammatory cytokines and inflammatory system	RS5 supplemented mice had significantly lower levels of interleukin-6, interleukin-1 beta, and tumor necrosis factor (TNF) alpha compared to positive control mice. Interleukin-10 production was equally significantly higher for RS2 and RS5 supplemented mice against positive control. Expression of toll-like receptor 4 (TLR4), TNF alpha, and interleukin-1 beta was significantly lower while occludin and zonula occludens-1 expression was significantly higher in mice supplemented RS2 or RS5 compared to positive control mice.

eWAT = epididymal white adipose tissue; HDL = high-density lipoprotein cholesterol; LDL = low-density lipoprotein cholesterol; NSD = No significant difference; SCFAs = Short chain fatty acids; TC = total cholesterol; TG = triglyceride.

Lee’s Index: formula for the assessment of obesity in mice calculated as the cubic root of body weight (grams)/naso anal length (cm) and multiplied by 1,000

Unless noted, production of RS5 was through a heated water-based method.

Decades of research on RS2, RS3, and RS4 have demonstrated the ability of RS to modulate the human gut microbiome and improve host health. Studies have shown that RS can enhance insulin sensitivity, improve glucose tolerance, decrease gut permeability through the mediation of epithelial tight junctions, and influence colonocyte gene expression and apoptosis [14,[127], [128], [129], [130], [131]]. However, RS5′s unique structure and guest molecule flexibility may offer synergistic benefits. Unlike other RS types, RS5′s specific effects on the gut microbiota and metabolic health can be fine-tuned by altering the guest molecule. Potentially, the net host health benefits are augmented by increased RS and SDS fractions, as well as transport protection and altered bioavailability of novel guest molecules that exert unique site-specific effects on the host. Realization of any synergistic health benefits are likely dependent on the specific host (i.e., genetics and enzyme expression), microbiome, RS5 guest and starch identity, and influence of the RS5 preparation method on guest and starch delivery rate and release locations. While assessment of gut health and RS5 combinations on a case-by-case basis is lacking, many guest molecules have already been studied extensively for health effects on their own. In this section we will only consider the case of phenolics and lipids (i.e., glycerol esters and medium- and long-chain fatty acids most relevant to RS5, such as glycerol monolaurate and oleic acid), as they are generally the most represented in RS5 studies. Phenolic compounds have generally been associated with anti-tumor, anti-inflammatory, and anti-obesity effects and resistance to human enzymatic digestion [132,133]. The role in gut health of RS5 complexes with polyphenols such as anthocyanins and epigallocatechin gallate (EGCG) have demonstrated enhanced butyrate production, modulation of energy metabolism, and support for carbohydrate degradation pathways [134,135]. As the low digestibility of phenolics implies high proportions of dietary phenolics make it to the colon even in the absence of a protective complex, most health benefits from its inclusion as the guest in an RS5 complex would likely be related to a mechanism involving targeted site delivery and altered release rates. However, the potential existence and benefit that would be attributed to this mechanism has not been clearly demonstrated and requires further investigation. Lipids have demonstrated properties that include antibacterial and probiotic effects, dose dependent weight loss, and (as with SCFAs) are activators of specific free fatty acid receptors (FFAR) that play an important role in mucosal barrier integrity with potential beneficial anti-inflammatory and anti-colorectal cancer effects [[136], [137], [138], [139], [140]]. Lipids interacting with the gut microbiota may also undergo modifications into potentially health beneficial compounds such as linoleic acid conversion to certain conjugated linoleic acids (CLAs) by members including Bifidobacterium, Clostridium, and Lactobacillus [138]. Health effects of RS5 made with lipid guests have included weight loss, favorable shifts in markers of inflammation (i.e., cytokines), and decreased serum triglycerides [122,126]. Dietary lipid digestion and absorption in healthy humans are primarily in the small intestine (∼90%), [141] with little reaching the colon. RS5 with a lipid guest can play an important role in augmenting dietary health benefits to the host by offering transport protection to lipids that themselves offer health benefits realized after reaching the colon. Without RS5 protection, these lipids would be largely lost to upstream host digestion and absorption along with any health benefits requiring colonic delivery. As the prior discussion of lipid and phenolic guest demonstrates, the potential mechanisms on how RS5 augment health can widely differ based on the guest properties such as digestibility. This variation indicates the value of subdividing RS5 into subtypes based on mechanism of how host digestion is altered as a means of clarifying RS5 role in gut health, once sufficient research has been completed in mechanism elucidation.

The fermentation of RS promotes the production of SCFAs, including butyric, acetic, and propionic acid, which together account for 90-95% of colonic SCFAs and are generated in a nearly constant molar ratio of 15:60:25, respectively [142]. It also generates carbon dioxide, hydrogen, methane (in certain people), and other organic acids, such as, lactic, hexanoic/caproic, and valeric acid [143]. Some SCFAs are absorbed through the intestinal wall and transported mainly to the liver (propionate and acetate) or circulated through the bloodstream to other organs such as the heart and brain (acetate), where they can affect metabolism and immune responses [144]. Butyric acid plays a vital role in maintaining gut barrier integrity, reducing inflammation, and serving approximately 70% of total energy requirements for colonocytes [124]. Propionate contributes to gluconeogenesis and is believed to reduce lipogenesis, lower serum cholesterol levels, and inhibit carcinogenesis in peripheral tissues [145]. Acetate suppresses appetite and participates as a minor energy source in the nervous system (in addition to glucose and lactate) [146,147]. RS5 fermentation generally occurs at a slower rate compared to other RS types. A slow fermentation process supports a gradual pH drop and spreads the distribution of SCFAs, enabling nourishment of colonocytes throughout the entire length of the colon.

5. Future directions and conclusion

While research on RS5 is still in its early stages, it offers promising potential for gut microbiota modulation and health improvement. Its unique structure, slow fermentation rate, and ability to enhance SCFA production and beneficial bacterial populations distinguish it from other resistant starch types. Future studies should explore the health effects of RS5 compared to RS without a guest in more relevant human clinical feeding trials, which are lacking. Additionally, attention should focus on further elucidating properties of RS5 inclusion complexes and the finer details of RS5’s degradation by gut microbiota. Such information would aid in developing a classification system for types of digestion resistance mechanisms related to microbiota and various classes of RS5 constructs (i.e., polyphenols, amino acids, lipids). A classification by microbial resistance mechanism types would benefit development of functional foods by simplifying communication on what a given RS5 does to the gut thereby improving industry and consumer understanding and facilitating selection of an appropriate RS5 to achieve a desired effect. Lastly, the simplicity and cost of RS5 creation and novel health attributes lend themselves well to incorporation into functional food development for consumer bases intrigued by new developments in health foods. Reformulating products to utilize RS5 could especially stand to benefit both consumer health, public health, and companies that enter the market early.

In conclusion, resistant starch, particularly RS5, represents a valuable dietary intervention for improving gut health and metabolic outcomes. Its low cost and ease of production, potential to modulate the gut microbiota, enhance SCFA production, and alter the bioavailability location and rate of nutrient and non-nutrient guest molecules underscores the importance of continued research to unlock its full therapeutic and industry potential.

CRediT authorship contribution statement

Raju Ahmmed: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis. Andrew Paff: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis. Lingyan Kong: Writing – review & editing, Supervision, Resources, Methodology, Investigation, Conceptualization. Songnan Li: Writing – review & editing, Investigation. Darrell W. Cockburn: Writing – review & editing, Supervision, Resources, Methodology, Investigation, Funding acquisition, Conceptualization. Libo Tan: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Investigation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.