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REVIEW DOI: 10.1111/nbu.12244 Health effects of resistant starch S. Lockyer* and A. P. Nugent† *British Nutrition Foundation, London, UK; † University College Dublin, Dublin, Ireland Abstract The merits of a fibre-rich diet are well documented. Resistant starch (RS) is a form of starch that resists digestion in the small intestine and, as such, is classified as a type of dietary fibre. RS can be categorised as one of five types (RS1–5), some of which occur naturally in foods such as bananas, potatoes, grains and legumes and some of which are produced or modified commercially, and incorporated into food products. This review describes human evidence on the health effects of RS consumption, with the aim of identifying any benefits of RS-rich foods and RS as a functional ingredient. The reduced glycaemic response consistently reported with RS consumption, when compared with digestible carbohydrate, has resulted in an approved European Union health claim. Thus, RS-rich foods may be particularly useful for managing diabetes. There appears to be little impact of RS on other metabolic markers, such as blood pressure and plasma lipids, though data are comparatively limited. Promising results on markers of gut health suggest that further research may lead to the classification of RS as a prebiotic. Microbial fermentation of RS in the large intestine to produce short-chain fatty acids likely underpins some of its biological effects, including increasing satiety. However, effects on appetite have not resulted in notable changes in bodyweight after long-term consumption. Emerging research suggests potential for RS as an ingredient in oral rehydration solutions and in the treatment of chronic kidney disease. Overall, RS possesses positive properties as a healthy food component. Keywords: amylose, fibre, glycaemia, resistant starch, satiety, short-chain fatty acids Introduction The importance of dietary fibre has been recognised by health authorities globally for many years (IOM 2002; Mann et al. 2007; EFSA Panel on Dietetic Products Nutrition and Allergies 2010a). As such, recommendations for fibre intakes feature in national and international dietary guidelines. However, the details of the recommendations differ between nations Correspondence: Dr. Stacey Lockyer, Nutrition Scientist, British Nutrition Foundation, Imperial House, 15-19 Kingsway, London WC2B 6UN, UK. E-mail: [email protected] © 2016 British Nutrition Foundation (Lockyer et al. 2016). A recent review linked higher fibre intakes with a reduction in the risk of developing type 2 diabetes, cardiovascular disease (CVD) and colorectal cancer (SACN 2015). Biological effects consistently demonstrated in randomised controlled trials (RCTs) include decreased transit time and increased faecal bulk (SACN 2015). Dietary fibre is almost universally defined as the material isolated by methods approved by the Association of Official Analytical Chemists (AOAC). Older AOAC methods measured non-starch polysaccharides (NSP), lignin, some inulin and some resistant starches and non-digestible oligosaccharides, but not all 1 2 S. Lockyer and A. P. Nugent (Prosky et al. 1988; Lee et al. 1992). The current AOAC method (2009.01) measures all carbohydrates that are neither digested nor absorbed in the small intestine and have a degree of polymerisation of three monomeric units or more (i.e. NSP, non-digestible oligosaccharides, all resistant starches and polydextrose, plus lignin; McCleary et al. 2012). Until recently in the UK, dietary fibre was defined as NSP [measured using the Englyst method (Englyst et al. 1994)] and dietary intakes were reported in this way but food labels list AOAC values in alignment with European Union (EU) law (European Commission 2011). In 2015, the UK Scientific Advisory Committee on Nutrition (SACN) recommended that the AOAC definition should now be used for all purposes (SACN 2015) and revised (higher) reference values were set, reflecting the strengthening of evidence since recommendations were last made in 1991. These include reference intakes for children of different ages. Resistant starch (RS) is a fraction of starch that resists digestion within the small intestine, reaching the large intestine intact. RS is then fermented by microbes in the large intestine, producing short-chain fatty acids (SCFAs). The term ‘resistant starch’ was coined by Englyst and colleagues in the 1980s (Englyst et al. 1982). An array of health benefits have been attributed to RS, and this paper serves as an update to our 2005 review of this topic (Nugent 2005). The main areas covered are gut health, glycaemia and plasma lipid effects, satiety and bodyweight. Prominent human studies published in English from January 2005 to July 2016 have been considered. Starch types Starch is a compound present in plants for the storage of glucose and is made up of two polymers: amylose, which is a linear molecule, and amylopectin, which is branched. Distinct crystalline forms of amylose and amylopectin have been identified in starch granules. Type A is characteristic of cereals, type B is more commonly present in potatoes and bananas, and type C is an intermediate form found in legumes. Starch is digested by a-amylase in the mouth, pancreatic amylase, and glucoamylase and sucrose-isomaltase embedded in the membrane of microvilli within the small intestine. The resultant glucose is then absorbed. Starch can be classified into rapidly digestible, slowly digestible and RS. RS resists hydrolysis by the digestive enzymes a-amylase and pullulanase in vitro beyond 120 minutes, unlike both rapidly digestible and slowly digestible starch (Englyst et al. 1982). Classification of resistant starches and factors affecting resistance to digestion There are five types of RS. • RS1 – inaccessible to digestive enzymes due to the physical barriers formed by cell walls and protein matrices. Less resistant to digestion compared with other types. • RS2 – starches protected from digestion due to their crystalline structure. • RS3 – retrograded starch formed when starchy foods (e.g. potatoes, pasta) are cooked then cooled. Long-branched chains of amylopectin form double helices that cannot be hydrolysed by digestive enzymes. • RS4 – chemically modified starch formed by crosslinking, etherisation or esterification. • RS5 – two different components have been proposed as RS5. The first comprises amylose–lipid complexes, which either form during processing and reform after cooking or can be created artificially and added to foods (Seneviratne & Biliaderis 1991; Hasjim et al. 2010, 2013; Lau et al. 2016). The second is resistant maltodextrin, which is processed to purposefully rearrange starch molecules (Mermelstein 2009). The majority of publications describe RS5 as amylose– lipid complexes. Amylose is digested slowly, whereas amylopectin is digested rapidly. Therefore, in general, the RS content of starch is positively associated with the amount of amylose present. Other factors that affect the resistance of starch include the size and type of starch granules; the physical form of grains and seeds; plant genotype and mutations [genetic engineering can be used to increase RS content (Wei et al. 2010; Carciofi et al. 2012)]; crop-growing location; associations between starch and other food components [i.e. lipids, proteins, sugars, gums, other fibre types and plant bioactives that inhibit a-amylase (Lochocka et al. 2015)]; and food processing methods such as milling, cooking, annealing (physical modification of starch in water at temperatures below gelatinisation), high-pressure processing, autoclaving, cirradiation, extrusion and storage time (Seneviratne & Biliaderis 1991; Englyst et al. 1992; Muir & O’dea 1992; Chung et al. 2009; Chung & Liu 2010; Rohlfing et al. 2010; Singh et al. 2010; Pollak et al. 2011; Linsberger-Martin et al. 2012; Dundar & Gocmen 2013). Grinding of grains during processing can reduce RS1, due to the breakdown of cell walls, and cooking starchy foods in water can lead to gelatinisation, which reduces resistance to digestion. In © 2016 British Nutrition Foundation Health effects of resistant starch contrast, cooking and cooling before eating (e.g. new potatoes for potato salad) can produce RS3. A recent study reported that cooked then chilled potatoes contained more RS than either hot or reheated potatoes. In addition, baked potatoes had higher RS contents than boiled and there was no impact of potato variety on RS (Raatz et al. 2016). However, a further study found that there were significant differences in RS content between potato varieties (Pinhero et al. 2016). Food sources of resistant starch and dietary intake Table 1 details the different types of RS and Table 2 provides estimates of the total RS content of a variety of common foods, although it is important to reiterate that values will vary considerably due to factors such as cooking and storage. The best natural sources may include less ripe bananas, pasta, pulses and potatoes. It is of note that wholegrain versions of starchy foods (e.g. wholewheat pasta, brown rice) contain more RS than refined versions. In the 1990s, Baghurst and colleagues suggested that 20 g of RS per day is needed to confer benefits related to gut health (Baghurst et al. 1996); however, there are no official intake recommendations for RS. At present, dietary intakes of RS are not routinely reported and RS is not typically included in food composition databases or national dietary surveys (e.g. the National Diet and Nutrition Survey) as a discrete entity. Accurate assessment of dietary intake is difficult due to factors that affect concentration in foods such as ripening, natural variation, losses, which occur during cooking and storage, and potential gains due to the formation of RS3 after cooking and cooling. These factors, plus others listed in the previous section and differences related to the measurement of RS (see ‘Methods of analysis of Table 1 Food sources of resistant starch (RS) types RS1 RS2 RS3 RS4 RS5 Coarsely ground or whole kernel grain products (e.g. bread, seeds and legumes) Raw potatoes, green bananas, high-amylose maize, ginkgo starch Cooked, then cooled starchy foods such as potatoes, bread (especially stale bread), pasta, rice, cornflakes Foods containing chemically modified starches, such as cross-linked starch and octenyl succinate starch (e.g. some commercially produced breads and cakes) Foods containing naturally occurring amylose–lipid complexes, such as bread containing fat as an ingredient, or foods containing artificially made amylose–lipid complexes, such as stearic acid-complexed high-amylose starch © 2016 British Nutrition Foundation 3 Table 2 Typical amounts (g) of resistant starch present in selected foods relevant to the UK diet White bread rolls Wholemeal bread rolls Mixed grain bread rolls Porridge (cooked) Breakfast cereal, wheat biscuits Bran breakfast cereal White rice (long grain, cooked)† Brown rice (cooked)† Egg pasta Wheat pasta (white, cooked) Wholewheat pasta (cooked) Bananas (ripe) Bananas (green) Sweet potato Green beans Sweetcorn (canned)† Peas Hummus Baked beans Kidney beans† Chickpeas Lentils (cooked)† Potato crisps† Hot potatoes (e.g. boiled, mashed, baked chips) Cold potato dishes (e.g. potato salad) Potato products [e.g. hash browns, wedges (cooked)] Per 100 g (range) Per serving* 0.87 0.87 0.97 0.17 1.12 1.22 1.2 (0–3.7) 1.7 (0–3.7) 0.88 1.1 1.4 1.23 8.50 0.08 0.14 0.30 0.77 0.66 1.40 2.0 (1.5–2.6) 2.08 3.4 (1.6–9.1) 0.21 (2.9–4.5) 0.59 0.44 0.42 0.54 0.27 0.45 0.49 2.16 3.06 2.02 2.53 3.22 0.98 6.80 0.06 0.11 0.24 0.62 0.33 1.12 1.60 1.66 2.72 0.07 1.03 0.63 0.54 1.07 0.62 Values provided represent total RS, individual RS types not stated. Ranges given were reported. Items with no superscript, values derived from Landon et al. (2012). † Values derived from Murphy et al. (2008). *Serving sizes as per Food Portion Sizes (FSA 2006) or 80 g for fruit, vegetables and pulses. resistant starch in foods’ section), mean that quantities present in identical food types can vary. Estimates indicate that intakes are low in developed countries. For example, in the 1990s in the UK, average RS intake was estimated to be 3 g per day, an analysis published in 2008 estimated average intakes in the US to be 5 g per day (Murphy et al. 2008) and a recent study in Serbia calculated average intakes to be around 6 g per day, with RS contributing 30% of total dietary fibre (Dodevska et al. 2016). In developing countries, intakes may be up to 30–40 g per day due to differences in dietary composition (Baghurst et al. 2001). 4 S. Lockyer and A. P. Nugent In 2013, the United Nations declared 2016 to be the ‘Year of the Pulses’ in order to raise awareness of and position pulses as a primary source of protein and other essential nutrients, with messaging focusing on the nutritional composition and health benefits (FAO 2016). If successful, this initiative may increase consumption of pulses and therefore RS. Methods of analysis of resistant starch in foods A variety of methods exist to measure either total RS or specific types of RS in vitro, aiming to mimic human physiological conditions by first grinding samples (as would occur with mastication) or even self-chewing ( Akerberg et al. 1998), removing hydrolysable starch with enzymes and incubating at body temperature (as during digestion). These processes have been described in detail elsewhere (Perera et al. 2010). The Megazyme RS assay kit (Megazyme 2015), based on the American Association of Cereal Chemists Method (AACC 32-40; American Association of Cereal Chemists 2000) and AOAC 2002.02 method (McCleary & Monaghan 2002), is widely used at present. Different methods yield different quantitative results due to varying sample preparation, enzymes used and length of incubation periods. For example, the RS content of cornflakes has been reported as 2.2% when using the Megazyme assay (McCleary et al. 2002) and 3% and 6.5% using two other methods (Englyst et al. 1992; Muir & O’dea 1992). There is evidence that the amount of RS available in the colon after food consumption varies from person to person due to individual difference in chewing habits (Englyst et al. 1992). RS can also be determined in vivo in studies of patients with ileostomies (McCleary 2013). Challenges in developing food products with added resistant starches As mentioned above, cooking and preparation of food products can greatly affect the RS content. For example, extrusion cooking followed by cooling has been reported to increase RS3 formation in some studies, but decrease RS3 formation in others (Rabe & Sievert 1992; Adamu 2001); therefore, manipulating RS naturally present within foods can be complex. Manufactured purified forms of RS are commercially available and have been listed elsewhere (Fuentes-Zaragoza et al. 2011; Raigond et al. 2015). However, some types of RS are not suitable as ingredients in certain food products because they may be destroyed by the food processing methods (e.g. high temperatures in the case of RS1 and RS2). In general, RS has good potential as a functional ingredient due to its fine particle size, pale colour, bland flavour, good extrusion and film-forming properties, high gelatinisation temperature, low calorific value (1.6–2.8 kcal/g) and low water binding capacity, which can improve shelf-life of dry products and prevent the formation of ice crystals in ice cream (Elia & Cummings 2007; Tulley et al. 2009; Raigond et al. 2015). Such properties have led to RS being successfully incorporated into a variety a foods, such as dairy products, baked goods and pasta (Noronha et al. 2007; Almeida et al. 2013; Aravind et al. 2013). The first of these products was introduced commercially in Australia in the mid1990s (Roberts et al. 2004). RS has also been used in the microencapsulation both of probiotics in dairy products (Vandamme et al. 2016), in an attempt to increase viability, and of fish oil to reduce odour and oxidation (Nasrin & Anal 2015). Nevertheless, the inclusion of ingredients within products to increase the RS content requires careful consideration because of the potential impact on functional and rheological properties. For example, adding RS3 to batter can increase the crispiness and golden colour of the product (Sanz et al. 2008), but adding green banana flour to instant noodles can produce a weaker dough and less firm noodles due to the dilution of gluten, although less oil may be needed for frying (Vernaza et al. 2011). In addition, bread loaf volume has been reported to decrease with the addition of high levels of RS2 and RS3 (Ozturk et al. 2009) and when high-amylose wheat flour is used in bread making (Van Hung et al. 2005). Furthermore, RS2 and RS3 added to muffins has been found to have a negative impact on texture, making them more dense and less springy (Sanz et al. 2009). Safety Under EU Regulation 1333/2008, native starches and starch modified by acid or alkali treatment, bleached starch, physically modified starch and starch treated by amylolytic enzymes are considered to be normal ingredients rather than food additives (European Parliament 2008). After assessment by the European Food Safety Authority (EFSA) as part of a novel food application (EFSA Panel on Dietetic Products Nutrition and Allergies 2010b), one type of chemically modified starch, phosphated distarch phosphate © 2016 British Nutrition Foundation Health effects of resistant starch (RS4), was deemed safe and unlikely to be allergenic in adults and children, at a maximum level of 15%, when added to low-moisture food products (such as baked goods, pasta, snacks and breakfast cereals). An unpublished study submitted as part of the application reported no adverse effects in ten volunteers consuming 60 g of potato starch or five chemically modified starches, including phosphated distarch phosphate on 4 days per week for 6 weeks. Other studies testing both lower and higher doses of RS (up to 100 g/day) have reported increased gastrointestinal symptoms, such as diarrhoea in some subjects (Grabitske & Slavin 2009), but such high doses are far in excess of what would normally be (and could easily be) consumed by the general population. In February 2016, EFSA called for submission of technical data relating to certain starches and celluloses, including types of RS4 such as hydroxypropyl distarch phosphate (E1442) and acetylated distarch adipate (E1422), so that the safety of these additives can be reassessed (EFSA Food Ingredients and Packaging Unit 2016). Labelling and health claims RS meets the EU definition of fibre specified by Commission Directive 2008/100/EC (European Commission 2008), which refers to a statement produced by EFSA’s Panel on Dietetic Products Nutrition and Allergies (2007). As such, the energy value of RS from a labelling perspective is 2 kcal/g (European Commission 2011). Material constituting dietary fibre is defined as carbohydrate polymers with three or more monomeric units, which are neither digested nor absorbed in the human small intestine, and is either naturally occurring in food; obtained from food raw material by physical, enzymatic or chemical means (and with a beneficial physiological effect demonstrated by generally accepted scientific evidence) or edible synthetic carbohydrate polymers (that have a beneficial physiological effect demonstrated by generally accepted scientific evidence) (European Commission 2011). Declaring the quantity of fibre present in a food is not a mandatory component of nutrition information on food labels, but can be stated voluntarily in the format set out within the EU Food Information for Consumers Regulations (European Commission 2011). However, fibre values are not permitted on front-of-pack nutrition labels. Manufacturers can voluntarily claim foods as a ‘source of fibre’ if it contains at least 3 g of fibre per 100 g or 1.5 g of fibre per 100 kcal, or © 2016 British Nutrition Foundation 5 ‘high in fibre’ if it contains at least 6 g per 100 g or 3 g per 100 kcal. Where these claims are used, the fibre content must be listed in the nutrition information on the back-of-pack (European Commission 2006). A relevant health claim has been approved by EFSA with the wording ‘Replacing digestible starch with resistant starch induces a lower blood glucose rise after a meal’ (EFSA Panel on Dietetic Products Nutrition and Allergies 2011b). This health claim can be applied to RS from all sources used to replace digestible starch in high carbohydrate-baked foods (at least 14% of the total starch content must be RS). Health claim applications relating to RS and digestive health have not been authorised, as the biological effects in the submitted dossiers were deemed to be general, non-specific and not in reference to any specific health claims (EFSA Panel on Dietetic Products Nutrition and Allergies 2011b). According to EFSA guidance, physiological effects considered to be beneficial for the general population with regard to gastrointestinal (GI) health include reducing GI discomfort, which is considered an indicator of improved GI function, and the maintenance of normal defecation, defined as increasing the frequency of bowel movements and faecal bulk, and loosening the consistency of stools (e.g. as indicated by the Bristol Stool Form Scale) and decreasing transit time. Evidence for sustained health benefits with continuous consumption of the food/constituent over extended periods of time (e.g. 4–8 weeks) should be provided (EFSA Panel on Dietetic Products Nutrition and Allergies 2016). Claims relating to the maintenance of normal defecation have been evaluated with a favourable opinion for other fibre types, such as wheat bran (EFSA Panel on Dietetic Products Nutrition and Allergies 2010d), rye (EFSA Panel on Dietetic Products Nutrition and Allergies 2011d), oat and barley grain fibre (EFSA Panel on Dietetic Products Nutrition and Allergies 2011c) and the prebiotic inulin from chicory (EFSA Panel on Dietetic Products Nutrition and Allergies 2015). Unfavourable opinions were given to health claim applications relating to resistant maltodextrins and a reduction in postprandial glycaemic responses, maintenance of normal blood LDL-cholesterol concentrations, maintenance of normal (fasting) blood concentrations of triglycerides and changes in bowel function. This was because most of the references provided in the applications were unavailable in an EU language and, as such, causality could not be established (EFSA Panel on Dietetic Products Nutrition and Allergies 2011a). 6 S. Lockyer and A. P. Nugent Key points • RS is a type of dietary fibre. It passes through the small intestine intact and is then fermented in the large intestine, producing SCFAs. • RS provides ~2 kcal/g. • There are five different types of RS (RS1, RS2, RS3, RS4 and RS5). • RS is present in grain products, seeds, pulses, bananas, potatoes and in commercially purified forms, which can be added to food products. • RS intake is estimated to be around 3 g/day in the UK and is likely to be similar in other countries with Western-style diets. Resistant starch and colonic health As a fibre type, RS would be expected to confer benefits to gut health, particularly in the large intestine where RS is fermented and can result in the release of gases (methane, hydrogen, carbon dioxide), SCFAs (formate, acetate, propionate, butyrate and valerate) and smaller amounts of organic acids (lactate and succinate) and alcohols (methanol and ethanol). Described in detail elsewhere (Birt et al. 2013), this process involves several key bacterial groups, such as amylolytic gut bacteria (including Firmicutes, Bacteroidetes and Actinobacteria), which mediates starch (amylose) breakdown; butyrogenic archaea (including Eubacterium rectale), which is involved in butyrate production; and methanogenic archaea, which is necessary to produce methane. In general, studies that have investigated the influence of RS on gut health have focused on endpoints such as gas production, SCFAs and gut bacterial composition. Although less is known about the influence of RS on other breakdown products, the SCFA butyrate is of particular interest given its role as fuel for the cells lining the colon (colonocytes) and there is growing interest in the potential influence of gut bacteria on health outcomes, both locally in the gut and systemically (Kataoka 2016). The following section will examine the influence of RS on important markers of gut health and the gut microbiome. Short-chain fatty acid production As mentioned, the SCFA butyrate is the preferred fuel for colonocytes, increasing colonic blood flow, lowering luminal pH and helping prevent the development of abnormal colonic cell populations (Topping & Clifton 2001). The concentrations of all SCFAs are typically higher in the proximal colon where fermentation is greatest and the amount present relates to the supply of carbohydrates in the diet (Topping et al. 2003). In humans, SCFA abundance is typically acetate > propionate > butyrate (Schwiertz & Lehmann 2002). Dietary interventions that increase the amount of SCFAs, particularly butyrate, in the colon are thought to be beneficial to gut health, with such SCFA levels commonly used as a marker of fermentation and colonic health. However, faecal SCFAs are poor markers of colonic production as it is estimated that approximately 95% of SCFAs produced are absorbed by the colon (Cummings et al. 1987). Furthermore, faecal SCFAs may not be wholly reflective of fermentation at the luminal surface and may exhibit significant inter-individual variation (McOrist et al. 2011). Isotopic dilution has been suggested as the gold standard (Sakata et al. 2003). EFSA’s NDA Panel does not consider changes in SCFA production in the gastrointestinal tract to be a beneficial physiological effect per se, and states that any changes would need to be linked to a beneficial physiological or clinical outcome (EFSA Panel on Dietetic Products Nutrition and Allergies 2011e, 2011f). Earlier studies describe the ability of RS to modulate SCFA production in vitro and in animal and human feeding studies; this research has been reviewed elsewhere (Nugent 2005; Birt et al. 2013). While a number of human studies found increased faecal excretion of SCFA (concentrations and molar ratio), specifically butyrate, following supplementation with RS (Young & Le Leu 2004; Nugent 2005; Birt et al. 2013), not all studies conclusively show that all types of RS increase SCFA production in an equal manner. This is perhaps due to differences in study design and/ or control vehicle and given that SCFAs are both produced and absorbed by the colon (Cummings et al. 1987; see Table S1). For example, human colonic microbiota cultured with a laboratory generated RS3 increased butyric acid production (Lesmes et al. 2008). However, in interventions with healthy young adults, there were no changes in total faecal SCFA production following supplementation with RS3, in the form of a heat- and moisture-treated high-amylose maize starch (HAMS), at 12 g/day for 14 days (Stewart et al. 2010) or 20 g/day for 7 days (Klosterbuer et al. 2013), albeit that faecal propionate decreased by 8% (P < 0.05) in one study (Stewart et al. 2010) with a tendency (P > 0.05) for an increase in the proportion of butyrate. In another example, in middle-aged adults selected to have lower-than-average faecal butyrate concentrations, no change in faecal SCFA © 2016 British Nutrition Foundation Health effects of resistant starch concentrations was found following supplementation with RS2 (HAMS; 20 or 40 g/day for 14 days) (Clarke et al. 2011). In contrast, faecal SCFAs (acetate, propionate, butyrate and succinate) decreased relative to an NSP-rich diet or control diet following RS3 supplementation (26 g/day for 3 weeks) in 14 overweight males (Salonen et al. 2014). Esterified or acylated forms of RS (e.g. acetylated, butyrylated or propionylated RS4) offer specificity in SCFA delivery. This is because specific SCFA are esterified to a carrier starch and released only in the large intestine leaving the residual starch available for fermentation (West et al. 2013). Some initial studies have looked at the effects of butylated forms of HAMS or acetylated, propionylated or butyrylated HAMS in healthy adults and humans with ileostomies. In general, these studies report increases in free faecal butyrate (Clarke et al. 2007), free, bound and total butyrate and propionate (West et al. 2013) or delivery to the ileostomy site (Clarke et al. 2011). However, the lack of appropriate control groups (Clarke et al. 2011) and small sample sizes necessitate further study in well-designed, randomised controlled trials. The ability of naturally occurring RS to alter SCFA production is also of interest, with recent work chiefly focusing on barley that contains RS in addition to other fibre types (e.g. NSP). A number of studies from a Swedish research group took measurements in the morning following an evening meal of barley kernel bread, naturally rich in RS, or a white wheat flour bread control. This study design allowed adequate time for fermentation and study of ‘second meal effects’. Significant increases in fasting breath hydrogen (an indirect measure of colonic fermentation) of 140–160% (Nilsson et al. 2008, 2015; Sandberg et al. 2016), serum total SCFAs and acetate (both by 18%; Nilsson et al. 2015) and total plasma SCFAs, acetate and butyrate by 10–30% (Nilsson et al. 2010; Sandberg et al. 2016) were reported. Other work involving barley has focused on a novel high-amylose barley variety (Himalaya 292) rich in RS due to the absence of activity of a key enzyme in starch synthesis, starch synthase IIa. For example, a study was carried out in middle-aged overweight volunteers who consumed 103 g of foods rich in the Himalaya 292 grain or a wholegrain wheat cereal or refined cereal for four weeks; though exact amounts of RS consumed were not defined (Bird et al. 2008). Ninety-one per cent higher faecal excretion of butyrate and 57% higher faecal total SCFA excretion were reported compared with the refined cereal control (Bird et al. 2008). In a further study using Himalaya 292, the influence of RS © 2016 British Nutrition Foundation 7 from a combination of sources on faecal butyrate concentrations and excretion was examined (McOrist et al. 2011). For 4 weeks, 46 healthy adults consumed either 25 g of NSP or 25 g of NSP plus 22 g of RS from a mix of sources [Himalaya 292, canned legumes and HAMS (RS2; HI-MAIZEâ 260 Resistant Starch1)]. Overall, acetate, butyrate and total SCFA concentrations were significantly higher in the RS group, compared with baseline levels and control (NSP only group). However, there was a large degree of interindividual variation in responses, with differences in concentration and excretion also noted by gender (McOrist et al. 2011). It is perhaps unsurprising that effects of RS on SCFA production reported in vitro and in animal models are not replicated easily in human studies. Type and dose of RS, esterification and the food form in which RS is incorporated should be considered, given that RS is not a homogenous group. Recent in vitro experiments, examining the fermentation of RS3 from different plant sources by different bacteria, reported very different SCFA concentrations (Purwani et al. 2012). As mentioned, the majority of SCFAs produced are absorbed by the colon, with mechanisms thought to include non-ionic diffusion and transporter-mediated mechanisms coupled with sodium (Cummings et al. 1987; Scheppach 1994; Binder 2010). Hence, such effective utilisation by colonocytes may not allow for the detection of (subtle) differences in SCFA production between treatments (Anson et al. 2011). Furthermore, it has been suggested that faecal SCFAs (the main outcome measure of many studies) may only reflect production of SCFAs in the distal colon. Hence, positive outcomes may be more likely to arise with RS forms with more delayed fermentation rates (e.g. RS4; Upadhyaya et al. 2016) or those present in combination with other more slowly fermentable fibres (e.g. wheat bran; James et al. 2015). Factors affecting faecal SCFA concentrations other than site of digestion include transit rate and the moisture content of the digesta (Salonen et al. 2014). Other measures of SCFA production include peripheral blood (plasma/serum) concentrations and/or breath hydrogen. However, both are indirect measures and may lack sensitivity to detect treatment differences. In acute feeding studies, timing of measurements should also be considered. For example, in one study using isotopic dilution, increases in 13C-acetate were only observed <6 hours and increases in 13C1 The HI-MAIZE, NOVELOSE and INGREDION marks and logo are trademarks of the Ingredion group of companies. 8 S. Lockyer and A. P. Nugent propionate and 13C-butyrate only observed >6 hours post-consumption of labelled intact barley kernels (rich in RS and NSP; Verbeke et al. 2010). There appears to be large inter-individual variability in butyrate production and responsiveness to RS interventions, likely influenced by differences in microbiota composition between individuals. Such variability may be difficult to capture when using small sample sizes, short study durations (Cummings et al. 1996) or smaller doses of RS (<20 g/day; Topping & Clifton 2001). In one of the largest studies published to date [n = 46 with 25 g/day RS delivered via various foods (cereal, legumes, commercial RS preparations) for 4 weeks], approximately 10% variability in baseline total and individual SCFA production was noted, with body mass index (BMI) explaining 27% of variation in butyrate production (McOrist et al. 2011). Furthermore, differences in response were observed by gender and the greatest responses to RS supplementation were observed for individuals with the lowest habitual intakes. The greatest decrease in butyrate production was noted when baseline levels were high. Future studies should factor such considerations into their design. Influence of resistant starch on gut bacteria and the microbiome The influence of gut bacteria and the microbiota on human health is well recognised. Gut bacteria may influence immune function and nutritional acquisition (Sekirov et al. 2010); appetite control mechanisms [as demonstrated in mice (Lyte et al. 2016)]; disease states such as mental health disorders (Flowers & Ellingrod 2015) and obesity and its associated metabolic imbalances, including CVD (Arora & Backhed 2016; Kobyliak et al. 2016). Diet is believed to influence the microbial communities of the gastrointestinal tract (Birt et al. 2013) and so the potential of RS to influence gut bacteria is of interest. Prior to advances in DNA sequencing, the influence of RS on gut microbiota was inferred through its effects on colonic pH, SCFA composition, reductions in harmful metabolites (e.g. bile acids, phenols and ammonia) and enzymatic activity associated with bacterial degradative pathways. Early human intervention trials suggested reduced faecal ammonia, phenols, pH and secondary bile acid concentrations in faecal water with RS intake [for review, see Nugent (2005)]. More recent human studies (post-2005, see Table S1) typically show no statistical differences in stool pH following supplementation with RS (Martinez et al. 2010; Stewart et al. 2010; Clarke et al. 2011; Klosterbuer et al. 2013), with only one study (using the Himalaya 292 grain) reporting reduced stool pH (Bird et al. 2008). However, reduced faecal p-cresol concentrations (an end-product of protein breakdown) of a magnitude of 20–33% have been reported (Bird et al. 2008; Clarke et al. 2011), and higher ammonia concentrations, but not excretions, have been reported in males only (McOrist et al. 2011). Advances in sequencing platforms and culture-independent molecular methods, based on analysis of 16S ribosomal RNA, have allowed more detailed investigations into how bacterial communities interact with the different starch forms. For example, while one study reported no change in faecal pH, changes in the faecal microbial communities were described that were dependent on RS type (Martinez et al. 2010). In a double-blind crossover trial, ten healthy young adults consumed crackers containing approximately 30 g of RS2 (HI-MAIZEâ 260 Resistant Starch) or RS4 (FibersymTM) or control (4 g of fibre) for 3 weeks with a 2-week wash-out period. During this short intervention, both forms of RS increased representatives of the Actinobacteria and Bacteroidetes phyla and decreased Firmicutes. However, while RS2 increased the abundance of Ruminococcus bromii (Firmicutes) and E. rectale (Firmicutes), RS4 was associated with increased Bifidobacterium adolescentis (Actinobacteria) and Parabacteroides distasonis (Bacteroidetes), with the differential activities thought to reflect differences in substrate binding. Furthermore, there was a significant inter-individual variation in response among the ten volunteers (Martinez et al. 2010). Ruminococcus bromii has also been implicated as abundant in humans and significant in the fermentation of complex carbohydrates including RS2 (HIMAIZEâ 260 Resistant Starch; Abell et al. 2008), while P. distasonis reportedly facilitates the release of esterified butyrate from butyrylated HAMS (RS2; Clarke et al. 2011; West et al. 2013). The influence of approximately 25 g of RS3 or NSP (wheat bran) or a weight-loss diet on colonic microbiota in overweight men was studied (Walker et al. 2011). Using 16S ribosomal RNA gene sequencing, bacterial profiles were found to be consistent over time within an individual for a given diet, but to alter substantially between diets. Although lacking a true control group, R. bromii increased the most on the RS diet (17% of total bacteria vs. 3.2% after the NSP diet), while increases were also observed for the Oscillibacter (Oscillibacter guillermondii) bacteria during the RS diet and during the weight loss component. © 2016 British Nutrition Foundation Health effects of resistant starch Eubacterium rectale increased on the RS diet to approximately 10% of total bacteria. Subsequent indepth microarray analysis of this cohort revealed an overall reduced total number and diversity of microbiota following the RS diet (Salonen et al. 2014). In addition to confirming the changes in R. bromii and Oscillibacter guillermondii, they also noted changes in other lower abundance bacterial groups [e.g. increases in Ruminococcaceae (Sporobacter termitidis, Clostridium leptum and C. cellulosi)], Bacteroidetes (Alistipes spp.) and decreases in bacteria related to Papillibacter cinnamivorans. Furthermore, while diet explained 10% of the total variance in microbiota composition, as reported elsewhere (Martinez et al. 2010; McOrist et al. 2011), individual SCFA response was highly variable. In a follow-up in vitro study, R. bromii was identified as a key species for RS breakdown in the colon, facilitating RS fermentation by other species, even bacteria that are weak RS2 and RS3 fermenters in isolation (e.g. E. rectale and B. thetaiotaomicron; Ze et al. 2012). The influence of a wheat-derived RS4-enriched flour or a control flour on gut bacteria and health outcomes was recently studied in 20 individuals with metabolic syndrome (Upadhyaya et al. 2016). The authors reported increases in B. adolescentis, P. distasonis and the newly described species, Christensenella minuta, and no difference in R. bromii in response to 12 weeks’ consumption. The authors completed associational analysis, linking bacterial species with SCFA production and with markers of metabolic health, such as cholesterol, cytokines and adiponectin. For example, RS4-specific significant inverse correlations were observed between total cholesterol and abundance of Bacteroides plebeius (r = 0.46), Blautia producta (r = 0.49) and Prevotella stercorea (r = 0.45). Further research is needed to validate the associations observed in this cohort and whether they translate to healthy populations. In summary, a number of studies have investigated the influence of RS on gut bacteria. Advances in technology implicate key species such as R. bromii and E. rectale; however, considerable work remains to understand the number of species involved (including lower abundance groups), their interactivities and their subsequent host interactions, both locally in the gut and systemically (Lyte et al. 2016). Caution should be applied when interpreting animal and human studies as results are not always consistent. For example, only 10% of total variance in human gut microbiota composition has been ascribed to dietary RS (Salonen et al. 2014), yet the comparable value from murine © 2016 British Nutrition Foundation 9 studies is 60% (Zhang et al. 2010; Faith et al. 2011). Future studies in humans should examine the influence of the various RS types on gut microbiota response. All future studies should have suitable control groups and be of adequate length to allow for microbiota response (Wu et al. 2011), given that changes in microbiota composition (expressed as alternative enterotype states) are usually only achieved with dietary modifications lasting at least 10 days (Wu et al. 2011). Other influential factors include sex, genotype, medical history, medication use, habitual diet, bodyweight status (obesity), level of glycaemic control (diabetes), geographical location and the high degree of inter-individual variation apparent when studying RS– gut microbe interactions. Such variation in response is perhaps unsurprising given that each person has a distinct and highly variable intestinal microbiota (at least at a species level), although it has been suggested that a stable core of intestinal microbiota and genes (microbiome) are shared by individuals and may be related to intestinal function (Martinez et al. 2015). It has been proposed that any dietary advice on consumption of non-digestible carbohydrates may need to be personalised (Walker et al. 2011). Understanding of how RS interacts with gut bacteria is rapidly advancing. For example, new research comparing gut microbiota metabolites and the host metabolome in urban vegans and omnivores in the US suggests that diet has a large impact on metabolome, but a more modest impact on gut microbiota (Wu et al. 2016). The authors suggested diet as a substrate affecting bacterial metabolome rather than as a factor regulating gut bacteria community membership. Future studies investigating the influence of RS on gut bacteria should consider the inclusion of tools such as metabolomics to try to understand the complexities of how this fermentable starch may influence bacterial metabolome and biomarkers of bacterial activity, as well as focusing on bacterial community membership. Pre- and probiotics Prebiotics have been defined as ‘selectively fermentable ingredients that allow specific changes in the composition and/or activity of the gastrointestinal microbiota that allow benefits to the host’ (Gibson et al. 2010). Although various Bifidobacterium strains have been reported to hydrolyse RS (Crittenden et al. 2001) and benefits of RS appear related to SCFA production by colonic bacteria, studies in humans regarding the potential of RS as a prebiotic compound are limited 10 S. Lockyer and A. P. Nugent (Martinez et al. 2015). There are three classification guidelines proposed for prebiotic labelling: (i) resistance to gastric and gastrointestinal digestion; (ii) ability to be fermented and used by gut microbiota; and (iii) ability to selectively stimulate activity of one, or a limited number of, gut bacteria with health properties (Roberfroid et al. 2010). The current literature on RS does not allow for clear fulfilment of all three of these criteria. RS by nature is resistant to digestion in the small intestine and is fermented in the colon where it may be used by gut bacteria. However, it is not a homogenous group and it is probable that resistance to digestion, and most probably fermentation rates, varies by RS type (Purwani et al. 2012; Zaman & Sarbini 2016). Currently, the greatest challenge in defining RS as a prebiotic is proving its ability to selectively stimulate beneficial microorganisms (Martinez et al. 2015). Additional considerations include the significant inter-individual variations in gut microbiota and diet-induced responses, as well as factors such as age and geography (Br€ ussow 2013). Nevertheless, interest persists regarding the potential of RS to act as a fermentable substrate for growth of probiotic microorganisms; to act in concert with other prebiotics in a synergistic fashion; and/or to enhance the stability of probiotics, perhaps as part of an encapsulation process or by acting symbiotically with probiotics (Fuentes-Zaragoza et al. 2011). For example, RS has been coupled with short-chain fructo-oligosaccharides or inulin to increase prebiotic effects (Younes et al. 2001); co-encapsulated in the region of 1–2% as a HAMS to enhance the viability of probiotic cultures (Sultana et al. 2000; Iyer & Kailasapathy 2005); and used to extend the shelf-life of probiotic cultures in frozen dairy products (Homayouni et al. 2008). However, further research is needed before RS can be defined as a prebiotic. This evidence base should include well-designed human intervention trials involving molecular-based techniques. Finally, it has been proposed that there is a need for global consensus on standard procedures to confirm prebiotic potential using a combination of in vitro and in vivo testing (Zaman & Sarbini 2016). Such an initiative would support and standardise the study of RS as a prebiotic (Zaman & Sarbini 2016). Colon cancer Considerable evidence exists suggesting a protective role of high-fibre diets in colon cancer (SACN 2015). Fibre is suggested to reduce colon cancer risk by increasing faecal bulk, reducing transit time and diluting faecal contents (Aune et al. 2011; Murphy et al. 2012). In addition to these general benefits, RS is suggested as a positive influence on colon cancer risk by stimulating SCFA production, particularly butyrate. High butyrate production is reported to reduce colon cancer risk and butyrate treatment of cultured colon cancer cells can blunt the proliferation of the cancer cells and stimulate apoptosis (Macfarlane & Macfarlane 2003). Animal studies suggest that butyrate may reduce colorectal carcinogenesis by enhancing the apoptotic response to methylating carcinogens (Clarke et al. 2012) and that green banana flour (RS2) may prevent DNA damage through desmutagenesis and bio-antimutagenesis (Navarro et al. 2015). Proposed mechanisms of action include G-protein activity (GPR 43) and genetic and epigenetic modulation of the Wnt signalling pathway (e.g. inhibition of histone deacetylation, reduced DNA methylation and altered expression of miRNA; Fung et al. 2012; Malcomson et al. 2015). Despite a number of in vitro and animal studies suggesting that RS reduces colorectal cancer risk, there is less information available from human trials (Malcomson et al. 2015). Focusing on cell proliferation as a primary endpoint, two earlier studies with small numbers of healthy volunteers (n < 14) reported no effect (Wacker et al. 2002) or decreased cell proliferation (van Munster et al. 1994) following consumption of RS2 (amylomaize at doses up to 59.7 g/day). Similarly, in a randomised, placebo-controlled 4-week crossover trial of 20 healthy volunteers, no robust change in cell proliferation or DNA methylation was reported following consumption of 25 g of HAMS (12.5 g of RS2; Worthley et al. 2009). In contrast, reduced cell proliferation in the upper colonic crypt and differential expression of key cell cycle regulatory genes in 65 adult patients with colorectal cancer has been reported following consumption of a 30 g/day blend of RS2 (NOVELOSEâ 240 Resistant Starch) and RS3 (NOVELOSEâ 330 Resistant Starch) for 4 weeks (Dronamraju et al. 2009). Such differences in response may relate to the health status of the tissue under study whereby butyrate may increase proliferation in healthy colonic cells (Malcomson et al. 2015), but suppress proliferation in cancer cells (Williams et al. 2003). The DNA mismatch repair status of cells may also determine proliferative response (Dronamraju et al. 2009). Furthermore, it has also been suggested that focusing on proliferation in the crypt (one of the earliest detectable pre-malignant changes) may be more sensitive than focusing on total cell proliferation (Malcomson et al. 2015). © 2016 British Nutrition Foundation Health effects of resistant starch To date, two major randomised controlled trials have investigated the influence of RS on colon cancer incidence and risk, the CAPP-1 (Burn et al. 2011) and CAPP-2 studies (Mathers et al. 2012). Both studies were large multi-centre, placebo-controlled trials in groups at risk of colon cancer. CAPP-1 involved 133 young people (aged 10–21 years) with familial adenomatous polyposis who were randomised to consume aspirin (600 mg/day) and/or 30 g of RS2 (1:1 blend of potato starch and HAMS; Hylon VII). CAPP-2 involved over 900 individuals with hereditary colorectal cancer (Lynch syndrome) who were randomised to consume either 600 mg of aspirin or aspirin placebo and 30 g of a RS source (RS2: NOVELOSEâ 240 Resistant Starch and RS3: NOVELOSEâ 330 Resistant Starch; final dose 13.2 g of RS/day) or a starch placebo (Amioca waxy starch). The median follow-up for CAPP-1 was 17 months and for CAPP-2 52.7 months. In both studies, RS intake had no clinical effect on polyp number and size (Burn et al. 2011) or on cancer development (Mathers et al. 2012). Such studies highlight the difficulty with translating science from controlled in vitro and animal studies to human clinical trials and the need to reach consensus on gold standard biomarkers for the indication of colorectal cancer risk. As yet, the influence of RS on cancer risk in the general population is unknown. Consideration should be given to the type of RS and dose in any future clinical trial, as at least 20 g of RS/day may be needed to increase stool levels of SCFA (Topping & Clifton 2001). Furthermore, future studies should account for the influence of the gut microbiome on the development of colon cancer, as different microbial populations have been identified in observational studies of patients with colorectal cancer and healthy controls (McCoy et al. 2013). Diets rich in red meat have been linked to an increased risk of colon cancer, particularly in the distal region (Bouvard et al. 2015). Following high meat consumption and in the absence of fermentable carbohydrates, it is suggested that red meat may undergo fermentation in the colon and alter the microbiota composition (Le Leu et al. 2015). Hence, a high-protein, reduced carbohydrate diet may alter the colonic microbiota favouring a more pro-inflammatory microbiota profile and decreased SCFA production. The potential of RS to offset this risk, by yielding greater fibre fermentation in the distal colon and attenuating red meat-induced colorectal DNA lesions, has recently been explored (Le Leu et al. 2015). In this study, 23 © 2016 British Nutrition Foundation 11 healthy volunteers consumed 300 g/day of cooked red meat with or without 40 g of a butyrylated HAMS (RS4) for 4-week periods in a randomised crossover design. Inclusion of butyrylated RS4 prevented the increase in the pro-mutagenic DNA adduct O6-methyl-2-deoxyguanosine in rectal epithelial cells observed following the high-red meat diet. Furthermore, inclusion of RS increased stool SCFA concentrations, reduced p-cresol concentrations and induced more favourable changes in composition of the gut microbiota (Le Leu et al. 2015). This suggests that inclusion of RS in the high-meat diet facilitated a switch from fermentation of protein substrates to carbohydrate substrates (SCFAs), leading to a decrease in the production of pro-mutagenic adducts that arise during protein fermentation (Yao et al. 2016). A second proposed mechanism was via increased telomere length, which can protect against DNA damage. The potential for RS to attenuate disease risk associated with high-protein (meat) diets merits further study. Conditions associated with disturbances in gastrointestinal function There is considerable interest in the influence of RS on disturbances in gastrointestinal function (e.g. constipation and diverticulitis), as well as conditions such as irritable bowel syndrome (IBS) and inflammatory bowel diseases (IBD; e.g. ulcerative colitis and Crohn’s disease). As a component of dietary fibre, RS can contribute to daily fibre intakes deemed adequate for normal laxation in adults [25 g/day according to EFSA (EFSA Panel on Dietetic Products Nutrition and Allergies 2010a) and 30 g/day according to SACN in the UK (SACN 2015)], with associated benefits such as increasing stool bulk (faecal dilution) and consistency and decreasing transit time and stool pH [for review, see Maki et al. (2009); Abellan Ruiz et al. (2015); Nugent (2005)]. For individuals with constipation, diverticulitis or IBS, for whom fibre intake is an important part of dietary therapy, RS-containing foods may make a useful addition to total dietary fibre intakes. Disruptive changes in gut microbiota (dysbiosis) and intestinal host–microbe interactions are reported in IBD and colorectal cancer, including reduced microbial diversity, particularly in butyrate-producing bacteria (Hu et al. 2016). Furthermore, studies in healthy children have revealed significant differences in microbial profiles between native African children consuming fibre-rich traditional foods vs. children living in Europe (De Filippo et al. 2010). 12 S. Lockyer and A. P. Nugent Animal models suggest that RS3 intake reduces ileal and colonic inflammatory lesions in wild-type mice and mice with induced colitis (IL-10-knockout mice), with roles suggested for IL-10, IFN-c and the transcription factor PPARc (Bassaganya-Riera et al. 2011). In addition, rats fed RS (HAMS, RS2) at 10% of total diet or in combination with green tea extract (0.5% GTE) had reduced inflammation assessed by the expression of COX-2, NF-KB, TNF-a and IL-1b mRNA and cell proliferation (Hu et al. 2016). However, mixed results have been obtained from two human randomised controlled trials designed to evaluate the effect of RS on immune function. In one of these studies, decreases in the cytokines IL-10 and TNF-a in physically active adults were reported following consumption of a butyrylated HAMS for 4 weeks (West et al. 2013), whereas there were no differences in a panel of other cytokines assessed. Similarly, no influence was seen on high-sensitivity Creactive protein or a panel of 11 cytokines in a trial involving 17 older adults who consumed 25 g/day HAMS (HI-MAIZEâ 958 Resistant Starch, ~12.5 g of RS2) alone or in combination with a probiotic for 4 weeks (Worthley et al. 2009). More recently, the hypothesis that low-dose RS intake (RS2, Hylon VII 70%, 8.5 g/day for 4 weeks) could decrease intestinal inflammation was tested in Malawian 3-5 year-old children (Ordiz et al. 2015). Modest changes in SCFA production and bacterial populations were noted (increased Actinobacteria and reduced Firmicutes), although inflammation was not reduced, along with increased concentrations of calprotectin and up-regulation of the bacterial lipopolysaccharide biosynthesis pathway, both indicators of increased inflammation (Ordiz et al. 2015). However, there was no control group in this study and there were no clinical manifestations of increased inflammation. Hence, the influence of RS on immune function in healthy populations and in children with existing disease is unclear. The benefits of using dietary fibre and RS in the clinical management of IBD are uncertain. To address this, fibre intakes and colonic fermentation in patients with ulcerative colitis in remission following consumption of their habitual diet and when dietary fibre was increased were compared with the responses of matched healthy subjects (James et al. 2015). The intervention involved consumption of foods containing a high wheat bran–RS mix [12 g of wheat bran and 15 g of RS (RS1 and RS2) per day] or a low wheat bran–RS mix (2–5 g each of wheat bran and RS per day) over a 17-day period. RS was combined with wheat bran to delay fermentation and butyrate production from the proximal to the distal colon and rectum (fermentation at the distal and rectal sites being of most potential benefit in ulcerative colitis). Total fibre intake was lower in the ulcerative colitis patients than in the healthy controls. Encouragingly, the ulcerative colitis patient group tolerated the RS mix and showed some normalisation of gut transit post-intervention. However, they also displayed a reduced ability to ferment dietary fibre, with intake of the RS and wheat bran mix having little effect on faecal fermentation patterns or microbiota structure. More studies specific to this population group are needed (James et al. 2015). Oral rehydration solutions Diarrhoea is a common feature of many gastrointestinal conditions and severe diarrhoea can cause dehydration, with associated morbidity and mortality, particularly in children with acute infectious diarrhoea. Since the 1960s, oral rehydration solutions have been effectively used to treat dehydration associated with diarrhoea. In their simplest form, oral rehydration solutions are isomolar, glucose–electrolyte solutions with added base designed to correct dehydration and metabolic acidosis (Series 1978). However, simple glucose-based oral rehydration solutions do not reduce the duration or severity of diarrhoea as they can only correct water loss occurring in the small intestine (Binder et al. 2014). In contrast, RS may be useful in the treatment for diarrhoea as it is known that SCFAs can enhance fluid and sodium absorption in the colon, which is an important regulator of water absorption (Ramakrishna et al. 1990; Monira et al. 2010; Food and Drug Administration 2015). Hence, potential exists to use RS as an adjunct and/or replacement for glucose in oral rehydration solutions, providing a low-osmotic source of digestible glucose and taking advantage of the normal function of microbial fermentation and SCFAs in electrolyte and water absorption (Binder 2010; Food and Drug Administration 2015). To date, there have been two clinical trials in adults with cholera, one study in children with non-diarrhoeal cholera and one in severely malnourished children with dehydrating cholera (three involving isomolar and one a hypomolar RS-containing oral rehydration solution). All tested the effects of HAMS RS2 on dehydration (Ramakrishna et al. 2000, 2008; Raghupathy et al. 2006; Alam et al. 2009). In three studies, the oral rehydration solutions containing HAMS were associated with a 30–50% reduction in time of © 2016 British Nutrition Foundation Health effects of resistant starch occurrence of the first formed stool (Ramakrishna et al. 2000, 2008; Raghupathy et al. 2006). While benefits were noted in the study of Alam et al. (2009), they were not statistically significantly different from regular oral rehydration solutions. However, it is of note that this study population included severely malnourished children who also received nutritional supplements (Alam et al. 2009). Other RS forms, particularly acylated or esterified starches (RS4), for the treatment of diarrhoea are being considered for study due to their ability to target SCFA stimulation in the colon (Binder et al. 2014). Currently, the US authorities have awarded a ‘Generally Recognised as Safe’ status to the use of HAMS as an ingredient in oral rehydration solutions (Food and Drug Administration 2015). Key points • RS increases the production of SCFAs in the gut but there appears to be significant inter-individual variation in responses. • RS modulates the composition of gut microbiota, specifically those involved in amylose breakdown and butyrate and methane production, but responses are variable and health implications remain to be elucidated. • There is some evidence that RS can counteract the detrimental effects of high red meat intake on colorectal cancer risk. • Emerging evidence suggests a role of RS as an ingredient in oral rehydration solutions. Role of resistant starch in metabolic responses – lipid and glucose metabolism, insulin resistance and prevention of cardiovascular disease, type 2 diabetes and metabolic syndrome This section describes the most recent developments regarding the influence of RS on markers of metabolic health in humans. Obesity and type 2 diabetes have been associated with metabolic and cardiovascular disturbances such as insulin resistance, high blood pressure, chronic low-grade inflammation and abnormal blood lipid profiles (Stanner 2005). Risk factors for metabolic health can include genetics and ethnicity, as well as lifestyle attributes including physical activity and the diet. The influence of RS on markers of metabolic health is summarised below. EFSA has recently reviewed the evidence relating to non-digestible starch (EFSA Panel on Dietetic © 2016 British Nutrition Foundation 13 Products Nutrition and Allergies 2014a); a commercial RS, HAMS (RS2; EFSA Panel on Dietetic Products Nutrition and Allergies 2011b); and a soluble fibre with resistant maltodextrin mix (Nutriose 06TM; EFSA Panel on Dietetic Products Nutrition and Allergies 2014b). These Scientific Opinions sought to find out whether ‘sufficient evidence exists to establish a cause and effect relationship between the consumption of foods/beverages containing non-digestible carbohydrates (including RS) and a reduction in postprandial glycaemic responses, as compared with foods/beverages containing glycaemic carbohydrates’. The reviews all concluded that a cause and effect relationship had been established between the consumption of foods/beverages containing non-digestible carbohydrates and a reduction in postprandial glycaemic responses, compared with the effect of foods/ beverages containing glycaemic carbohydrates. As described earlier, conditions of use with respect to the RS claim include a threshold of 14% replacement of total starch with RS for a target population of ‘people wishing to reduce their postprandial glycaemic responses’. It remains to be established whether RS functions independently of simply reducing the total amount of available carbohydrate; whether glycaemic effects are equal across the various RS types (and food vehicles); whether there are differences in response of individuals with normal glycaemic responses and those with type 2 diabetes; and whether effects on metabolic health go beyond glucose and insulin metabolism (Robertson 2012a; de la Hunty & Scott 2014). Studies on the effects of RS on metabolic health published after the EFSA opinions are detailed in Tables S2–S4; the majority of these have focused on the commercial ingredient HAMS-RS2. A smaller number of studies have investigated the influence of RS from bananas/plantain, brown beans, barley, wheat breads, porridges or rice (Table S3) or novel RS4 or RS3 ingredients (Table S4). Broadly speaking, the papers could also be classified into those that matched the intervention and control foods for available carbohydrate content and those that did not (see Tables S2–S4), with the majority having a crossover design, subjects acting as their own controls and short follow-up periods. Most studies involved healthy individuals without insulin resistance/type 2 diabetes. The doses of RS ranged from as low as ~1.4 g of RS contained in a serving of bread to ~25 g of RS3 (NOVELOSEâ 330 Resistant Starch) and ~48 g of commercial RS2 (HAMS-RS2) in a powder form to be consumed each day. 14 S. Lockyer and A. P. Nugent In agreement with the EFSA opinion, in studies where the available carbohydrate content was not matched, and irrespective of the food vehicle or RS type involved, RS consumption was generally associated with improved glycaemic outcomes (i.e. reduced blood glucose and insulin responses). Only three of the studies that were unmatched for available carbohydrate content failed to see a statistically significant change in glucose concentrations (Penn-Marshall et al. 2010; Gargari et al. 2015; Sarda et al. 2016). In all instances, low doses of RS were used (5, 6 and 7.4 g of RS/day, respectively) over periods of 6–8 weeks, with intakes at this level unlikely to meet the 14% of total starch threshold suggested by EFSA. In contrast, in studies where available carbohydrate contents were matched the findings were less consistent. Overall, supplementation with RS can be said to improve markers of glycaemic control. However, the magnitude and direction of effect is not always consistent, even within a particular study design. Regarding blood glucose, effects reported included reduced fasting blood glucose concentrations, reduced postprandial responses (reduced area under the curve) and enhanced skeletal muscle uptake of glucose (AlTamimi et al. 2010; Johnston et al. 2010; Hallstr€ om et al. 2011; Karupaiah et al. 2011; Rosen et al. 2011; Bodinham et al. 2012, 2014; Robertson et al. 2012; Ekstrom et al. 2013; Ames et al. 2015; Lin et al. 2015; Nilsson et al. 2015; Boll et al. 2016; Gower et al. 2016; Oladele & Williamson 2016; Sandberg et al. 2016). However, less clear evidence of benefits were noted by others (Robertson et al. 2005; Bodinham et al. 2010, 2013; Johnston et al. 2010; Maki et al. 2012; Lobley et al. 2013). Improvements in insulin secretion and/or sensitivity have also been noted (Robertson et al. 2005, 2012; Al-Tamimi et al. 2010; Bodinham et al. 2010, 2013; Haub et al. 2010; Johnston et al. 2010; Karupaiah et al. 2011; Maki et al. 2012; Ekstrom et al. 2013; Lin et al. 2015; Nilsson et al. 2015; Gower et al. 2016; Sandberg et al. 2016), but not consistently (Johnston et al. 2010; Bodinham et al. 2012, 2014; Johansson et al. 2013; Lobley et al. 2013). Overall, differences in study design, primary outcome and study population make drawing conclusions on efficacy difficult. Acute feeding studies are helpful in understanding the direct effects of RS on postprandial glycaemic response to a specific meal (Robertson et al. 2005; Al-Tamimi et al. 2010; Bodinham et al. 2010, 2013; Haub et al. 2010; Hallstr€ om et al. 2011; Rosen et al. 2011; Ames et al. 2015; Lin et al. 2015; Nilsson et al. 2015; Boll et al. 2016; Oladele & Williamson 2016; Sandberg et al. 2016). Chronic feeding studies (longer-term interventions ranging from 4 to 12 weeks) in both healthy populations and individuals with insulin insensitivity/type 2 diabetes are helpful in understanding the influence of RS on fasting and postprandial glycaemic response (Robertson et al. 2005, 2012; Johnston et al. 2010; Penn-Marshall et al. 2010; Bodinham et al. 2012, 2014; Kwak et al. 2012; Maki et al. 2012; GarcıaRodrıguez et al. 2013; Lobley et al. 2013; Gower et al. 2016). The results of these studies describing the impact of RS on glycaemic health are summarised in Tables S2–S4. The majority of the studies used HAMS-RS2 with doses ranging from 15 to 48 g, but mainly as 40 g of RS from HAMS-RS2 (or 67 g of the parent product, HI-MAIZEâ 260 Resistant Starch) and compared with 27 g of a highly digestible control starch (Amioca). Forty grams was deemed to be the maximum RS dose which could be delivered without adverse effects on taste or texture (Bodinham et al. 2010). Most of the papers using HAMS-RS2 were published by a single research group (from the University of Surrey, UK). RS2 was consumed in these studies as sprinkles (powder), in baked goods (e.g. breads, crackers or muffins) and in dessert items (e.g. mousse). The influence of RS types other than HAMS-RS2 on glycaemic control, matched for available carbohydrate content, is less well characterised, though studies have been published on a novel RS4, a novel retrograded starch, unripe plantains, barley tortillas and a brown rice variant (Al-Tamimi et al. 2010; Haub et al. 2010; Karupaiah et al. 2011; Lin et al. 2015; Oladele & Williamson 2016). RS doses used in these studies were typically much lower than used for HAMS-RS2 (ranging from 1.4 g/serving to approximately 20 g of RS/ meal), and, in general, positive effects on glycaemic responses were observed vs. glucose controls, including the one study that compared RS4 with RS2 (HAMS; Haub et al. 2010). Similar findings have been reported in acute studies where rye, wheat or barley grains naturally containing RS were incorporated into breads or porridge, with generally positive effects on glycaemic responses vs. glucose controls. Again, RS doses provided were lower than those typically used with HAMS-RS2 (Hallstr€ om et al. 2011; Rosen et al. 2011; Johansson et al. 2013; Nilsson et al. 2015; Boll et al. 2016; Sandberg et al. 2016). In the papers identified, responses to RS were smaller in magnitude than those to oligosaccharides (Boll et al. 2016) or b-glucans (Ames et al. 2015), but appeared to act synergistically with guar gum (Ekstrom et al. 2013). Hence, the potential may exist for pure RS ingredients to act © 2016 British Nutrition Foundation Health effects of resistant starch in concert with other fibre types to improve glycaemic control. Of the studies identified, which included participants at risk of the metabolic syndrome, insulin resistance and/or type 2 diabetes, the majority were longterm in nature and used HAMS-RS2, although one study used a novel RS4 and another an RS3 ingredient (NOVELOSEâ 330 Resistant Starch). The effects of RS consumption on glucose and insulin metabolism as primary endpoints were variable, but no adverse effects on glycaemic management were observed (Penn-Marshall et al. 2010; Bodinham et al. 2012, 2014; Kwak et al. 2012; Robertson et al. 2012; Garcıa-Rodrıguez et al. 2013). Greater improvements in glycaemic control were noted following modest weight loss than following a high-fibre diet that included either RS (25 g of RS3/day) or NSP (42 g/ day; Lobley et al. 2013), indicating that bodyweight control remains the primary modifiable factor in glycaemic management. Currently, the influence of RS on insulin and glucose responses, independent of carbohydrate availability, is not well established. This is partly due to the methodological challenges in capturing glucose absorption, clearance and hepatic release (Robertson 2012a). Furthermore, different methodological approaches can make inter-study comparisons difficult (e.g. use of minimal model vs. euglycaemic–hyperglycaemic clamps for assessing insulin sensitivity). There is no evidence to suggest adverse effects of RS on glycaemic control in healthy subjects or in those with type 2 diabetes, with studies either reporting a benefit or no response (Nugent 2005). More comprehensive studies in humans, accounting for normal and insulinresistant phenotypes and responses during challenge tests, are required before definitive effects and doses can be defined. Future studies should continue to account for the fat and protein composition of the test diets, in addition to available carbohydrate content, given that these macronutrients can influence glycaemic response (Nugent 2005; Evans 2015). It would also be helpful to understand whether the benefits observed with HAMS-RS2 extend to other RS types in foods with differing physicochemical properties (e.g. rice and plantain). It is also unknown whether simultaneous consumption of the various RS forms has any additive or synergistic effects on glycaemic control, or indeed the exact influence of RS when consumed alongside other fibre forms. There is some suggestion that the influence of RS on glycaemic control may be confounded by other constituents in foods (Oladele & Williamson 2016) and further research is © 2016 British Nutrition Foundation 15 needed to understand the influence of the food matrix and subsequent interactions between the RS types, food matrix and other fibre forms in foods as consumed. Influence of resistant starch on other markers of metabolic health Metabolic health is influenced not only by glucose metabolism but also by circulating lipids, hormones and immune mediators. The balance of evidence does not suggest a role of RS in mediating lipid metabolism via total, LDL- or HDL-cholesterol (Robertson et al. 2005, 2012; Johnston et al. 2010; Shimotoyodome et al. 2011; Bodinham et al. 2012; Kwak et al. 2012; Gower et al. 2016). However, a recent study found a significant reduction in total cholesterol and nonHDL-cholesterol after the consumption of a RS-rich diet vs. a fibre-rich diet for 12 months (see Table S3; Dodevska et al. 2016). Most studies do not demonstrate an influence of RS on circulating triglycerides in healthy adults (either fasting or postprandially; Robertson et al. 2005, 2012; Johnston et al. 2010; Shimotoyodome et al. 2011; Bodinham et al. 2012; Kwak et al. 2012; Garcıa-Rodrıguez et al. 2013; Edwards et al. 2015; Dodevska et al. 2016; Gower et al. 2016). RS affected triglyceride concentrations in two studies with type 2 diabetics, albeit in opposing directions. In the first of these studies, TAG concentrations were reduced by 15% (i.e. associated with reduced risk; Gargari et al. 2015), while the other study reported higher fasting TAG in the RS group (Bodinham et al. 2014; see Table S2). Using sophisticated MRI scanning, the latter study also identified increased skeletal muscle cellular TAG (soleus intramyocellular triglycerides) after RS consumption, indicative of increased skeletal muscle uptake of fatty acids. The increase in soleus intra-myocellular triglycerides occurred despite improvements in glucose tolerance. As noted by the authors, the clinical significance of the effect is unknown, molecular mechanisms unclear and sample size very small (n = 12). Non-esterified fatty acids (NEFA) are vehicles by which triglycerides, stored in adipose tissue, are transported to the site of utilisation and so are indicative of lipolysis. Circulating NEFA concentrations are typically reduced by insulin (e.g. after a carbohydrate-containing meal); hence, any improvements in insulin function may be associated with reductions in NEFAinduced lipolysis. There is some evidence of reduced circulating NEFA concentrations following RS intake. For example, one study reported that 12 weeks of 16 S. Lockyer and A. P. Nugent 40 g of RS2 (HAMS-RS2) consumption by participants with type 2 diabetes resulted in reductions in plasma NEFA concentrations (both in the fasting state and postprandially), with NEFA levels being inversely related to soleus intra-myocellular triglycerides (Bodinham et al. 2014). Another study reported that consumption of 30 g of RS2 for 12 weeks by healthy adults resulted in no change in fasting NEFA levels, but a decrease in postprandial NEFA concentrations (Robertson et al. 2005), while a later study reported decreased skeletal muscle uptake of NEFA in individuals with insulin resistance after an 8-week intervention of 40 g of RS2/day (Robertson et al. 2012). In other studies using barley bread, reduced NEFA concentrations were reported the morning after the RS-rich meal (Johansson et al. 2013), while a similar study reported the opposite effect (Nilsson et al. 2008). Typically, other studies reported no effects (see Tables S2– S4; Shimotoyodome et al. 2011; Kwak et al. 2012; Maki et al. 2012; Garcıa-Rodrıguez et al. 2013; Edwards et al. 2015; Gower et al. 2016). In summary, taking the totality of the evidence into account, conclusions cannot be made about how RS influences NEFA activity. However, there are suggestions from more comprehensive studies that RS may, at least in those with type 2 diabetes, improve glucose tolerance through mechanisms involving increased muscle uptake of free fatty acids. There is a need to examine this idea further by comparing individuals with normal and impaired glycaemic responses. Finally, it should be noted that there is limited evidence suggesting that RS does not influence vascular function, including peripheral or vascular stiffness (Johnston et al. 2010), or blood pressure (Kwak et al. 2012; Nichenametla et al. 2014). Furthermore, there does not appear to be a role of RS in mediating effects through the activity of the adipose tissue derived hormones, adipokines, such as adiponectin (Robertson et al. 2005, 2012; Maki et al. 2012; Bodinham et al. 2014; Gower et al. 2016; Sandberg et al. 2016), leptin (Johnston et al. 2010; Maki et al. 2012; Robertson et al. 2012) or resistin (Johnston et al. 2010). Proposed mechanisms/sites of action of resistant starch Attempts have been made to understand how RS influences glycaemic control, with putative mechanisms summarised in Figure 1 and described below. Statistical modelling suggests that HAMS-RS2 supplementation increases first-phase insulin secretion by over a third (36%; Bodinham et al. 2012). This is significant given that loss of first-phase insulin secretion is wellcharacterised in type 2 diabetes and targeted by pharmaceutical treatment for this disease. Other studies have investigated whether RS may benefit peripheral or hepatic insulin clearance. Two papers suggest that a reduced postprandial response may be due to increased hepatic insulin clearance based on higher C-peptide/insulin ratios (Robertson et al. 2005; Bodinham et al. 2010). However, later studies have not found evidence for this effect (Bodinham et al. 2012, 2013). Furthermore, Robertson and colleagues have reported that RS improves peripheral glucose uptake in the periphery (forearm skeletal muscle), rather than influencing hepatic production, independently of changes in bodyweight or macronutrient/energy intake (Robertson et al. 2005, 2012). However, in a subsequent study by the same group and using the same methodology, albeit in a diabetic population, there was no significant effect on hepatic or peripheral Resistant starch Unmatched for available carbohydrate content Decreased glycaemic response due to less available carbohydrate content Matched for available carbohydrate content • Influence on glucose and insulin metabolism; increased glucose uptake in periphery • Possible influences of short-chain fatty acids post-fermentation: • ↓ lipolysis • ↓ endotoxaemia and inflammation • gut hormone production (e.g. ↑ GLP-1 and insulin secretion) Figure 1 Schematic overview of putative mechanisms of action of resistant starch on glycaemic control © 2016 British Nutrition Foundation Health effects of resistant starch insulin sensitivity and only a trend (P = 0.07) for greater glucose uptake in the forearm muscle; greater effects were observed in terms of muscle uptake of fatty acids (Bodinham et al. 2014). It should be noted that the latter cohort involved a group of 17 individuals with well-controlled type 2 diabetes using a combination of medications. Additional work is required to tease out the influence of RS on insulin secretion and/ or sensitivity and establish whether RS mediates effects peripherally (Bodinham et al. 2014). RS may have beneficial effects on glycaemic control through colonic production of SCFAs, then subsequent absorption, by exerting anti-lipolytic activity; affecting the activity of gut hormones, such as the incretins (which stimulate insulin secretion; Delzenne 2005); and reducing endotoxaemia, thereby reducing markers of inflammation (Cani et al. 2007). SCFAs, propionate and acetate (the generation of which is described in the section on colonic health) are absorbed from the gut into the circulation and are taken up into adipose tissue and skeletal muscle tissue where they are directly associated with improved insulin sensitivity. It has also been proposed that they stimulate adipose tissue receptors (FFA2/3) and lower NEFA concentrations (and reduced lipolysis) as described above. Attempts have been made to elucidate how RS may influence lipolysis at a transcriptional level and there are some suggestions of increases in genes coding for enzymes involved in adipocyte differentiation and mobilisation. For example, in 15 individuals with insulin resistance, HAMS-RS2 (40 g for 8 weeks) consumption increased adipose tissue gene expression (~twofold) for lipoprotein lipase, adipose triglyceride lipase, perilipin and hormone-sensitive lipase, but not for PPARc or visfatin (Robertson et al. 2012). In contrast, in a study of ten healthy adults, intake of 30 g of RS from HAMS-RS2 did not influence the expression of genes measured in adipose tissue or skeletal muscle, including lipoprotein lipase. In this second study, only increased expression of hormone-sensitive lipase (involved in the release of stored free fatty acids in adipose tissue) and insulin receptor substrate-1 (involved in insulin signalling pathways) was noted (Robertson et al. 2005). Perhaps this lack of effect is unsurprising given the insulin-sensitive nature of the group. More comprehensive mechanistic studies are needed that account for level of insulin resistance and any potential site-specific differences between skeletal muscle and adipose tissue. Animal studies suggest that RS increases glucagonlike peptide-1 (GLP-1) production (Zhou et al. 2008). In human studies, there are some reports of increased plasma GLP-1 concentrations following consumption © 2016 British Nutrition Foundation 17 of RS-containing foods (barley or rye kernel breads; Nilsson et al. 2008, 2015; Sandberg et al. 2016) and HAMS-RS2 by individuals with well-controlled type 2 diabetes (Bodinham et al. 2014), though the most comprehensive study to date does not support a role of GLP-1 in enhancing insulin sensitivity following HAMS-RS2 intake in healthy humans (Bodinham et al. 2013). Interestingly, in a study of 17 individuals with type 2 diabetes (Bodinham et al. 2014), decreases in fasting GLP-1 concentrations were observed after consumption of HAMS-RS2 (40 g/day RS2) for 12 weeks. However, the opposite effect (i.e. increased GLP-1) was observed during a postprandial challenge. Given that this increase in GLP-1 occurred alongside improved glucose disposal and without influencing insulin response, the authors suggest that this improved meal handling in people with type 2 diabetes occurred via insulin-independent effects of GLP1 (Bodinham et al. 2014). Such insulin-independent effects include muscle glucose uptake (Ayala et al. 2009), increased nitric oxide levels altering microvascular recruitment (Chai et al. 2012) and endothelial function (Nystrom et al. 2004). However, replication of these results is needed. A number of considerations have been proposed when studying gut hormone activity in humans. For example, many gut hormones have short half-lives and are rapidly inactivated (Kim & Egan 2008). Furthermore, gut hormones do not act independently (Klosterbuer et al. 2012) and so reliance on a single biomarker may not reveal the complete picture. In addition, most researchers measure total GLP-1 rather than the active fragment (GLP-1 7– 36; Bodinham et al. 2013). Other potential influencing factors when assessing GLP-1 activity in humans may include inter-individual differences in circulating concentrations as a result of defects in the GLP-1 synthesis pathways (Jin 2008) and the influence of high-fibre diets (Freeland et al. 2010). Overall, unlike animal studies, the balance of evidence from human studies does not suggest a role of GLP-1 in mediating glycaemic control, perhaps in part due to differences in physiology between species but perhaps also due to inter-individual differences in response and baseline levels of glycaemic control. Perhaps such variability is unsurprising given inter-individual responses to SCFA production after consumption of RS, as noted in the gut health section of this review. A third proposed mechanism of RS fermentation on glycaemic control is reduction in Gram-negative bacteria. It has been suggested that such bacteria can increase host exposure to lipopolysaccharides, allowing these to enter the bloodstream via a ‘leaky gut’ 18 S. Lockyer and A. P. Nugent and cause low-grade inflammation and insulin resistance [for review, see Keenan et al. (2015b)]. Currently, the only available information relates to animal studies (Shen et al. 2011), with limited transferability to humans. Overall, the literature on RS and metabolic health mainly focuses on insulin and glucose metabolism where, in general, RS consumption has been found to improve glycaemic control. As yet, consistent effects over and above reductions in total available carbohydrate content require confirmation and studies in populations with normal and impaired glucose tolerance (including those with type 2 diabetes), accounting for mechanisms of action where possible. RS does not appear to cause any adverse effects on metabolic health in any population group. Given that RS-derived SCFAs affect insulin sensitivity and lipolysis, and gut health studies suggest large inter-individual differences in SCFA production, future studies should capture inter-individual variability in glycaemic response, as well as in SCFA production. Furthermore, there is a need to understand the influence of the food matrix on glycaemic response and how the various RS types may act independently, and synergistically, to improve glycaemic control. The influence of RS on individuals with impaired glucose tolerance and/or diabetes merits further study, especially over longer periods of time. Key points • It is well accepted that postprandial glycaemic responses to RS are reduced compared with digestible carbohydrates. • In the EU, there is an approved health claim that baked products containing at least 14% RS in place of digestible starch reduce postprandial glycaemia. • There may be synergism between RS and other fibre types in reducing glycaemic responses. • More long-term studies are required to fully ascertain the effect of RS consumption on plasma lipids. Resistant starch and appetite regulation Satiation describes the within-meal decline in hunger and increase in fullness that lead to the inhibition of further eating, and satiety describes the extent to which appetite is suppressed between meals (Blundell et al. 2010). Many factors influence the amount of food consumed at an eating occasion, including environmental cues, such as portion size; sensory cues, such as palatability; cognitive factors, such as previous experiences with that particular food; and metabolic signals between the gut and the brain, which involve stretch receptors in the stomach and gut hormones (Blundell et al. 2010). Peptide YY (PYY), pancreatic polypeptide (PP), GLP-1 and cholecystokinin (CCK) are produced in response to eating and suppress appetite and decrease food intake (i.e. increase satiety; Chaudhri et al. 2006; Troke et al. 2014). Conversely, ghrelin, known as the ‘hunger hormone’, increases food intake, is released in the fasted state and suppressed after a meal. The inclusion of foods or ingredients in the diet that decrease hunger and promote fullness has the potential to prevent weight gain or even aid weight loss (if the result is lower calorie intake overall), as hunger is cited as a barrier to the success of diets undertaken with the aim of losing weight (L opez-Nicolas et al. 2016). Human satiety studies often use a ‘preload’ design, which involves consumption of the test food or ingredient as a ‘preload’ (the effects of which can be compared with a control preload), which is followed sometime later by an ad libitum test meal. The effect of the preload on satiety is assessed by measuring subsequent subjective sensations of appetite and food intake at the test meal. Sometimes, physiological measurements, such as plasma levels of gut hormones, are also taken. Ideally, food intake over the rest of the day (and possibly even the following day) is also measured in order to capture any delayed effects. Dietary fibre and satiety There is some evidence that dietary fibre can enhance satiety, with proposed mechanisms including increased stomach distension, reduced rate of gastric emptying and modulation of gut hormone production due to the formation of a gel in the stomach by some fibre types (Chambers et al. 2015a). A systematic review published in 2011 reported that in 43% of high-fibre vs. low-fibre control comparisons, fibre consumption reduced appetite by at least 10% (Wanders et al. 2011) and a later systematic review reported similar findings, with 39% of high-fibre treatments significantly reducing appetite compared with low-fibre controls (Clark & Slavin 2013). Overall, these two reviews suggest that around 60% of studies demonstrate that fibre does not significantly impact on appetite. These same reviews, respectively, indicated that 54% and 22% of fibre interventions reduced subsequent energy intake in an acute setting vs. a control. Health claim applications submitted to EFSA related to dietary fibre and effects on satiety, weight management and fat absorption have been rejected on the © 2016 British Nutrition Foundation Health effects of resistant starch grounds that dietary fibre is not sufficiently characterised in relation to the claimed effects (EFSA Panel on Dietetic Products Nutrition and Allergies 2010c). In its Scientific Opinion, EFSA commented that the references provided demonstrated varying effects of different fibre types (i.e. soluble, insoluble, viscous, non-viscous) on appetite and subsequent food intake. Resistant starch, satiety and acute energy intake In terms of RS, the aforementioned systematic reviews were in agreement that RS does not influence subjective satiety ratings, but does impact on subsequent energy intake (Wanders et al. 2011; Clark & Slavin 2013). In general, the numbers of studies testing the satiating effects of RS tended to be smaller than those on other fibre types. The Clarke and Slavin review included four acute RS studies (Nilsson et al. 2008; Willis et al. 2009; Anderson et al. 2010; Bodinham et al. 2010), and Wanders et al. included three (Raben et al. 1994; Willis et al. 2009; Bodinham et al. 2010). Studies in this area conducted since our last review (Nugent 2005) are summarised in Table S5, with some studies also described in Tables S2–S4 (marked with an asterisk). The number of subjects ranged from 10 to 90, and RS doses ranged from ~2 to 48 g (though in some papers exact doses are not stated). Most studies assessed the impact of RS consumption on satiety and food intake over the course of several hours (up to 1 day) and some studies measured the effect of RS intake on these parameters on repeated occasions (Stewart et al. 2010; Sarda et al. 2016). Reported short-term effects of RS on satiety-related outcomes have been mixed, with some studies indicating positive effects (i.e. increased satiety scores, reduced hunger and/or reduced energy intake; Quilez et al. 2007; Nilsson et al. 2008, 2013; Anderson et al. 2010; Bodinham et al. 2010; Johnston et al. 2010; Rosen et al. 2011; Chiu & Stewart 2013; Harrold et al. 2014; Sandberg et al. 2016), some reporting no effect of RS on self-reported appetite compared with a control (Nilsson et al. 2008; Stewart et al. 2010; Karalus et al. 2012; Klosterbuer et al. 2012; Ekstrom et al. 2013; Garcıa-Rodrıguez et al. 2013; Bracken et al. 2014; Gentile et al. 2015; Nilsson et al. 2015), others reporting no effect on subsequent energy intake compared with a control (Stewart et al. 2010; Klosterbuer et al. 2012; Karalus et al. 2012) and, in one study, lower energy intake (~322 kcal over 24 hours) after RS consumption, but no effect on appetite scores (Bodinham et al. 2010). © 2016 British Nutrition Foundation 19 Very small differences in RS content between intervention and control foods (around 1 g or less) may provide an explanation for the null findings in some of the satiety studies (Keogh et al. 2006; Ames et al. 2015). Studies with greater doses of RS suggest that satiety increases with intake of RS. The largest study identified here (n = 90; Harrold et al. 2014) demonstrated that satiety was sustained for longer following consumption of 30 g of a composite product [with an assumed RS content of ~21 g (Ingredion Incorporated 2014)] served within a smoothie vs. an energymatched smoothie containing only 20 g of the same product (~14 g of RS). In a further study, wheat bread enriched with 8 g of RS2, barley kernel bread (9.5 g of RS) and high-amylose barley bread (22 g of RS), given at evening meals, had no effect on self-reported satiety vs. standard white wheat bread (1.33 g of RS), whereas high-b-glucan barley bread (~14% b-glucan, 31 g of RS) resulted in significantly lower satiety ratings after a standard breakfast the next morning (Nilsson et al. 2008). The higher b-glucan content in this intervention introduces a confounding variable, a feature that also limits the interpretation of some of the other studies described here in which RS was intentionally combined with other fibre types, such as guar gum or pullulan, or where intervention foods were not matched for these components making it difficult to tease out any effects of RS alone (Keogh et al. 2006; Klosterbuer et al. 2012; Ekstrom et al. 2013; Harrold et al. 2014; Ames et al. 2015). A number of potential mechanisms may explain the effects of RS on satiety and food intake observed in some studies. A delay in gastric emptying rate (GER), and possibly the associated delayed and prolonged presence of glucose in the blood, has been linked to an increase in satiety (Bergmann et al. 1992; Holt et al. 1992), though this mechanism may be more relevant to viscous and soluble fibres (RS is neither viscous nor soluble; Marciani et al. 2001; Hoad et al. 2004). Data relating to RS and GER are scarce. The GER (measured by ultrasonography) of a second meal preceded by a breakfast containing 13 g of RS was found not to differ significantly from meals containing just 1 g of RS, though breath hydrogen values were significantly higher at 6–10 hours (Brighenti et al. 2006). However, in a study testing eight different bread types, served at an evening meal, the type with the highest RS content (30.9 g) produced a significantly delayed GER (measured by serum paracetamol) and significantly higher satiety scores and breath hydrogen after a standard breakfast the following day. However, the test meals were also higher in b- 20 S. Lockyer and A. P. Nugent glucan, a soluble fibre (Nilsson et al. 2008). Furthermore, paracetamol is suggested not to be an accurate measure of gastric emptying (Bartholome et al. 2015). The effect of RS on GER is therefore unclear at present. PYY, PP, GLP-1 and CCK are thought to delay gastric emptying (Troke et al. 2014). RS has been noted in some studies to increase postprandial GLP-1 (Nilsson et al. 2008, 2015; Johnston et al. 2010; Sandberg et al. 2016), but not others (Bodinham et al. 2013; Garcıa-Rodrıguez et al. 2013; Ames et al. 2015). Two studies have observed an increase in postprandial PYY after RS consumption in healthy subjects (Nilsson et al. 2015; Sandberg et al. 2016), whereas two have found no impact (Garcıa-Rodrıguez et al. 2013; Ames et al. 2015). Most RS studies have measured only a few selected gut hormones rather than a broad range, with very few examining CCK and PP, meaning that full understanding of the cascade of gut hormone release in response to RS intake is a long way off (Klosterbuer et al. 2012). Almost all studies summarised here that measured ghrelin have found that RS intake has no effect of ghrelin release, apart from one in which RS consumed in liquid form decreased ghrelin (Garcıa-Rodrıguez et al. 2013), an interesting finding considering that a systematic review concluded that fibre consumed in liquid form is more satiating (Wanders et al. 2011). The satiety-enhancing effects of RS have been measured over short time periods (<3 hours; Quilez et al. 2007; Anderson et al. 2010; Harrold et al. 2014), and it has been proposed that signals generated by the colonic fermentation of RS, indicated by increased breath hydrogen or the appearance of SCFAs, could account for satiety effects observed at later time points via the reduction of GER, facilitated by gut hormone release (Nilsson et al. 2008; Harrold et al. 2014). A series of studies demonstrated that the SCFA propionate stimulates the release of PYY and GLP-1 from gut cells in vitro and consumption of 10 g/day inulin-propionate ester in humans increased plasma PYY and GLP-1 and reduced acute energy intake (Chambers et al. 2014). Furthermore, intake over a 24-week period was observed to have beneficial effects on bodyweight, adiposity and appetite ratings compared with a control (Chambers et al. 2014). While these results may not be directly applicable to RS, they may be of relevance due to the reported (though varied) production of propionate after RS consumption (see colonic health section). SCFAs have been shown to act as ligands for specific receptors that are located on colonic endocrine cells (known as L-cells), along with other cells such as adipocytes, and secrete PYY and GLP-1 (Chambers et al. 2015b). A positive correlation has been found between plasma acetate and propionate levels and mean GLP-1 concentrations after the consumption of wholegrain rye kernel bread vs. a control (Sandberg et al. 2016). If this mechanism explains the action of RS, the duration of some of the studies described here may have been too short to capture any effects, as measurements ended before SCFAs would have been generated (see colonic health section). Therefore, for studies that provide RS at an evening meal or at breakfast, measuring effects on appetite and food intake for up to 15 hours may be necessary (Verbeke et al. 2010). In addition, because the formation of SCFAs is so variable between individuals due to differences in gut bacteria and other factors such as transit time (Chambers et al. 2015b), smaller studies may lack statistical power to detect an effect. Interestingly, a portion of boiled rye kernels containing 7.5 g of RS had more of an effect on satiety and subsequent food intake than a portion of boiled wheat kernels containing the same amount of RS and similar amounts of available carbohydrate (Rosen et al. 2011). The increased satiating effects of rye could partly be due to the larger portion size (227 g vs. 172 g of wheat kernels), as food volume can influence satiety (Keller et al. 2013). Also, the glycaemic profile value (defined in this study as the duration of the glucose curve divided with the incremental glucose peak) of the rye was almost double that of the wheat kernels (i.e. the presence of glucose in the bloodstream was more prolonged). A high glycaemic profile was associated with reduced ghrelin levels at 270 minutes post-consumption and a lower desire to eat, which were both associated with lower energy intake at an ad libitum meal. A high glycaemic profile was also inversely related to insulin responses including insulinaemic index (defined as the incremental positive area under the blood insulin curve after a test product, expressed as a percentage of the corresponding area after an equi-carbohydrate reference product taken by the same subject) and incremental insulin peak, and a low postprandial insulin response correlated with increased satiety and lower energy intake. Reduced glucose and/or insulin responses coupled with either increased satiety, reduced food intake and/or increases in satiety hormones after the consumption of RS vs. a control have also been reported elsewhere (Bodinham et al. 2010; Johnston et al. 2010; Luhovyy et al. 2014; © 2016 British Nutrition Foundation Health effects of resistant starch Nilsson et al. 2015). This may be explained by a prolonged release of gut hormones in response to prolonged presence of glucose in the blood. Overall, compared with digestible carbohydrate, there is some evidence that RS is more satiating and decreases short-term energy intake, with several plausible mechanisms having been identified, but there are also a substantial number of studies in which no significant differences have been observed. Inconsistencies in study design, including type and dose of RS, mean that it is difficult to draw conclusions from the evidence base and an effective minimum dose is yet to be identified. When interpreting studies in which foods with intrinsically higher RS contents are compared with foods with lower RS contents, it must be remembered that components other than RS (such as protein, other fibre types) may contribute to observed biological effects. While such studies are informative as they represent real-life food choices, studies comparing purified RS-containing products with products otherwise identical in composition but without added RS are required to understand the specific effects of different types of RS on satiety. Such information could aid product innovation. Resistant starch and bodyweight It seems plausible that if RS is at least as satiating, if not more satiating, than refined carbohydrates, consumption of RS in part replacement of refined carbohydrates could reduce bodyweight simply because it is lower in energy. Some animal data indicate that an RS-rich diet may lead to weight loss (Keenan et al. 2015a). However, the 2015 report from SACN, Carbohydrates and Health (SACN 2015) identified only three human studies investigating the relationship in humans (De Roos et al. 1995; Heijnen et al. 1996; Jenkins et al. 1998) and concluded that there is no significant effect of long-term RS2 and RS3 intake on overall energy intake (meta-analysis revealed energy intake differences of +4.4 and 24.6 kcal/day, respectively). SACN did not identify any prospective cohort studies examining RS intake and health outcomes in its review. Studies published since 2005 examining the effect of long-term RS consumption on bodyweight are summarised in Table S6, with some also detailed in Tables S2 and S3. The majority reported no change in bodyweight in response to longer term intake of RS, with doses ranging from 5 to 40 g of RS per day for a duration of 2–6 weeks. One study provided participants (n = 86; 47% of whom had metabolic © 2016 British Nutrition Foundation 21 syndrome) with RS4-enriched flour (24 g/100 g, providing ~100 kcal less per 100 g than the control flour) to use in place of usual flour for the preparation of foods such as bread, noodles and dumplings for 12 weeks. Consumption of the RS4-enriched flour resulted in no significant changes in overall bodyweight, but in the metabolic syndrome-free participants a significant reduction in fat mass (0.5%), a significant increase in fat-free mass (1%) and a 2.6% reduction in waist circumference occurred (Nichenametla et al. 2014). A significant limitation of this study is that the mean daily intake of RS was not reported but the length of the intervention period is a strength, with EFSA guidance recommending 12 weeks for weight loss intervention studies (EFSA Panel on Dietetic Products Nutrition and Allergies 2012). A significant effect of RS on bodyweight (a reduction of 1.6 kg) was reported following consumption of 24 g of native banana starch (dissolved in 240 ml water, providing 8.16 g of RS) for 4 weeks by 28 participants who were obese and had type 2 diabetes (Ble-Castillo et al. 2010) vs. a control (24 g of soy milk in 240 ml water). There was also a significant reduction in BMI (0.6 kg/m2), but no change in percentage body fat or waist-to-hip ratio, which suggests that the weight loss may have been due to reduced body water or loss of lean mass. The energy content of the intervention and control foods was not provided but the soy milk is assumed to be more calorific as it contained 46.2% fat. Soy is not ideal to use as a control food due to its reported bioactive effects including weight loss, perhaps due to the presence of phytoestrogens (Zhang et al. 2013). The authors reported no changes in diet or exercise during the intervention periods, but the study had no wash-out period. Overall, it is difficult to drawn firm conclusions from this study due to limitations in study design. A study in which overweight and obese subjects were advised to consume a diet rich in RS-containing foods for 12 months reported reductions of 4.4 kg, 1.42 kg/m2 and 3.8 cm in weight, BMI and waist circumference, respectively, compared with equivalent reductions of 2.4 kg, 0.61 kg/m2 and 3.1 cm in the group following a more general fibre-rich diet (Dodevska et al. 2016). Subjects in the RS group consumed on average 142 kcal less per day than at baseline compared with a 120 kcal reduction in the fibre group. This study suggests a positive effect of increasing RS (and indeed all dietary fibre) in the diet over a long time period on bodyweight. 22 S. Lockyer and A. P. Nugent Animal studies have suggested that replacing digestible starch with RS can result in lower body fat and adipocyte size (Higgins 2014; Keenan et al. 2015b). Several mechanisms have been suggested, including increased energy expenditure and increased fat oxidation (Keenan et al. 2015b). RS has been observed to increase postprandial resting energy expenditure (by more than 70% in the first 3 hours) and fat oxidation measured by indirect calorimetry in one human study, an effect that was coupled with a reduction in the gut hormone GIP (Shimotoyodome et al. 2011). GIP is an incretin released from the small intestine upon meal ingestion, which stimulates the secretion of glucagon and may have a role in lipid metabolism and therefore bodyweight, though the latter effects remain to be established (Holst et al. 2016). Most human studies have found either no effect of RS on postprandial energy expenditure (Howe et al. 1996; Keogh et al. 2006; Sands et al. 2009) or a reduction (Tagliabue et al. 1995), although the study durations may have been too short to capture any longer-term effects following gut fermentation. There has been evidence of increased fat oxidation in humans after the consumption of RS-enriched foods compared with digestible carbohydrates (Tagliabue et al. 1995; Higgins et al. 2004; Gentile et al. 2015). At present, there is little evidence that RS consumption can induce weight loss in humans. Due to the existence of positive results from animal studies and some evidence of increased satiety and reduced shortterm energy consumption in humans, this area may be worthy of further investigation. Further studies should be at least 12 weeks in duration. In addition, data from animal studies have suggested that small decreases in bodyweight can be obscured by the concomitant increase in the mass of gut microbiota, which may occur with RS supplementation. This, coupled with the impact of losing water weight, means that body composition should ideally be measured rather than total bodyweight in order to provide an accurate assessment of whether or not RS consumption can result in fat loss. Other health benefits of resistant starch A small amount of evidence suggests that RS may have immune modulatory effects, though at present most data available are from animal and in vitro studies (Nofrarıas et al. 2007; Bassaganya-Riera et al. 2011; Haenen et al. 2013; Bermudez-Brito et al. 2015). Some human studies have observed that RS consumption lowers circulating inflammatory cytokines such as IL-6, IL-18 and TNF-a (Nilsson et al. 2008, 2013; Bodinham et al. 2014; Gargari et al. 2015), whereas others have reported no change in these or other inflammatory markers such as CRP and PAI-1 (Worthley et al. 2009; Johnston et al. 2010; Penn-Marshall et al. 2010; Kwak et al. 2012; Maki et al. 2012; Sandberg et al. 2016). It has been proposed that the mechanism for reduced inflammation is an increase in GLP-2 induced by RS consumption, which decreases intestinal wall permeability and thus blocks the entry of endotoxins (Nilsson et al. 2013). A study conducted in subjects with impaired glucose tolerance or newly diagnosed type 2 diabetes reported that 4-week consumption of rice, delivering 6.51 g of RS/day, reduced markers of oxidative stress (plasma malondialdehyde and urinary F2-isoprostanes), which was associated with improved endothelial function measured by RH-PAT (Kwak et al. 2012). It was hypothesised that the RS-related reduction in postprandial glycaemia caused reduced production of oxygen-derived free radicals (superoxide dismutase activity tended to increase after RS consumption), which led to increased serum nitric oxide. However, in another study, 40 g of RS2 supplementation for 12 weeks had no effect on arterial stiffness (another measure of vascular function) measured by a gold standard methodology, pulse wave velocity (Johnston et al. 2010). More research is clearly needed in this area. Emerging research indicates that RS may be useful in treating chronic kidney disease. In a small study of nine patients, 40 g of fermentable carbohydrate per day for 5 weeks shifted nitrogen excretion from urine into faeces, therefore decreasing uraemia (raised blood levels of urea and other nitrogenous waste compounds that are normally eliminated by the kidneys; Younes et al. 2006). Many uraemic substances are generated by the gut microbiota, and altered microbial composition and increased intestinal permeability are seen in renal disease (Vaziri 2012). It has been proposed that RS consumption may yield benefits via the generation of SCFAs and increased proliferation of SCFA-producing bacteria, which may in turn boost gut barrier integrity and result in reduced production of waste solutes (Vaziri et al. 2014). In a study of 28 patients receiving haemodialysis, consumption of 15 g of high-amylose corn starch (60% RS) per day for 6 weeks resulted in a reduction in plasma levels of one such solute, indoxyl sulphate (Sirich et al. 2014). © 2016 British Nutrition Foundation Health effects of resistant starch Key points • There is some evidence that RS can decrease appetite and short-term food intake. • Potential mechanisms include an increase in the release of gut hormones that promote feelings of satiety, stimulated by SCFAs. • There is little evidence that RS can decrease adiposity in humans. • RS may have a role in treating chronic kidney disease. Conclusion From a food technology perspective, RS is an established ingredient used in a range of food products including baked goods, pasta, beverages and dairy products, as well as for the microencapsulation of components such as probiotics. In addition, current evidence points towards RS as a dietary component with several potential health benefits and no harmful effects, besides possible gastrointestinal symptoms in some individuals at high intakes. The wealth of evidence linking RS to reduced postprandial glycaemic responses has resulted in an EUapproved health claim, which clearly sets out a minimum amount that is needed to replace digestible carbohydrates in order to produce the desired effect. There is also some evidence of glycaemia-related benefits of RS over and above simply replacing digestible carbohydrates, but interpretation of these findings is complicated by differences in study design, study populations, doses and forms of RS used. The role of the food matrix appears important, where lower doses of RS may have an effect when naturally present within foods that contain other types of fibre, rather than as a purified form. More research among subjects with diabetes could lead to RS being recommended to this group as an aid to glycaemic control, especially as no detrimental effects on glycaemia have been identified. There is little evidence of effects of RS on lipid metabolism, markers of immune function or adipokines, although research in these areas is scant at present. It is clear from animal and in vitro studies that RS modulates many outcomes related to gut health but there is difficulty in translating this to humans, perhaps due to inadequacies in the methodologies used at present. There is evidence that RS can increase the production of SCFAs in the human gut but there appears to be a significant degree of variability in response to RS intervention, most likely influenced by differing gut microbial compositions between © 2016 British Nutrition Foundation 23 individuals, habitual intake, RS type and presence or absence of other RS and other fibre types. Furthermore, SCFAs use by the host within the colon prior to excretion can be influenced by sex, BMI and gut microbiota. More data in this area would be useful as this is an important point for researchers to consider when undertaking power calculations for studies using RS. Esterified RS4 may be more effective at generating SCFAs than native RS. There is good evidence that RS influences gut microbial communities involved in amylose breakdown and butyrate and methane production, but there is a significant variability in responses. Advances in gut microbe profiling and metabolomics may help us to understand the complexity of this variation. If stimulatory effects are found to be selective in favour of bacteria that promote health benefits, this could lead to the classification of RS as a prebiotic which is not possible at present. The potential for RS to act synergistically with other fibre types and probiotics merits further attention. While there is no evidence for an effect of RS on colorectal cancer occurrence or polyp number and size in individuals at high risk, there is evidence of a reduction in disease risk markers at a molecular level and for mitigation by RS intake of the detrimental effects of red meat on colorectal cancer. Emerging research also indicates a role of RS in treating dehydration. There is some evidence that RS can positively influence satiety and short-term food intake and plausible biological mechanisms have been demonstrated, but results have not been consistent. Studies that have measured appetite as a secondary endpoint have sometimes used flawed study designs. For example, information on the effect of RS on glycaemic responses has been provided by matching an RS-rich test food and control food for carbohydrate content, but this can make comparisons of appetite effects difficult due to the introduction of other factors that impact on satiety, such as differences in portion sizes. There is some evidence of potential synergistic effects of RS with other fibre types leading to enhanced satiety effects, which requires further study. The duration of RS satiety studies published to date varies significantly, with some studies providing RS in an evening meal and measuring next day effects on appetite to allow time for SCFA generation, while others have taken measurements for only a few hours after RS consumption. Individual differences in SCFA production should also be taken into account in these types of studies. The EU-funded SATIN (SATiety INnovation) project is looking into the effect of food 24 S. Lockyer and A. P. Nugent components, including RS, on satiety and weight loss, with the aim of increasing understanding of the biopsychological mechanisms underpinning satiety (www.satin-satiety.eu). Outputs from this project could help to inform methodologies for future studies examining the impact of RS and appetite regulation. Currently, there is very little evidence in humans that any satiating effects of RS translate into weight loss. This is an area worthy of investigation with welldesigned studies of sufficient duration and robust dietary assessment. There may be a lot still to be discovered in terms of the health benefits of RS. At the time of writing, upcoming trials investigating the effect of RS on bodyweight, gut health, glycaemia, insulin sensitivity, CVD risk markers, appetite and colorectal cancer prevention, in a variety of populations including patients with Parkinson’s disease, type 2 diabetes, renal diseases and those undergoing transplantation, have been registered on clinicaltrials.gov. At present, most RS research relates to RS2. A wider spread of evidence relating to all RS types, via the testing of whole foods, would be useful in informing innovation, the selection of grains/varieties of RS-rich foods and perhaps in future, a recommended intake value for RS. Conflict of interest The British Nutrition Foundation is grateful to Ingredion Incorporated for financially supporting some of the time spent on the preparation of this review. The views expressed are those of the authors alone. References Abell GC, Cooke CM, Bennett CN et al. 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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Overview of studies post 2005 exploring the effect of resistant starch on gut health in humans. Table S2. Summary of acute, semi-acute and chronic studies (post 2005) investigating the influence of high amylose maize starch (HAMS) on markers of metabolic health in humans. Table S3. Summary of acute, semi-acute and chronic studies (post 2005) investigating the influence of resistant starch from banana, bread, beans and rice on markers of metabolic health in humans. Table S4. Summary of acute, semi-acute and chronic studies (post 2005) investigating the influence of RS3 and RS4 on markers of metabolic health in humans. Table S5. Studies (post 2005) investigating the effect of resistant starch on appetite and short-term energy intake in humans. Table S6. Studies (post 2005) investigating the effect of resistant starch on bodyweight in humans. © 2016 British Nutrition Foundation