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Associate Professor Ma Hao, Shaanxi Normal University, et al.: Research progress on multi-scale structural characteristics of different types of resistant starch and the regulatory function of intestinal microbiota

 Last Update: 2022-11-04
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Resistant starch (RS) refers to the general term
for starch and its degradation products that cannot be digested and absorbed by the small intestine of healthy humans.
As shown in Figure 1, RS can generally be divided into five categories
.
Physically embedded starch (RS1) refers to starch particles that are difficult to be contacted by amylase due to the embedding of the cell wall or the blocking effect of protein components, mainly found in intact or partially ground grains, seeds and legumes; Natural starch granules (RS2) refer to starch granules that are not hydrolyzed by amylase due to their compact conformation or structure, mainly found in raw potatoes and green bananas; Aging starch (RS3) refers to the recrystallized components formed by heating gelatinization, cooling and repolymerization, mainly found in ready-to-eat breakfast cereals, bread, and potatoes cooled after cooking; Chemically modified starch (RS4) mainly refers to a type of starch that introduces new chemical functional groups into the starch molecule through chemical methods such as etherification, esterification and crosslinking, so that the molecular structure changes and produces enzyme resistance; Amylose-lipid complex (RS5) is a newly discovered class of resistant starch in recent years, under the action of adherent conditions, amylose forms a left-hand spiral cavity structure, and lipids enter the helical cavity of amylose to varying degrees of compounding
.

As a new type of dietary fiber, resistant starch can regulate the composition of intestinal flora, promote the proliferation of beneficial bacteria and inhibit the proliferation of pathogenic bacteria, and produce short-chain fatty acids (SCFAs) such as acetic acid, propionic acid, and butyric acid, which play an important role
in protecting the intestinal barrier and reducing intestinal permeability.
In addition, resistant starch also participates in the regulation of glycolipid metabolism, and plays a beneficial role
in controlling body mass, lowering lipids, and preventing diabetes.
Jinxiu Zhang, Xinzhong Hu and Hao Ma from the College of Food Engineering and Nutritional Sciences, Shaanxi Normal University* introduced the apparent morphological structure and multi-scale structure characteristics of different types of resistant starch, compared the effects of different types of resistant starch on intestinal flora, SCFAs production, intestinal health and glycolipid metabolism, and analyzed and discussed
the potential association between resistant starch types and intestinal microbiota regulatory function.
1.
Multi-scale structural characteristics of resistant starch

Epimorphological and structural characteristics of different types of resistant starch

The apparent morphology and structure of resistant starch can generally be observed and analyzed
by scanning electron microscopy (SEM), environmental scanning electron microscopy (ESEM), laser scanning confocal microscopy (CLSM) and atomic force microscopy (AFM).
RS1 and RS2 are naturally occurring resistant starches, and natural starch granules usually appear as irregular spherical or ellipsoidal shapes
with smooth surfaces.
High-amylose cornstarch has been reported to be smooth spherical and slender stick-like granularity
.
RS3 generally appears as an irregular flaky polymeric structure
.
For example, the high-linear corn starch treated with high temperature and high pressure treatment and pullulan enzyme debranching can obtain resistant starch (RS3) after cooling and regeneration, and the results show that the untreated high linear corn starch is a mixture of round particles and multi-angle particles, while the apparent structure of resistant starch becomes loose, rough, broken, and the birefringence crossover phenomenon disappears
。 The RS3 starch granules prepared by ultrasonic high-pressure cooking showed a pore-like structure, which may be due to the cavitation of ultrasonic waves.
The surface of RS3 prepared by high-pressure cooking method and enzyme high-pressure treatment method has shallow bands; The surface of RS3 prepared by microwave treatment has deep bands forming dense streak-like ravines, which may be the rapid thermal conductivity mechanism of microwaves causing its surface to be rougher
.
The RS3 prepared from mixed bean raw material presents an irregular, compact lumpy accumulation form with a rough and delaminated surface, which may be a continuous matrix composed of starch granules swelling and some components overflowing during pressure heat treatment, resulting in rearranged molecular chains and new crystalline structures when resistant starch recrystallization

Multi-scale structural characteristics of different types of resistant starch

X-ray diffraction (XRD) can be used to characterize the crystal form of resistant starch and quantify its relative crystallinity, but has certain limitations and is often used in combination
with techniques such as high-resolution 13 C solid-state nuclear magnetic resonance (¹³C NMR) and small-angle X-ray scattering (SAXS).
XRD can reflect the long-range ordered structure of starch particles (i.
e.
, the accumulation of double helixes), and the crystal form of starch particles can be analyzed by diffraction peaks in XRD maps, and the ratio of the area of the diffraction peak to the total area is often used to characterize the relative crystallinity
of resistant starch.
Generally, the short-range ordered structure of starch (that is, the short-chain ordered structure and overlapping crystals in the subcrystalline region and amorphous region, as well as the double helix structure in the amorphous region) is analyzed by high-resolution ¹³C NMR technology, and the crystalline structure characteristics of starch can not only be obtained by ¹³C NMR spectra, but also the relative crystallinity, amorphous phase ratio and single/double helix content
of starch can be further fitted and calculated 。 Fourier transform infrared spectroscopy mainly reflects the relevant information such as interatomic telescopic vibration, intermolecular rotation and short-range ordered structure inside starch molecules, and the changes of starch grain orderliness and double helix can be further determined by analyzing and calculating the peak-intensity ratios of
995/1 022 cm-1 and 1 047/1 022 ^cm-1.
High performance zeolite exclusion chromatography, gel permeation chromatography, high performance anion exchange chromatography and fluorophore-assisted carbohydrate electrophoresis can be used to determine the molecular mass distribution and chain length distribution of resistant starch
.
Combined with these techniques, the structural characteristics of
resistant starch can be comprehensively analyzed.
According to the XRD map, the crystal types of resistant starch can be divided into 4 categories, namely A, B, C and V types
.
Type A crystals consist of parallel left-handed double helix chains with closely arranged monoclinic unit cells; Type B crystals consist of parallel left-handed double helix chains with hexagonal unit cells that contain more water molecules in their structure; V-type crystals differ from type A and B crystals in that they are made of a single amylose helical of many complex fragments compounded with endogenous lipids; The composite of type A and B crystals makes the XRD pattern appear as C-type crystals
.
The crystalline form of resistant starch granules is usually related to
its source and processing method.

2.
The regulation function of different types and different structures of resistant starch on intestinal flora

Major gut microorganisms with the ability to degrade resistant starch

Ruminococcus bromii and Bifidobacterium adolensentis are widely recognized as major starch-resistant degraders
.
Ruminococcus brucei is the main member of the human intestinal flora, has a unique amylase structure, can form a multi-enzyme complex attached to the cell surface, antagonistic starch has special activity, is a key strain
for the degradation of resistant starch.
The sugars and acetic acid released by Ruminococcus brucei degrading resistant starch can be used as substrates for other gut microorganisms that do not have the ability to degrade resistant starch, so that resistant starch can play a beneficial role
.
Bifidobacterium juvenile has a similar effect to Ruminococcus brucei, degrading resistant starch to produce lactic acid and sugar
.
Resistant starch-degrading bacteria themselves cannot produce butyric acid, and the butyric acid producing properties of resistant starch are produced
by other gut microorganisms.

Effects of different types of resistant starch on intestinal flora

The fermentation of resistant starch in the intestine can selectively regulate the level of specific intestinal flora, and the influence of different types of resistant starch on the structure and dynamics of intestinal flora is different
.
The results showed that after 24 h of in vitro fermentation of resistant starch isolated from natural, pullulan enzyme debranching and acid hydrolysis pea starch, the relative abundance of firmicutes in the enzymatic hydrolysis treatment of resistant starch (RS3) group increased significantly, and the relative abundance of proteobacteria was the lowest.
The relative abundance of actinomycetes increased significantly in all treatment groups, among which natural pea resistant starch (RS2) was the most obvious
.
Liang Dan et al.
took RS1, RS2, RS3 and RS4 as the research objects, and after 24 h of in vitro fermentation, the composition of the intestinal flora of all resistant starch groups changed significantly, and the ratio of Firmicutes/Bacteroides decreased significantly (P<0.
05), among which the RS2 group was the lowest<b12>.
In addition, the relative abundance of Bifidobacterium genera in the RS4 group increased significantly, while the RS2 group promoted the proliferation
of Megamonas and Prevotella species.

As shown in Table 1, researchers have conducted a lot of in vivo studies
on antagonistic starch.
Kieffer et al.
fed rats with high amylose cornstarch RS2 to the chronic kidney disease model, and the results showed that the relative abundance of actinomycetes and proteobacteria in the intestine increased significantly, the relative abundance of firmicutes decreased significantly, and the ratio of Bacteroides to firmicutes increased
.
Another study showed that the intestinal bacterial diversity level of mice fed RS3 was lower than that in the normal group and the high-orthochain corn starch group, and the number of starch-using bacteria such as lactic acid bacteria, bifidobacteria, pilospiridae, and rumen bacteria in the intestines of mice increased, while the number of Riken bacteria and purple monas decreased
.
Kawakami et al.
found that anaerobic levels of cecal contents in rats fed with the feed supplement RS2 were higher than in rats
fed with the feed supplement RS3.

Effects of different types of resistant starch on SCFAs formation

Lehmann et al.
found that when RS2 was used as the substrate, the content of SCFAs after in vitro fermentation was 370~1 800 μmol/g, and when RS3 was used as the substrate, the content of SCFAs after in vitro fermentation was 890~2 100 μmol/g, and RS3 fermentation produced more butyric acid
.
Studies have shown that RS3 affects the production of total SCFAs and lactic acid in mouse feces, and can ferment lactic acid to butyric acid
through the intestinal flora.
Liang Dan et al.
carried out in vitro fermentation of different types of resistant starch (RS1~RS4), and during the whole fermentation process, the pH value of all resistant starch groups showed a similar downward trend, and the contents of acetic acid, propionic acid and butyric acid in each group increased significantly, among which the acetic acid content of RS4 group was higher than that of other groups, the amount of butyric acid produced by RS2 and inulin group was higher than that of other groups, and the content of propionic acid in RS2 group and RS3 group was higher than that of other groups
。 Sorndech et al.
pointed out that compared with RS2 and RS4, RS3 has the highest utilization rate after 24 h fermentation, lower pH value, and produces a large number of SCFAs (especially butyric acid), while RS2 has the lowest substrate utilization, which may be due to the more compact particle structure of RS2, which limits the degradation activity
of bacteria in feces 。 Another study of in vitro fermentation of RS2, RS3 and RS5 showed that the pH values of RS2, RS3 and RS5 groups gradually decreased, but RS3 and RS5 reduced the pH of fermentation cultures to a greater extent than RS2, indicating that RS3 and RS5 could be fermented more efficiently by intestinal flora.
In addition, compared with RS2 and RS5, RS3 has a higher relative crystallinity and produces the most lactic acid, while the RS5 group has the highest
butyric acid content.
Different sources and fine structural differences of the same type of resistant starch will also lead to different
SCFAs content produced by fermentation.
In vitro fermentation experiments showed that the concentration of butyric acid produced by RS1 fermentation increased with the weakening of cell wall integrity
.
It was reported that there was a difference in the content of SCFAs in potato RS2 and corn after fermentation, with the content of butyric acid and total SCFAs increasing significantly after fermentation in the former, while the content of butyric acid in the latter did not change
significantly 。 Heat treatment of ordinary corn starch and high amyriad corn starch to prepare resistant starch (RS3), heat treatment can reduce the original ordered structure of ordinary corn starch molecules, the generated resistant starch belongs to the rapid fermentation substrate, after fermentation will produce a large amount of lactic acid and acetic acid, and the synthesis of butyric acid will be inhibited; For high straight-amylose corn starch, heat treatment has no obvious effect on starch structure, resistant starch still retains a high molecular structure order, and its fermentation mode belongs to the slow fermentation type, which is conducive to the production of high concentration of butyric acid
.

Effects of different types of resistant starch on gut health

Intestinal flora (rumen coccus, bifidobacteria, etc.
) use resistant starch fermentation to produce SCFAs, which can reduce the pH value of the intestinal environment, thereby inhibiting the growth and reproduction of pathogenic bacteria with poor acid resistance in the intestine, while the gas produced by the degradation of resistant starch can also make the volume of feces fluffy, promote intestinal peristalsis, maintain intestinal health, and reduce the incidence
of colon cancer.
Dietary supplementation of resistant starch plays an important role
in improving the body's intestinal function, preventing or treating colitis, and reducing the incidence of intestinal diseases such as colon cancer.
Different types of resistant starch have different fermentation patterns in the gut and therefore have an identical
effect on the intestinal environment.
Apply resistant starch to animal models to study the effects of
different types of resistant starch on gut health.
With the addition of 15% resistant starch (RS2, RS3 and RS4) to mouse feed, the total mass of cecum, cecum wall mass and wet mass of cecum contents in the three resistant starch groups increased significantly, among which the RS3 group had the highest index level, and resistant starch could change the physiological structure of the small intestine and cecum, such as significantly reducing the villi height and mucous membrane thickness of the cecum, and significantly increasing the muscle thickness of the cecum, etc.
, but compared with the control group mice, it did not cause detectable pathological changes in the small intestine and cecum
。 Another study of RS2, RS3 and RS4 showed that rats in the RS4 group had the smallest increase in body mass in terms of controlling body mass.
In terms of regulating intestinal metabolites in rats, RS3 has the best effect; In terms of regulating blood lipids, RS3 has the most significant effect on serum triglycerides, with an average reduction level of about 30%, while RS4 has the best
effect on serum cholesterol.

The effect of different types of resistant starch on glycolipid metabolism

In recent decades, with the improvement of people's living standards and changes in lifestyle, intestinal health problems have not only brought severe tests to clinical medicine, but also the incidence of type 2 diabetes mellitus (T2DM) has also risen sharply, becoming a major threat to
global human health.
It is estimated that by 2030, there will be 439 million
people with type 2 diabetes worldwide.
Obesity as a predisposing factor for health problems and chronic diseases such as type 2 diabetes, cardiovascular disease and cancer has also been on the rise globally in recent years
.
In addition to drug treatment, dietary intake of high content of resistant starch can repair pancreatic damage, improve liver glycogen synthesis, and then control blood sugar levels, which is of great significance
for the prevention of obesity and type 2 diabetes mellitus.
In addition, the relationship between intestinal microbiota and obesity and diabetes has attracted increasing attention, and it has been reported that the regulation of glycolipid metabolism by resistant starch may be mediated by microorganisms
.
Si Xu et al.
used resistant starch in a high-fat diet-induced obesity rat model to study the anti-obesity effects
of high amylose (RS2) and esterified high amylose (RS4).
Compared with the control group, the RS2 and RS4 treated mice had significantly lower blood glucose levels and insulin levels, serum triglycerides, total cholesterol and malondialdehyde concentrations, which increased total antioxidant capacity, superoxide dismutase level and glutathione peroxidase activity, and RS4 was superior to RS2
in lowering blood sugar, lowering blood lipid levels and enhancing liver function 。 Similar studies were carried out by Shimotoyodome et al.
, and mice in the RS4 group had lower body mass, visceral fat and insulin levels than the RS2 group, and improved the oxidation capacity of liver fatty acids, indicating that RS4 had a more significant
effect in regulating blood lipid levels and improving liver lipid metabolism in rats.
It was reported that the blood glucose value of mice fed corn RS3 decreased by 14.
70%, indicating that corn RS3 could also effectively reduce the blood glucose value
of mice with type 2 diabetes.
Wang Qi et al.
obtained similar results
.
Studies have shown that RS4 can improve the intestinal flora disorder caused by high-fat diet, alleviate obesity caused by high-fat diet, and improve the blood sugar regulation ability of obese mice, thereby improving blood lipid disorders
.

conclusion

Resistant starch of different types or structures is conducive to the proliferation of different types of microbiota and can be used as a unique gut microbiota substrate to regulate the composition of intestinal microbiota, thereby affecting the production
of SCFAs.
In addition, studies of the human microbiota have shown that even in healthy people, the diversity and abundance of the gut microbiota vary greatly
.
Therefore, the initial microbiota composition of individuals plays a crucial role in improving the structure of intestinal flora and the level of metabolites, and future research still needs to pay special attention to individual differences
.
Even the same type of resistant starch may lead to significant changes in the composition of intestinal flora and SCFAs, and the classification system
of resistant starch can be reconstructed according to the regulation function and health effect of intestinal flora in the future.
In addition, in order to better understand the potential mechanism of resistant starch regulating intestinal flora, it is also necessary to conduct a comprehensive study on the relationship between the structural characteristics of resistant starch and the regulatory function of intestinal flora, and accurately regulate the intestinal flora by changing the structure of resistant starch and carrying out targeted and personalized design to improve its nutritional value or health benefits
.

Expert profiles

Ma Hao Associate Professor, School of
Food Engineering and Nutritional Science, Shaanxi Normal University.
Doctor of Engineering, McGill University, Canada, postdoctoral
fellow in Agriculture and Agri-Food Canada.
His research interests include the structure and function of
complex carbohydrates.
As the first or corresponding author, he has published nearly 30 SCI papers in food journals such as Journal of Agricultural and Food Chemistry, Critical Reviews in Food Science and Nutrition, Food & Function, Food Hydrocolloids, etc.
, including 2 cover articles and 2 highly cited papers in the top 1% of ESI
。 He has published and co-edited 5 monographs in English and Chinese published by Elsevier Academic Press, Wiley and Science Press
.
He has presided over the completion of national, provincial and ministerial projects such as the National Natural Science Foundation of China, the Key R&D Program of Shaanxi Science and Technology Plan, the Natural Science Foundation of Shaanxi Province, and the Scientific Research Start-up Fund for Returnees Studying Abroad of the Ministry of Education, and won the "Shaanxi University Science and Technology Association Youth Sponsorship Talent Program" in 2016, the "Excellent Academic Backbone of Shaanxi Normal University" in 2019 and the first prize of the Science and Technology Award of Shaanxi Provincial Colleges and Universities
in 2020.
He serves on the editorial boards of Frontiers in Nutrition and Legume Science, and is a reviewer for
more than 20 international mainstream journals.

This paper, "Research Progress on Multi-scale Structural Characteristics of Different Types of Resistant Starch and the Regulation Function of Intestinal Microbiota" is from Food Science, Vol.
43, No.
17, pp.
24-35, 2022, authors: Zhang Jinxiu, Hu Xinzhong, Ma Hao
.
DOI:10.
7506/spkx1002-6630-20220401-007
。 Click to view information about
the article.

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