Effect of Resistant Starch on Symptom Improvement and Intestinal Microbiome in Patients With Functional Constipation
Phase: NA
Conditions: Constipation
Sponsor: Huaping Xie

Study Protocol and Statistical Analysis Plan
（Effect of resistant starch on symptom
improvement and gut microbiota in patients
with functional constipation）
2023.9.21
Main researcher: Huaping Xie
Research unit: Tongji Hospital affiliated to Tongji Medical College,
Huazhong University of Science and Technology

1、Research background
The
human
gut
microbiota
is
one
of
the
most
densely
popupated
microbial
communities on earth and contains highly diverse microbial communities. They
provide metabolic, immune, and protective functions and play a vital role in human
health[1-3].Gastrointestinal microbiota is influenced by a variety of factors, including genetics,
host physiology (host age, disease, stress, etc.) and environmental factors, such as living
conditions and drug use. At the same time, diet is considered to be a key environmental factor
mediating the composition and metabolic function of gastrointestinal microbiota[4]. In view of
the fact that it is difficult to be digested and absorbed by the human body, probiotics can enter the
intestinal tract through the digestive tract, so as to improve intestinal microecology and promote
lipid, protein and mineral metabolism.
Food rich in anti-digestible starch has many functions, such as controlling body weight,
reducing blood lipids and blood sugar, regulating intestinal flora and so on, which has attracted the
interest of many scholars and become a new field of international food research in recent years.
Resistant starch (RS) is the anti-digestion part of compound polysaccharide starch. According to
the conditions of enzymatic hydrolysis resistance and starch source, the digestible resistant starch
was mainly divided into four types, namely RS1, RS2, RS3 and RS4. From the cause of resistance,
RS1 and RS2 have natural resistance to amylase, and the resistance can disappear after
gelatinization, while the resistance of RS3 and RS4 is formed by the transformation of starch in
the process of food processing or food production. RS1 can coexist with RS2 or RS3 in the same
kind of food, and the existence of RS4 can increase the food intake of RS3. Only a small part of
the indigestible starch can be digested and absorbed in the small intestine, providing a very low
utilization rate of glucose. When most of the rest of the undigested starch enters the colon, it is
fermented by the intestinal microflora, mainly producing short-chain fatty acids (SCFAs): acetate,
propionate and butyrate[5]. Although both acetate and propionate have health effects, butyrate is
particularly thought to improve health and is the fatty acid with the largest increase in resistant
starch intake. Butyrate plays an important role in human intestinal health, including reducing
inflammation, reducing the risk of colon cancer and improving intestinal barrier function[6]. In vivo
and in vitro, resistant starch diet and butyrate significantly increased the proportion of ChAT
immunoreactive intermuscular neurons, intestinal neurons expressed monocarboxylic acid
transporter 2 (MCT2), small interference with RNA silenced MCT2, and prevented butyrate-
induced increase in the proportion of ChAT immunoreactive neurons. Butyrate and trichostatin A
increased the acetylation of histone H3 in intestinal neurons. Src signal pathway inhibitors blocked
the effect of butyrate. Resistant starch diet increased colonic transport, and butyrate increased
cholinergic-mediated contraction of colonic circular muscles in vitro[7]. Although resistant starch
has been shown to be one of the best fibers to raise butyrate levels in the population, it is clear that
not everyone can get the same benefits, and some people do not respond to
resistant starch

supplements[8]. This suggests that differences in individual microflora play an important role in
determining the outcome of resistant starch consumption, and we need to explore more deeply the
mechanism of resistant starch digestion and how it leads to the production of butyrate. In addition,
more work needs to be done to understand the effects beyond butyrate levels that may affect
health through resistant starch consumption.
Due to the complex structure of
resistant starch, some bacteria are needed to initiate the
degradation of this semi-crystalline material. Ruminococcus bromii and Bifidobacterium
adolescence are the two known human intestinal microorganisms with the ability to degrade
resistant starch[9]. Ruminococcus bromii has attracted attention because of its role as a key species
in
resistant starch metabolism, feeding and / or enabling other members of the intestinal
microbiome to obtain the substrate[9]. The amylolytic enzyme of Ruminococcus bromii has a
unique tissue structure and forms a multi-enzyme complex. Through the adhesion protein and
dockerin module, it is attached to the cell surface through the scaffold protein in the cellulose
body, so it is called amylasome
[10]. This system has been found in a variety of human
Ruminococcus, and the key enzyme structure of its amylase is highly conserved among
strains[11]. Despite its incredible ability to degrade resistant starch, Ruminococcus bromii itself
does not seem to win or dominate other species in the competition, but plays a beneficial role by
cross-feeding other species by releasing sugars and acetates of different lengths[12, 13]. Resistant
starch supplementation increased fecal butyrate concentrations in healthy young adults from 8 to
12 mmol/kg wet feces, but responses varied widely between individuals[8]. A follow-up study
by the same team found that people with increased Ruminococcus bromii abundance
in the microbiome were more likely to have a higher butyrate response to potato
starch[14], the results showed that the correct combination of primary degrading
bacteria and resistant starch was needed to increase the yield of butyrate.
Constipation is one of the most common gastrointestinal dysfunction in clinical practice.
About 11-20% of adults worldwide suffer from constipation[15].In clinic, it is called constipation
when the frequency of defecation is reduced, or the defecation is laborious, unsmooth, difficult
and the stool is dry[16]. Intractable constipation is tricky to treat and over-reliance on laxatives
often leads to water-electrolyte imbalance, gastrointestinal dysfunction, colonic darkening and
anal sphincter relaxation[17-19], It even leads to colorectal cancer, diabetes, anorexia nervosa and
other complications in some cases[20]. Therefore, it is very important to find a safe and effective
laxative medicine or dietary therapy to improve and relieve the symptoms of constipation.
Resistant starch plays a health-promoting role mainly due to its short-chain fat and gas produced
by microbial fermentation in the colon, and its role in the prevention of colorectal cancer and
some diet-related chronic diseases is stronger than that of dietary fibre, and it can effectively
overcome the drawbacks of food fortification with dietary fibre, such as bad odour, rough texture
and poor quality.From the analysis of research data, as a natural, safe, "medicinal and food" food
resources, like dietary fibre, has a very important role in human health. It has important industrial

application value and broad market development prospects, and opens up a new field of functional
food research, greatly compensating for the drawbacks of traditional dietary fibre.
In our previous analysis, stool microorganisms of 20 constipation patients and 20 healthy
people were found to be very different in structure. The difference analysis indicated that
Ruminococcus was abundant in healthy people. A classification model of AUROC 0.967 was
established using Lasso algorithm, and important features of the classification model were as
follows: Ruminococcus is the feature with the highest weight. SPINGO notes that the genus
Ruminococcus contains species, in which Ruminococcus bromii has the highest relative abundance.
A search of data from the GMrepo database revealed that Ruminococcus bromii were found in
28,796 trials, belonging to 93 phenotypes. A total of 40,795 valid runs belonged to these
phenotypes. The relative abundance of Ruminococcus bromii in healthy people is significantly
higher than that in constipated people. Therefore, the purpose of this clinical trial is to
supplement
resistant
starch
for
constipation
patients
with
low
abundance
Ruminococcus bromii, and (1) Observe whether the symptoms of constipation patients
are improved; (2) To analyze the changes of intestinal microbes in patients with
constipation; (3) Verify whether the relative abundance of Ruminococcus bromii
increases and
analyze the correlation between the relative abundance of R. bromii
in
intestine
and
the
improvement
of
constipation
symptoms
in
patients
with
constipation..
2.The purpose of the study
In this study, a group of patients with functional constipation were recruited to take resistant
starch: (1) to observe whether the symptoms of patients with constipation improved; (2) to
analyze the changes of intestinal microorganisms in patients with constipation; (3) to verify
whether the relative abundance of Ruminococcus bromii increased and
analyze the
correlation between the relative abundance of R. bromii in intestine and the improvement of
constipation symptoms in patients with constipation.
3.Research and design
This study was a non-randomized controlled study without placebo. A total of 30 patients
with functional constipation were recruited.
4.Subject recruitment
Inclusion criteria
Within two years, colorectal tumors were excluded by colonoscopy, and the clinical
manifestations were constipation, which met the diagnostic criteria of Rome IV constipation.
Health status was assessed by having the study subjects fill out a questionnaire to assess
bowel health related questionnaires and those who did not fulfil the requirements were
excluded.
Exclusion criteria

(a) Patients with other gastrointestinal diseases; (b) Patients with previous abdominal surgery,
cardiovascular disease, or serious medical conditions; (c) Participants have used medications
(e.g., probiotics, prebiotics, antibiotics, laxatives, prokinetic medications) in the past month
that could affect the results of the study.; (d) Pregnant women; (e) Patients participating in
other clinical studies.
5、Research process
For recruited patients with functional constipation, stool samples were first collected and 2
sachets of resistant starch were administered per day, each pack being 10 g. This could be
divided into 2 doses or 2 packs could be taken at a time. The resistant starch was brewed in
200 ml of warm water. The administration was continued for 14 days, in which fecal samples
were collected from the volunteers on day 0, day 7 and day 14. On day 0 and day 14 patients
filled
out
the
questionnaire
which
is
shown
in
the
attached
table.
A questionnaire was used to find out whether the symptoms of the constipated patients
improved, while stool samples were collected and analysed by macrogenomic sequencing to
investigate what changes occurred in the gut microbes of the patients before and after the
resistant starch intervention, with a special focus on whether there was an increase in the
relative abundance of Ruminococcus bromii. The study was conducted without recruitment
advertisements and the recruitment of subjects was completely voluntary, with informed
consent signed by the subjects after explaining the possible risks involved in the trial and
obtaining their consent to join the study. Volunteers who agreed to join the trial were given a
questionnaire to obtain their gut health and other health conditions, and were screened
according to the above criteria for selection and exclusion of study participants. The samples
collected are named with a number, which does not reveal the subject's personal information,
and the subject's identity is kept confidential throughout the study, with only the number and
disease phenotype visible. Subjects can withdraw from the study at any stage.

In this study, the composition and structure of the intestinal flora of the subjects will be
analyzed, and the subjects can keep abreast of the progress of the test and analysis and obtain
their own relevant data.The resistant starch used in this study (HiMaize260) is produced by
Ingredion. HI-MAIZE®260 resistant starch is a dietary fiber derived from Ingredion's
proprietary high-amylose corn starch that enhances the nutritional content of everyday foods
such as white bread, muffins, cookies, cakes and pasta.
The HI-MAIZE®260 standard is GB31637-2016, which conforms to the national standards
for food safety. The HI-MAIZE®260 resistant starch contains approximately 53% resistant
starch (dietary fiber) and 40% digestible starch and can be easily added to standard
formulations by partially replacing plain flour. In addition, because HI-MAIZE®260 resistant
starch contains fewer calories than flour, it enhances the nutritional content of food.
6、Possible risks and preventive measures
During the study, if the patient had poor defecation, Kaisailu could be used to record the date
and time of use.
7、Statistical Analysis Plan
The results of the questionnaire filled out by the patients were collected and statistically analyzed,
and the scores of constipation symptoms and PAC-QOL (patient assessment of constipation
quality of life questionnaire) were tested by paired t-test. The higher the score, the more serious
the constipation symptoms, and the lower the score means the relief of constipation symptoms.
The minimum score of the scale is 0, the maximum score is 30, and a score of more than 15 can be
regarded as constipation. The PAC-QOL is a specific scale for assessing the quality of life of
patients with chronic constipation. The PAC-QOL consists of 28 items divided into four
dimensions: worries and concerns (11 items), physical discomfort (4 items), psychological
discomfort (8 items), and satisfaction (5 items). Each item was scored on a 5-point scale, and the
more severe the illness, the higher the score.
The experimental data are expressed in the form of mean ±standard error (mean ±SD). The
difference of intestinal flora was analyzed by Wilcoxon paired test, and it was considered that the
data had significant difference. Statistical analysis uses GraphPadprism and R (version 4.1.2,
https://www.r-project.org/).
Macrogenomic data were analysed using the bioBakery 3 process
to obtain microbial species
abundance, gene abundance and metabolic function abundance results.
(1) Data quality control: firstly, the quality of sequencing raw data was checked by FastQC
software, then Trimmomatic software) was used to excise junctions, low-quality bases and filter
low-quality sequences, and then host-sequence comparisons were performed by Bowtie2 to
remove host sequence contamination in the sequencing data, and then finally, sequence quality
test to confirm that the sequence quality had met the analysis standard.

2) Species annotation: Species annotation was performed using MetaPhlAn software, and the
sequences after quality control were compared to the marker gene database using Bowtie2 to
obtain the microbial community composition.
3) Gene annotation: select all the microbial genomes that have been detected by MetaPhlAn 3.0,
construct the ChocoPhlAn pan-genome database, compare the sequences after quality control to
the ChocoPhlAn pan-genome database by Bowtie2 to obtain the gene annotation information of
all the known species, and then compare the sequences that have not been compared to the
ChocoPhlAn pan-genome database by Diamond. ChocoPhlAn pan-genome by Diamond, and then
translated and aligned the sequences not aligned to ChocoPhlAn pan-genome to the UniRef90
protein database, to obtain the gene annotation information of unknown species.
4) Functional annotation: according to the gene annotation results, the ko gene family was
corresponded to the KEGG database to obtain the metabolic function annotation information.
5) Visual analysis: according to the species abundance table, gene abundance table and metabolic
pathway abundance table of each sample, analyse the α-diversity and β-diversity of each period,
and carry out the difference between groups Anosim, Wilcoxon analysis.
8、Reference
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balance studies reveal associations between gut microbes, caloric load, and nutrient absorption
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[3]
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K. E. Bach Knudsen, H. N. Laerke, M. S. Hedemann, T. S. Nielsen, A. K. Ingerslev, D. S.
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Attachment
Attachment 1. Cleveland Constipation Scoring System（CCS)
No.
frequency
score
score
1
Defecate frequency
1-2times/1-2day
0
2times/week
1
1time/week
2
＜1time/week
3
＜1time/month
4
2
Defecation time: squatting
time for each defecation
(minutes)
<5
0
5-10
1
10-20
2
20-30
3
>30
4
3
Difficulty:
Painful
defecation
never
0
very few
1
sometimes
2
often
3
always
4
4
Assisted defecation: type
of assistance
no
0
Stimulant laxative
1
Finger defecation or enema
2
5
Empty: Incomplete Empty
Sense
never
0
very few
1
sometimes
2
often
3
always
4
6
Defecation
failure:
the
number
of
times
defecation fails every 24
hours
never
0
1-3
1
3-6
2
6-9
3
>9
4
7
Pain: abdominal pain
never
0
very few
1
sometimes
2

often
3
always
4
8
History:
Course
of
constipation (years)
<1
0
1-5
1
5-10
2
10-20
3
>20
4

Attachment 2 .Quality of Life Scale for Constipated Patients（PAC-QOL）
The following questions are related
to symptoms of constipation. In the
last 2 weeks, the severity or intensity
of the following symptoms
not
at
all
a little
usual
more
serious
extremely
serious
1
2
3
4
5
1.bloat
2.feel heavy
The
following
questions
about
constipation domain daily life . How
much time in the last fortnight ......
never
very
few
sometimes
often
always
1
2
3
4
5
3.discomfort
4.Difficulty in passing stools despite
having the urge to do so
5.Feeling uncomfortable with others
6.eating less and less because of
constipation
The
following
questions
about
constipation and daily life . In the
past
2
weeks,
the
severity
and
intensity of the following problems
not
at
all
a little
usual
more
serious
extremely
serious
1
2
3
4
5
7.Must be concerned about what to
eat
8.loss of appetite
9.Concerns about not being able to
pick food at will (e.g., at a friend's
house)
10.Feeling
uncomfortable
about
spending
too
much
time
in
the
bathroom when you're out and about
11.Feeling
uncomfortable
about
going to the bathroom too often
when you are out and about.
12.Always worried about changing
habits (e.g. travelling, going out, etc.)
The following questions relate to
feelings of constipation. In the past 2
weeks, the frequency of time the
following symptoms have occurred
never
very
few
sometimes
often
always
1
2
3
4
5
13.Feeling irritable

14.thrill
15.It's always a problem
16.Feel nervous
17.Feel a lack of self-confidence
18.Feel that life is out of control
The following questions are related
to the feeling of constipation. In the
past
2
weeks,
the
severity
and
intensity of the following problems.
never
very
few
sometimes
often
always
1
2
3
4
5
19.Worrying
about
not
knowing
when to have a bowel movement
20.Worried about not having enough
bowel movements
21.Disruption of life due to lack of
bowel movement
The
following
questions
about
constipation and daily living . In the
past 2 weeks, how often have the
following symptoms appeared ......
never
very
few
sometimes
often
always
1
2
3
4
5
22.Worried it's getting worse.
23.Feeling physically unable to work
24.Stools
are
less
frequent
than
expected
The
following
questions
about
satisfaction . In the past 2 weeks, the
severity
and
intensity
of
the
following problems ......
very
satisfied
more
satisfied
Generally
satisfactory
a
little
upset
very
unsatisfactory
1
2
3
4
5
25.Are you satisfied with the number
of stools?
26.Are you satisfied with the rule of
defecation?
27.Are you satisfied with the time
when the food passes through the
intestines?
28.Are
you
satisfied
with
the
previous treatment?