Based on the Special Disease Management of Crohn's Disease Diet Studies --a Multicenter, Randomized, Controlled and Open Label Study
Conditions: Inflammatory Bowel Diseases
Sponsor: Second Affiliated Hospital, School of Medicine, Zhejiang University

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Based on the special disease management of Crohn's disease diet studies 
——a multicenter, randomized, controlled and open label study 
 
 
 
 
Research Program 
 
 
 
 
（English edition 2.2-August 01, 2021） 
IBD Center, Second Affiliated Hospital of Zhejiang University 
School of Medicine 
Chen Yan, Zhu Yongqun and Yu Qiao 
 
 
 
 
 
 
 
 
 
 
 

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1. Research background 
   Crohn's Disease (CD) is a chronic, segmental, full-thickness inflammatory bowel disease that involves 
the whole digestive tract, and is a type of inflammatory bowel Disease (IBD). Its clinical manifestations 
include diarrhea, hematochezia, abdominal pain and complications such as perforation, fistula formation 
and even cancer, which greatly affect the quality of life of patients[1]. CD patients need lifelong treatment 
and management, so the direct and indirect economic burden has become an unavoidable challenge for all 
countries in the world.  
The pathogenesis of CD is unclear, which may be caused by environmental, genetic, infectious and 
immune factors[2]. Studies have shown the incidence of CD in Asia has increased since the 21st century. 
Considering the huge population base in China, it is predicted that the total number of IBD patients in China 
is likely to surpass that in North America in the near future[3]. In the studies related to environmental factors 
and CD, it has been found that farm life, exposure to animals in childhood[4], use of antibiotics in childhood[5] 
and other factors are related to the incidence of CD. The reason may be that westernized diet and 
environment change intestinal flora, thus increasing the incidence of disease in susceptible 
population[6].  
    Enteral nutrition plays an important role in the treatment of CD, which can not only reduce surgical 
complications but also improve patients' quality of life with a low incidence of side effects. Exclusive 
Enteral Nutrition (EEN) is the only established dietary treatment for children with CD. EEN has received 
particular attention in Europe, where 80% of patients have achieved clinical remission and even intestinal 
mucosal healing [7-9]。Previous studies by our team at the IBD Center showed that EEN induced remission 
rate in adult patients with CD reached 67% [10]，However, the mechanism of action of EEN in treating IBD 
remains unclear [11-13]. So what effect does EEN have on the gut microbiota? Studies indicated that after 
EEN treatment, the levels of Bacteroides and Firmicutes in CD patients decreased, while the levels of 
Gammaproteobacteria and Actinobacteria and mucosa associated adhesive Escherichia Coli increased[14-
17]. The number of anti-inflammatory Faecalibacterium prausnitzii has also decreased[18]，In addition, 
tailed phage viruses[19] were also increased[20]. Changes in diet affect the amount of short-chain fatty 
acids and branched-chain fatty acids produced by changing the composition of intestinal flora, 
leading to changes in intestinal state.  
    As CD is an incurable lifelong disease, how to maintain disease remission is an extremely important 

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part of disease management. Although EEN has a good effect on CD induced remission, due to the long 
treatment cycle and strict condition control, patients cannot eat other food except fixed nutrient solution, 
which leads to low compliance of patients, especially adults[10]. Therefore, the promotion of EEN in clinical 
work is limited, and it is difficult for EEN to be used as the preferred method of CD maintenance treatment.  
     How to develop a safe, effective and inexpensive treatment for CD patients in China is of great 
clinical and scientific significance. In recent years, a large number of adjuvant therapies related to CD diet 
have emerged, such as specific carbohydrate diet therapy[21] , Low Fodmap diet [22] , allergen-free diet 
[23], etc. But there has been no consistent or convincing evidence on the efficacy of these diets. In 2019, an 
important study published in the journal Gastroenterology offered a promising diet. The prospective use of 
half-dose enteral nutrition in combination with a diet-based personalized nutrition regimen (CD-Treat) 
showed favorable outcomes and high patient compliance in patients with CD [24]. But the diet in the study 
was Western-style, and it was a small sample research. Through the long-term clinical observation of our 
team, most patients with CD can achieve stable remission by removing the processed food and screening 
the corresponding food by combining the characteristics of Chinese diet with the food intolerance 
detection method. So, what kind of diet is suitable for the treatment of CD patients in China? We refer to 
the CD-Treat program, replacing the Western diet with Chinese diet, so as to design an individualized 
program CD-Chinese-food (CD-C-food) that is consistent with the eating habits of Chinese patients with 
CD. By comparing the effects of this individualized diet with EEN on inducing and maintaining remission 
in CD patients. In addition, we used intestinal microbiome, bacterial metabolites, intestinal metabolomics, 
inflammatory factors and other technical means to deeply analyze the therapeutic mechanism of this new 
diet regimen for CD patients.  
To sum up, this project plans to develop a new diet therapy suitable for China -- CD-C-food. Based on 
the nutritional analysis of EEN, CD-C-Food is more in line with the common diet of Chinese patients' eating 
habits and economic conditions, and its expected therapeutic effect and influence on intestinal 
microorganism are similar to that of EEN. We will conduct a randomized control of adult subjects with a 
healthy CD-Chinese-food diet, treatment group of CD patients and animal model, using intestinal 
microbiome, intestinal metabolomics, bacterial metabolite analysis, inflammatory factors detection and 
other technical means. To explore the influence of intestinal microorganisms and their metabolites on the 
clinical remission effect and inflammatory response of patients with CD-C-Food CD, and to reveal the 

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possible internal mechanism; This study lays a foundation for further verification of larger samples in multi-
centers across the country, and provides a new approach for the development of CD diet therapy with similar 
efficacy as EEN, but better taste and higher patient compliance.  
References： 
1. Sartor RB, Wu GD. Roles for Intestinal Bacteria, Viruses, and Fungi in Pathogenesis of Inflammatory 
Bowel Diseases and Therapeutic Approaches. Gastroenterology 2017; 152(2):327-339.e324. 
2. Ng SC, Shi HY, Hamidi N, Underwood FE, Tang W, Benchimol EI, et al. Worldwide incidence and 
prevalence of inflammatory bowel disease in the 21st century: a systematic review of population-based 
studies. The Lancet 2017; 390(10114):2769-2778. 
3. Chi KR. Epidemiology: Rising in the East. Nature 2016; 540:S100. 
4. Ananthakrishnan AN. Epidemiology and risk factors for IBD. Nature Reviews Gastroenterology &Amp; 
Hepatology 2015; 12:205. 
5. Ungaro R, Bernstein CN, Gearry R, Hviid A, Kolho K-L, Kronman MP, et al. Antibiotics Associated With 
Increased Risk of New-Onset Crohn’s Disease But Not Ulcerative Colitis: A Meta-Analysis. American 
Journal of Gastroenterology 2014; 109(11):1728-1738. 
6. Kaplan GG, Ng SC. Understanding and Preventing the Global Increase of Inflammatory Bowel Disease. 
Gastroenterology 2017; 152(2):313-321.e312. 
7. Zheng L, T'Kind R, Decuypere S, von Freyend SJ, Coombs GH, Watson DG. Profiling of lipids in 
Leishmania donovani using hydrophilic interaction chromatography in combination with Fourier transform 
mass spectrometry. Rapid Commun Mass Spectrom 2010; 24(14):2074-2082. 
8. Zhang T, Watson DG, Wang L, Abbas M, Murdoch L, Bashford L, et al. Application of Holistic Liquid 
Chromatography-High Resolution Mass Spectrometry Based Urinary Metabolomics for Prostate Cancer 
Detection and Biomarker Discovery. PLOS ONE 2013; 8(6):e65880. 
9. Creek DJ, Jankevics A, Burgess K, Breitling R, Barrett MP. IDEOM: An Excel interface for analysis of 
LC-MS-based metabolomics data. Bioinformatics 2012; 28(7):1048. 
10. Xue M, Zhang H, Wang X, Xu D, Jin D, Li P, et al. Efficacy predictors of a 2-month exclusive enteral 
nutrition for inducing remission of active Crohn’s disease. European Journal of Clinical Nutrition 2018; 
72(8):1183-1186. 
11. Smith CA, Want EJ, O'Maille G, Abagyan R, Siuzdak G. XCMS:  Processing Mass Spectrometry Data 
for Metabolite Profiling Using Nonlinear Peak Alignment, Matching, and Identification. Analytical 
Chemistry 2006; 78(3):779-787. 
12. Scheltema RA, Jankevics A, Jansen RC, Swertz MA, Breitling R. PeakML/mzMatch: A File Format, 
Java Library, R Library, and Tool-Chain for Mass Spectrometry Data Analysis. Analytical Chemistry 2011; 
83(7):2786-2793. 
13. Klopfleisch R. Multiparametric and semiquantitative scoring systems for the evaluation of mouse model 
histopathology - a systematic review. BMC Veterinary Research 2013; 9(1):123. 
14. Kostic AD, Xavier RJ, Gevers D. The Microbiome in Inflammatory Bowel Disease: Current Status and 
the Future Ahead. Gastroenterology 2014; 146(6):1489-1499. 
15. Darfeuille-Michaud A, Boudeau J, Bulois P, Neut C, Glasser A-L, Barnich N, et al. High prevalence of 
adherent-invasive Escherichia coli associated with ileal mucosa in Crohn’s disease. Gastroenterology 2004; 
127(2):412-421. 
16. Lapaquette P, Glasser A-L, Huett A, Xavier RJ, Darfeuille-Michaud A. Crohn's disease-associated 

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adherent-invasive E. coli are selectively favoured by impaired autophagy to replicate intracellularly. 
Cellular Microbiology 2010; 12(1):99-113. 
17. Quévrain E, Maubert MA, Michon C, Chain F, Marquant R, Tailhades J, et al. Identification of an anti-
inflammatory protein from &lt;em&gt;Faecalibacterium prausnitzii&lt;/em&gt;, a commensal bacterium 
deficient in Crohn’s disease. Gut 2016; 65(3):415. 
18. Sokol H, Pigneur B, Watterlot L, Lakhdari O, Bermúdez-Humarán LG, Gratadoux J-J, et al. 
Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota 
analysis of Crohn disease patients. Proceedings of the National Academy of Sciences 2008; 105(43):16731. 
19. Norman Jason M, Handley Scott A, Baldridge Megan T, Droit L, Liu Catherine Y, Keller Brian C, et al. 
Disease-Specific Alterations in the Enteric Virome in Inflammatory Bowel Disease. Cell 2015; 160(3):447-
460. 
20. Sokol H, Leducq V, Aschard H, Pham H-P, Jegou S, Landman C, et al. Fungal microbiota dysbiosis in 
IBD. Gut 2017; 66(6):1039. 
21. Britto S, Kellermayer R. Carbohydrate Monotony as Protection and Treatment for Inflammatory Bowel 
Disease. Journal of Crohn's and Colitis 2019; 13(7):942-948. 
22. Damas OM, Garces L, Abreu MT. Diet as Adjunctive Treatment for Inflammatory Bowel Disease: 
Review and Update of the Latest Literature. Current Treatment Options in Gastroenterology 2019; 
17(2):313-325. 
23. Gunasekeera V, Mendall MA, Chan D, Kumar D. Treatment of Crohn’s Disease with an IgG4-Guided 
Exclusion Diet: A Randomized Controlled Trial. Digestive Diseases and Sciences 2016; 61(4):1148-1157. 
24. Svolos V, Hansen R, Nichols B, Quince C, Ijaz UZ, Papadopoulou RT, et al. Treatment of Active 
Crohn&#x2019;s Disease With an Ordinary Food-based Diet That Replicates Exclusive Enteral Nutrition. 
Gastroenterology 2019; 156(5):1354-1367.e1356. 
2. Research contents, research goals, and key scientific problems to be solved 
Research contents： 
In this study, CD-C-Food was developed to simulate the nutritional composition of EEN, and the 
efficacy of this diet treatment and the degree of replacement of EEN were tested in multiple populations 
including healthy adults, CD patients and animal models. Intestinal microbiome, intestinal metabonomics, 
bacterial metabolites analysis, inflammatory factors detection and other technical means were used to 
explore the influence of intestinal microbiome and its metabolites on the clinical relief effect and 
inflammatory response of patients with CD-C-Food, and to reveal the possible internal mechanism. 
Combined with the detection of inflammatory factors in the blood and inflammatory markers in the colon 
of patients with CD after treatment, the overall clinical effect was evaluated. In the case of certain 
affirmation of the treatment, a large sample of multi-center verification was conducted to complete the 
preliminary evaluation of the diet treatment program.  
Research goals： 

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This study aims to obtain a diet therapy that can simulate the effect of EEN treatment but has a higher 
degree of compliance after scientific evaluation of the dietary therapy program CD-C-FOOD, so as to 
provide more effective approaches for CD treatment and improve the quality of life of patients to a large 
extent. 
Key scientific problems to be solved： 
1. To reveal the influence of CD-C-food on the inflammatory response and clinical treatment effect of 
patients with CD through intestinal microorganisms and metabolites, as well as its internal mechanism, 
which can be studied by 16S rRNA sequencing mass spectrometry for metabolite detection and pathological 
observation of animal models  
2. To evaluate whether the therapeutic effect of CD-C-food on CD can achieve clinical remission effect. 
In the clinical pilot trial and subsequent large-sample verification of patients with CD, the study was 
conducted through observation of clinical inflammatory markers and comprehensive evaluation of clinical 
effect.  
3. To reveal the effective mechanism of CD-C-FOOD and EEN, to conduct in-depth analysis of the process 
test products of the experimental group, and to conduct specific studies by using 16S rRNA sequencing 
mass spectrometry and pathological observation of animal models.  
4. To evaluate whether the therapeutic effect of CD-C-food on CD can achieve clinical remission effect. 
In the clinical pilot trial and subsequent large-sample verification of patients with CD, the study was 
conducted through observation of clinical inflammatory markers and comprehensive evaluation of clinical 
effect. 
3. Research protocol 
1. Randomized controlled trials of healthy adults 
(1) Specimen collection 
A. Inclusion criteria: 
a. 
30 healthy adults over the age of 18; 
b. 
Excluded subjects with any acute or chronic medical conditions (i.e., conditions requiring regular 
medical attention);  
c. 
In the past month weight stable (plus or minus 2 kg);  
d. 
There is no history of intestinal surgery;  

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e. 
No antibiotics or steroids have been used in the past 3 months. 
B. Research procedure： 
a. 
The participants were randomly divided into two groups, group 1 and group 2 with 15 people in each 
group. During the experiment, dietary diary was recorded; 
b. 
During the first week, participants followed their usual eating habits； 
c. 
In the second week, group 1 received EEN and group 2 received CD-C-food for 7 days. In the CD-C-
food group, participants were first tested for blood FOOD intolerance IgG4 assay. The participants 
were given half a dose of enteral nutrient solution and were presented with a list of foods to choose 
from which they preferred, excluding intolerant foods and excluding wheat bran, lactose, alcohol, food 
additives and any worker's ingredients. Personalized meal plans provided by research nutritionists 
provide their daily energy needs. Energy needs were calculated from participants' estimated basal 
metabolic rate and self-reported levels of physical activity. EEN and CD-C-FOOD meals were 
provided free of charge to participants along with written preparation instructions.  
d. 
The third and fourth week was a cleanup period, during which participants ate their usual diet and 
restored their gut microbiome to baseline, avoiding interference with contamination bias. In the third 
week, they followed their usual eating habits, and in the fourth week, they followed the diet of the first 
week and monitored their weight.  
e. 
In the fifth week, the diet of the second week was repeated, but the diets of the two groups were 
switched, with group 1 receiving CD-C-Food and group 2 receiving EEN, for a total of 7 days  
f. 
Appetite, gastrointestinal symptoms, and adherence to the experimental diet were assessed by 
questionnaire, and food adherence was further assessed by food surplus and food diary.  
C. Sampling time: Fresh stool samples were collected at the end of the first, second, fourth, and fifth 
weeks. A total of four samples were provided by each participant. 
(2) Detection of routine fecal indexes and study of intestinal microorganism： 
A. The fresh Stool uses the Bristol Stool Form Scale to evaluate the shape and texture of the sample, and 
to measure the Stool water content, pH, and ammonia content.  
B. Fecal microorganism detection: microbial diversity detection (16S rRNA second-generation 
sequencing) and quantitative microflora detection (qPCR quantitative detection).  
(3) Detection of fecal bacterial metabolites 

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(4) Fecal metabolome study. Nontargeted metabolites of chloroform, methanol, and water (1:3:1) extracts 
were determined by high performance liquid chromatography-mass spectrometry (HPLC-MS). 
2. Open label trial for CD patients  
（1）specimen collection 
A. Inclusion criteria： 
a. 
200 patients with CD recurrence aged 6-15 years; 
b. 
Patients who had taken antibiotics within a month were excluded; 
c. 
Patients who changed CD treatment strategy within 1 to 3 months were excluded; 
d. 
The severity of the patient's illness required hospitalization; 
e. 
Exclude patients with FOOD allergies from CD-C-Food. 
B. Research procedure： 
a. 
Participants were treated with CD-C-Food for 8 weeks. The FOOD supply was provided during the 
study period. The FOOD provided to CD patients was different from that of healthy volunteers, whose 
FOOD was directly packaged. Participants were asked to record their daily intake of food and drink. 
b. 
Clinical examinations were conducted at two-week intervals, and no changes to the drug associated 
with CD therapy were allowed during the trial. If disease activity deteriorates at any time, or does not 
improve by 4 weeks, the patient discontinues the CD-C-Food treatment. Patients who responded 
clinically but did not enter clinical remission after 8 weeks had the option to continue CD-C-food 
treatment for 4 weeks.  
c. 
Clinical response and clinical response were assessed every two weeks. Feces and blood were collected 
once at baseline, once after 4 weeks, again after 8 weeks, and again if a patient had completed 12 weeks 
of treatment.  
d. 
The detailed procedures of intestinal microbiome study, bacterial metabolites and intestinal 
metabolomics of patients were referred to the previous experimental procedures.  
e. 
Patients were tested for inflammatory cytokines in blood cells at all stages: The expression of 
TNF-α, interleukin (IL)-6, IL-10, IL-1B and CXCL-1 in the samples were detected by Milliplex 
(Merck) liquid-phase suspension chip technology.  
（2）Clinical scores and clinical indicators: 
CDAI (Crohn’s disease activity index) scores were performed every two weeks, feces collected every four 

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weeks were tested for fecal calprotectin, and blood was tested for serum albumin and C-reactive protein. 
3. Animal experiment 
（1）samples： 
Adult (36-40 weeks of age) 12 Piebald Virol Glaxo (PVG) heterozygous HLA-B27 and 8 HLA-B7 
transgenic rats were studied. B27 rats express the human major histocompatibility complex class I HLA-
B27 gene and the related human β2-microglobulin gene and develop inflammation in their gastrointestinal 
tract (including ileum). They do not cause intestinal inflammation in a sterile environment, and antibiotic 
treatment can reduce the severity of ileitis. This model has been widely used in the study of inflammatory 
bowel disease, including with EEN intervention. The intestinal microbiota of B27 showed a similar 
phenotype to that of Cd, including an increase in proteobacteria and a decrease in sclerocytes. B7 rats did 
not have intestinal inflammation and were used as a control. Rats were placed in separate cages and raised 
under specific non-pathogenic conditions. Weight is monitored.  
（2）procedures: 
B27 rats was divided into three groups, which were CD-C-FOOD group, EEN group and conventional diet 
control group. B7 was divided into two groups, EEN group and normal diet control group. The net effect of 
EEN on their gut microbiota was studied in the B7 group setting without inflammation. There were 2 male 
rats and 2 female rats in each group, and there was no difference in body weight between groups.  
4. Data analysis 
The general linear model, Box-Cox transform and Fisher pair test were used for comparison between groups. 
The bacterial community diversity index among each group can be analyzed by alpha-diversity index. 
Community richness index (Chao and Ace) and Community diversity index (Shannon and Simpson) were 
calculated. The similarity and difference of microorganisms in different groups can be analyzed by Unifrac 
based PCoA. The differences between the groups were analyzed using NMDS (Nonmetric 
Multidimensional Scaling). PCA(Principal Component Analysis) principal component analysis can be used 
for grouping analysis between groups. Dietary pathology scores and metabolite markers were analyzed 
using SPSS, and fecal metabolites were analyzed using PCA, Unifrac based PCOA analysis, and 
permutation multivariate analysis of variance (PERMANOVA). Pearson test was used for correlation 
analysis. However, in metabolomic analysis, Spearman rank correlation test was used to compensate for the 
unbalanced influence of outliers, and Benjamin-Hochberg correction method was used to perform multiple 

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tests.