Örebro University School of Medicine Degree project, 15 ECTS January 2018
Association between butyrate-producing bacteria and
diet
Version 2
Author: Karolina Ågren Supervisors: Julia König, PhD & Savanne Holster, Doctoral student
Abstract
BackgroundButyrate is a short-chain fatty acid that has been found to have beneficial effects on gut and systemic health. It is produced by intestinal bacteria via fermentation of dietary fibers. Diet has an important impact on the ecosystem of the gut, providing selective growth advantages to certain bacteria. Even though it is hypothesized that dietary fibers increase the abundance of butyrate-producing bacteria in our intestine, more studies investigating which food compounds can affect these beneficial bacteria are needed.
Aim
The aim of the study is to investigate if there is a correlation between dietary components and butyrate-producing bacteria.
Method
Fecal samples were collected from 18 healthy individuals. 17 samples were analyzed using real-time qPCR investigating the abundance of butyrate-producing bacteria. The participants completed a food frequency questionnaire (FFQ) providing information on dietary intake. Correlations between diet and butyrate-producing bacteria were investigated using Pearson’s correlation test (SPSS).
Results
A negative correlation between protein intake and butyrate-producing bacteria was found (r=0.528, p=0.028). No statistically significant correlation between amount of butyrate-producing bacteria and fat, carbohydrates and fiber intake could be shown.
Conclusion
We found a negative correlation between amount of butyrate producing bacteria and protein intake. However, before drawing conclusions, further studies with larger numbers of
Table of contents
INTRODUCTION)...)4! AIM)...)5! MATERIAL)AND)METHODS)...)5! SUBJECTS!...!5! ABUNDANCE!OF!BUTYRATE2PRODUCING!BACTERIA!IN!STOOL!SAMPLES!...!5! DIET!...!6! STATISTICS!...!6! ETHICS!...!6! RESULTS)...)6! CALORIE!INTAKE!...!7! FAT!...!9! CARBOHYDRATES!...!10! PROTEIN!...!11! FIBER!...!12! DISCUSSION)...)12! CONCLUSION)...)14! ACKNOWLEDGEMENT)...)14! REFERENCES)...)15!Introduction
Butyrate is a short-chain fatty acid produced by bacteria via fermentation [1]. It is the
preferred energy substrate for enterocytes and accounts for 70% of their energy consumption. It has been shown to have anti-inflammatory properties and is a candidate for future treatment of Crohn’s disease [2]. Studies investigating treatment with butyrate enemas in ulcerative colitis showed that these enemas led to decreased disease activity [1,3]. In colonic cells, butyrate regulates proliferation [4] and differentiation [5], and it was suggested to have a protective roll in colorectal cancer [6,7].
The main substrates for bacterial fermentation in the colon are carbohydrates that are not absorbed in the small intestine. The main categories are resistant starch, non-starch
polysaccharides and oligosaccharides [8]. The correlation between carbohydrate intake and amount of butyrate-producing bacteria has been previously investigated. In a study comparing a high-protein/medium-carbohydrate diet and high-protein/low-carbohydrate diet, the amount of butyrate-producing bacteria was significantly lower as carbohydrate intake decreased [9]. In resembling studies similar results were found [10,11]. These studies suggest that it is mainly the presence of carbohydrates in the diet which influences the amount of butyrate-producing bacteria.
Little or no fat reaches the colon because it is absorbed in the small intestine and therefore fat has very little interaction with the microbiota. However, a high fat diet results in a higher production of bile acids. Bile acids are known to modulate the microbiota through their antibacterial properties, creating a strong selective force for the bacteria with lower sensitivity to bile [12,13]. How bile and a high fat diet influences the abundance of butyrate-producing bacteria is however not known.
Most protein is absorbed in the small intestines but some does reach the colon [14], especially if the protein intake is excessive [15]. Few studies have investigated the impact of protein intake on the microbiota. In one study rats fed with red meat had lower amounts of butyrate in cecal content compared to pea protein fed animals [8]. Metabolites with detrimental effect on colonic health have been found to increase with a high-protein diet [10], but how they
high-protein/low-carbohydrate diet the butyrate levels decreased but these results were likely to be due a diet low in carbohydrate rather than high in protein [9,10].
This study aims to further investigate the correlation between diet and the abundance of butyrate-producing bacteria. Although rather much is known about the influence of
carbohydrates on the microbiota, little is known about the impact of fat and protein intake on butyrate production.
Aim
The aim of the study is to investigate if there is a correlation between dietary components and butyrate-producing bacteria.
Material and Methods
Subjects
The aim of the study was to collect 20 fecal samples. Subjects were recruited via advertisement at Örebro University during May-June 2017. Inclusion criteria were age between 18-65 years and completely healthy. Exclusion criteria were present or previous gastrointestinal disease, previous gastrointestinal surgery, current infection, current or high risk of developing infectious disease like HIV, hepatitis A, B or C, consumption of antibiotics for 3 months previous to the study, completed tattoo or piercing during 6 months prior to the study and pregnancy or breastfeeding.
Abundance of butyrate-producing bacteria in stool samples
DNA was extracted from the stool samples using repeated bead beating and the Qiagen Stool Sample DNA extraction kit as previously described [16]. Faecal DNA was analysed for butyrate-producing microbiota by a semi-quantitative real-time PCR-based assay for
estimation of numbers of butyryl-coenzyme A (CoA) CoA transferase gene copies [17]. Most butyrate-producing bacteria in the human colon use this butyryl-CoA CoA transferase for the last step of butyrate formation, and it has been shown to be a good marker for quantification of the main butyrate-producing bacteria in the human colon [17].
Diet
The subjects completed a food frequency questionnaire (FFQ) [18], see attachment A, providing information on their dietary intake. Average total energy requirement was calculated using formulas from the Nordic Nutrition Recommendations 2012 [19]. Energy percentage (E%) was used when analyzing fat, carbohydrates and protein. E% measures the percentage of energy derived from a nutrient in relation to total energy intake. When
analyzing fibers, grams per day (g/day) was used as measurement. We used E% to look at intake ratios between the dietary compounds and g/day because the low energy utilization from fibers makes it not applicable to use E% for them.
Statistics
The data was analyzed using SPSS version 24. All data were tested for normality using the Shapiro-Wilk test and all data was log10-transformed to make it normally distributed.
Correlation was tested using Pearson’s correlation test. Statistical significance was defined as p < 0.05. The quantity of butyrate-producing DNA was used as the dependent variable. The independent variables were energy percentage (E%) of fat, carbohydrate and protein. Fiber intake was analyzed in grams per day.
Ethics
Ethical approval was obtained from the Central Ethical Review Board of Uppsala, Sweden (registration number 2017/072). Written informed consent was obtained from all study participants before the start. The study was registered at ClinicalTrials.gov (NCT03275467) on September 7, 2017. The subjects were not exposed to any harmful situations except maybe to inconveniences or discomfort during the collection of stool samples. Presentations of results were anonymized with key codes only accessible to members of the research team.
Results
Fecal samples were collected from 18 healthy subjects. One participant was excluded because of reported IBS symptoms. 7 males and 10 females aged 23-50 years were included in the study and the mean age was 29 years (see Table 1 and 2). Three subjects had taken antibiotics during the previous year. One subject followed a vegetarian diet and one excluded wheat from
the diet.
Table 1: Number of subjects in male, female and total numbers
Male Female Total
Number of participants
7 10 17
Table 2: Characteristics of study population and nutrient intake
Total mean (range) Male mean (range) Female mean (range) Average dietary intake in Sweden* Age (years) 29 (23-50) 29 (23-38) 30 (24-50) BMI (kg/m2) 23 (19-28) 24 (22-28) 22 (19-23) Calorie intake (kcal/day) 1530 (859-3000) 1916 (1005-3000) 1260 (859-2087) Fat (E%) 39 (30-50) 39 (30-47) 39 (30-50) 34 Carbohydrates (E%) 45 (32-58) 44 (32-58) 46 (35-55) 45-60 Protein (E%) 13 (8-17) 14 (10-17) 13 (8-16) 10-20 Fiber (g/day) 13 (7-24) 15 (8-24) 12 (7-18)
*The average dietary intake (E%) in Sweden 2003–2012 [19]. E% = Energy percentage, the
percentage of energy derived from a nutrient in relation to total energy intake. BMI = body mass index (kg/m2).
Calorie intake
Most subjects reported lower amount of total energy intake per day than expected from the average energy requirements. Only four subjects reported higher intake than the average energy requirement (see Figure 1). The reported mean calorie intake was 1530 kcal/day and the range was 859-3000 kcal/day (see Table 2).
Figure 1: Average total energy requirement (kcal/day) compared to reported total energy
intake (kcal/day) of the subjects.
Table 3: Correlation of amounts of butyrate-producing bacteria in stool samples and dietary compounds - Pearson’s correlation coefficient and p-value.
Dietary compound Pearson’s Correlation
coefficient P-value Fat -0.091 0.730 Carbohydrates 0.276 0.283 Protein -0.528 0.029 Fiber 0.101 0.699
Fat
Figure 2: Correlation between amount of butyrate producing bacteria (gene copy number,
Log10) and fat intake (E%, Log10). E% = energy percentage, the percentage of energy derived from a nutrient in relation to total energy intake. Each dot represents one subject.
No correlation between fat intake and butyrate levels was found (r=-0.091, p=0.730, see Figure 2 and Table 3). Mean reported fat energy percent (E%) was 39 E%, range 30-50 E% (see Table 2). Correlation tests between saturated-, mono- and poly saturated fat and butyrate-producing bacteria did not show significant results (data not shown).
Carbohydrates
Figure 3: Correlation between amount of butyrate producing bacteria (gene copy number,
Log10) and carbohydrate intake (E%, Log10). E% = energy percentage, the percentage of energy derived from a nutrient in relation to total energy intake. Each dot represents one subject.
No significant correlation result was found between butyrate levels and carbohydrate intake (r=0.276, p=0.283, see Figure 3 and Table 3). The mean reported energy percent (E%) of carbohydrates was 45 E%, range 32-58 E% (see Table 2).
Protein
Figure 4: Correlation between amount of butyrate producing bacteria (gene copy number,
Log10) and protein intake (E%, Log10). E% = energy percentage, the percentage of energy derived from a nutrient in relation to total energy intake. Each dot represents one subject. A negative moderate correlation was found between protein intake and butyrate levels (r=-0.528, p=0.029, see Figure 4 and Table 3). The mean energy percent (E%) reported was 13 E%, range 8-17 E% (see Table 2).
Fiber
Figure 5: Correlation between amount of butyrate producing bacteria (gene copy number,
Log10) and fiber intake (g/day, Log10). Each dot represents one subject.
No significant correlation was found between fiber intake and butyrate levels (r=0.101, p=0.699, see Figure 5 and Table 3). Mean reported fiber intake was 13 g/day, range 7-24 g/day (see Table 2).
Discussion
In this study, a negative correlation was found between high protein intake and butyrate producing bacteria in stool samples of healthy subjects. However, no correlation was found between carbohydrate or fiber intake and butyrate-producing bacteria. This is in contrast to previous studies (n=19 and n=17 respectively) in which intervention with two types of high protein diets with different amounts of carbohydrates and fat content showed that the abundance of butyrate-producing bacteria was mostly affected by the amount of
carbohydrates in the diet [9,10]. One reason for our conflicting results could be that we were looking at energy percentages (the percentage of energy derived from a nutrient in relation to total energy intake), however, we also checked for correlation with the total amount of the nutrients and found no correlations (data not shown).
Although not statistically significant, the intake of carbohydrates correlated positively with the amount of butyrate producing bacteria which is in alignment with previous research. What is notable is the weak correlation between the amount of butyrate-producing bacteria and fiber intake which is surprisingly small compared to previous studies [9]. One explanation to this could be that the fibers might not be well represented in the FFQ.
Not much is known yet about how fat and protein intake impacts the level of butyrate producing bacteria in the colon. In previous studies the amount of carbohydrates has varied together with variations in the amount of fat and protein, thus it has been difficult to discern if the impact has been due to a change in carbohydrate intake or a change in protein or fat ratio [9,10]. Intervention studies with diets equal in carbohydrates but with changes in fat or protein ratio could be an alternative for the future.
The subjects’ accuracy in answering the food frequency questionnaire (FFQ) is a weakness in this study. The reported calorie intake was lower than the estimated average intake for most of the subjects, suggesting that there might have been an underreporting of dietary intake (Figure 1). However, the ranges of energy percentage of the nutrients were similar to that of the average population, although fat was a little higher and carbohydrates on the lower range, implying that the subjects did report a somewhat correct ratio between the dietary compounds (see Table 2). For a more accurate assessment of dietary intake in future studies, the
participants could complete a food diary instead of a FFQ. A food diary provides more detailed information and gives a more accurate assessment of dietary intake since all meals are registered in a short period after food intake. We chose a FFQ as it is simple for the participants to complete and the data is easy to analyze.
In this pilot natured study, the low number of subjects made it challenging to find statistically significant results. In general, the non-significant p-values can probably be explained by the low number of subjects in this study. Our findings do implicate that there is a correlation between dietary intake and amount of butyrate producing bacteria, however a higher number
of participants is needed for further studies. Also, the study population of this study was a homogenous group of university educated individuals which makes it questionable if the results of this study can be generalized for the whole population.
Finding fecal transplant donors with high butyrate intake is an implication for the future. Fecal microbiota transplantation (FMT) is now recommended as treatment of recurring Clostridium difficile infections [21], and improvement of ulcerative colitis (UC) after FMT treatment has been seen in several case reports, indicating that FMT may in the future be used as a treatment for UC [22]. When searching for fecal transplant donors, it is an advantage to be able to look at dietary pattern to see what candidates are likely to have a high amount of butyrate-producing bacteria before making further costly and time-consuming tests. Donors could also be provided with a diet that promotes butyrate producers before giving fecal donations. In diseases in which a low amount of butyrate-producing bacteria play a role in the pathogenesis, dietary intervention increasing the butyrate levels might be a way to treat the disease.
Conclusion
We found a negative correlation between amount of butyrate-producing bacteria in stool samples and dietary protein intake. However, further studies with larger participant numbers are needed to confirm these results.
Acknowledgement
I would like to give a special thanks to my supervisors, Doctoral student Savanne Holster and Julia König, PhD for the support and guidance in the writing of this thesis. Part of this
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