Jurnal Gizi Indonesia (The Indonesian Journal of Nutrition)

Jurnal Gizi Indonesia Vol. 11, No. 2, June 2023 (119-127)

Submitted: 10 January 2023, Accepted: 06 February 2023
Online https://ejournal.undip.ac.id/index.php/jgi

COMPARISON BETWEEN METABOLIC PARAMETERS, FOOD INTAKE,

AND GUT MICROBIOTA IN TYPE 2 DIABETES AND NON-DIABETIC
INDONESIAN WOMEN
Ninik Rustanti1,3, Agnes Murdiati1, Mohammad Juffrie2, Endang Sutriswati Rahayu1,4*

ABSTRACT
Background: Globally, the increasing incidence of type 2 diabetes mellitus (T2D) has resulted in an upsurge in research
into this metabolic condition. Women, particularly in Indonesia, have a greater risk of T2D than males. The diversity of
the gut microbiota (GM) in T2D is regulated by the number of carbs, protein, fat, and fiber consumed.
Objectives: This study examined the comparison between metabolic parameters, food intake, and GM in T2D and non-
diabetic Indonesian women.
Materials and Methods: The cohort study included people who did not have T2D and those who did. On day 28 of
observations, anthropometric, metabolic parameters, food intake, physical activity, and feces were collected. Feces were
collected for pH, SCFA, and GM (L. plantarum, Bifidobacterium, and Prevotella) analysis.
Results: There were significant differences between non-diabetic and diabetic women in age, Waist Hip Ratio (WHR),
fasting blood sugar (FBS), and HbA1c. The two groups did not differ significantly in terms of their macronutrient intake
(calories, carbs, protein, and fat), total water, and dietary fiber. Fecal pH and GM did not statistically differ between the
control and T2D groups. Fasting blood sugar and HbA1c were positively associated with age, duration of T2D, WHR,
and total water consumption, but slightly negatively associated with dietary fiber intake. Fasting blood sugar was also
slightly negatively associated with Prevotella, meanwhile HbA1c with Bifidobacterium. Carbohydrate intake were
positively correlated with acetic, propionic, and butyric acid levels.
Conclusion: Macronutrient intake, fecal pH, SCFA, and GM did not differ because GM in T2D increased bacause
metformin consumption so that SCFA similar between two group.

Keywords : food, gut microbiota, short-chain fatty acid, diabetes, women

BACKGROUND
Diabetes Mellitus Type 2 (T2D) is a metabolic condition defined by elevated blood glucose levels
caused by a combination of inadequate insulin production and insulin resistance [1]. The International Diabetes
Federation (IDF) estimates that around 463 million (9.7%) adult persons aged 20–79 years had diabetes in
2019, with that number expected to climb to 700 million (10.9 percent) by 2045. Indonesia is one of the top
ten nations in the world with the greatest prevalence of diabetes, with 10.7 million adult diabetics in 2019 and
a projected increase to 16.6 million by 2045 [2]. The prevalence of diabetes among women is higher than
among men in Indonesia based on the national basic health research in 2018 [3]. Compared to men, women
had a greater relationship between diabetes mellitus and acute myocardial infarction, and chronic ischemic
heart disease [4]
T2D in Indonesia is determined by lifestyle, eating behavior, eating patterns such as smoking, obesity,
unhealthy diet, lack of physical activity, consumption of alcoholic beverages, hypertension, dyslipidemia, and
risk factors that cannot change, such as age and genetic factors [5–7]. Evidence suggests that dysbiosis of the
gut microbiota (GM) plays a critical role in the development and progression of T2D. Gut microbiota can
disrupt the host's glucose homeostasis [8]. A prior study revealed a link between Bifidobacterium and diabetes
mellitus development. Bifidobacterium were less represented in the microbiota of the diabetic group than the
non-diabetic group [9–11]. The diversity of GM is regulated by the number of carbs, protein, fat, and fiber
consumed, as well as the type of diet consumed [12–14]. Prevotella, which is primarily seen in persons in
developing countries or vegetarians, is very prevalent in the gut microbiome of most Indonesians, according
1
Department of Food and Agricultural Product Technology, Faculty of Agricultural Technology, Universitas Gadjah
Mada, Indonesia
2
Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Indonesia
3
Department of Nutrition Science, Faculty of Medicine, Universitas Diponegoro, Indonesia
4
Center for Food and Nutrition Studies, Universitas Gadjah Mada, Indonesia
* Correspondence: [email protected]

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to the previous study [15]. L. plantarum is also a dominating bacterium in the GM of the average Indonesian
[16]. Numerous metabolic products of GM, including short-chain fatty acids (SCFA) are implicated in the
control of glucose metabolism [17]. Therefore, GM and its correlation with SCFA, metabolic markers, food
intake has been considered as a suitable target for studying the T2D mechanisms. The purpose of this study
was to compare between metabolic parameters, food intake, and GM (L. plantarum, Bifidobacterium, and
Prevotella) in type 2 diabetes and non-diabetic Indonesian women.

MATERIALS AND METHODS

Study Design
This study was a cohort study compared two groups of subject participants: the non-T2D group and
the T2D group. The primary outcomes in this study are food intake, GM composition, and fecal SCFA, whereas
the secondary outcomes are demographic data, anthropometric data, metabolic markers, and physical activity.
Demographic data including age, and duration of T2D were obtained at the start of this study. Anthropometric
data, stool samples, and blood samples were collected at the end of this study. Further analysis of stool samples
was performed for GM and SCFA analyses, while blood samples were for metabolic markers analysis such as
HbA1c, fasting blood sugar (FBS), and cholesterol total analysis. For 28 days, food intake and physical activity
data were obtained using a semi-quantitative food frequency questionnaire (SQ-FFQ) and the International
Physical Activity Questionnaire (IPAQ). Based on the Indonesian Food Composition Data and Indonesians'
eating patterns, the FFQ included 165 food items in 9 food categories. The individuals disclosed their
consumption habits (never, daily, weekly, or monthly), as well as the quantity of every food item they had
consumed. For further analysis, the stated daily consumption of each food item was translated to grams. The
FFQ validated before to used. Consumption of macronutrients and micronutrients were determined using the
NutriSurvey 2007 application. (http://www.nutrisurvey.de/).

Location and Time

The research was conducted in public health centers in the Sleman regency, Yogyakarta, Indonesia,
and conducted from October 2019 to March 2020. FBS, HbA1c, and total cholesterol analysis were conducted
in Parahita Laboratory, Yogyakarta. Preparation of DNA extraction and SCFA analysis was done in
Biotechnology Laboratory and Waste Management Laboratory in the Faculty of Agricultural Technology,
Universitas Gadjah Mada. The PCR analysis of gut microbiota was carried out at the Laboratorium Penelitian
dan Pengujian Terpadu (LPPT), Universitas Gadjah Mada.

Subject Participants
The study compared two groups of subject participants: 12 non-diabetic women (the non-T2D group)
and 12 women with T2D (the T2D group). Subjects with T2D were obtained from 133 women with T2D and
14 non-T2D women who visited 3 public health centers in Sleman regency, Yogyakarta, Indonesia. Flow
diagram of study participant can be seen in Figure 1.
They were screened based on the inclusion criteria in this study. Subjects were recruited based on
puskesmas visitor data. Then the data is screened based on medical records. if they are eligible, the subject is
visited at his home to be interviewed and his nutritional status and fasting blood sugar are measured. if they
met the inclusion criteria, subjects were asked to sign an informed consent. The inclusion criteria for the T2D
group were as follows: women aged between 20 and 50 years old, BMI < 30, HbA1c ≥ 6.5%, not pregnant
and/or breastfeeding, not menopausal, not smoking, not drinking alcohol, not consuming antibiotic drugs, and
other drugs. The inclusion criteria for the non-T2D group were the same as for the T2D group, with except of
having an HbA1c level of 6.5%. The exclusion criteria were: going through probiotic and/or antibiotic therapy
within 28 days before drawing fecal samples, being pregnant, or withdrawal of consent during the study. At
the end of the field study, only 22 women finished the study: 11 non-T2D women and 11 women with T2D.
The subject who dropped out of the non-T2D group was excluded due to the consumption of antibiotics, while
the subject from the T2D group dropped out because the subject did not consume anti-diabetic medicine.

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Figure 1. Flow diagram of study partisipant

Ethical Approval
As a condition of participation, all of the subjects had to provide written informed consent. The
Medical and Health Research Ethics Committee, Faculty of Medicine, Public Health, and Nursing, Universitas
Gadjah Mada, authorized the study protocol, which followed the principles of the 1975 Declaration of Helsinki
(Protocol number: KE/FK/1356/EC/2019; Approval date: 18 November 2019). The Ethical Committee
approval document can be seen in Supplementary File 1.

Fecal Sample Collection

Before stool collection on day 28 (+1 day), each patient was given a fecal kit box and the method was
described. The participants were asked to defecate and were then placed in fecal tubes. A sample in a fecal
box containing ice bags was bought to the laboratory as soon as possible. After that, a fecal sample was
immediately transferred into another fecal tube containing 2 mL of RNA (Sigma-Aldrich; R0901; Saint Louis,
MO, USA). It was stored at frozen temperature (-18°C until -40°C) imamediately before it was used [15,18].

DNA Extraction
The sequencing process began with the extraction of DNA from the fecal sample. DNA was extracted
using a modified bead-beating technique previously described by Nakayama et al [15] with adjustments as
previously explained by Rustanti et al. [19].

Quantitative real-time qPCR Analysis

The microbiota analysis stage used the quantitative real-time PCR method, including DNA dilution
from the results of DNA isolation, making PCR master mix, reading, making standard curves, and calculating
the results of the total number of bacteria (log10 bacterial cells/g stool)[19,20]. The primers used had a DNA
base sequence, as shown in Table 1.
Table 1. The specific primers used in the study
Target Primer Sequence ( 5’ → 3’ )
Lactobacillus plantarum sg-Lpla-F CTC TGG TAT TGA TTG GTG CTT GCA T [20]
sg-Lpla-R GTT CGC CAC TCA CTC AAA TGT AAA
Bifidobacterium g-Bifid-F CTC CTG GAA ACG GGT GG [21]
g-Bifid-R GGT GTT CTT CCC GAT ATC TAC A
Prevotella g-Prevo-F CACRGTAAACGATGGATGCC [21]
g-Prevo-R GGTCGGGTTGCAGACC

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pH and SCFA Analysis

A pH meter was used to determine the pH of the feces (pH meter; Spear Eutech). Following
calibration, the probe was dipped immediately into the feces sample and measured until a stable value was
obtained [19].

Statistical Analysis
The SPSS 17 for windows was used for statistical analysis. Data presented as the mean ± SD. The data
normality test was carried out using the Shapiro Wilk with a significance of 0.05 because it has a sample size
of < 50. An independent t-test and non-parametric Mann-Whitney test were used to compare the groups with
a significance of 0.05. Spearman correlation was used to examine a few parameters. Corrplot figure showing
the correlation between two parameters analyzed using R.

RESULTS
Subject Characteristics
Subject characteristics after 4 weeks of observation are presented in Table 2.
Table 2. Characteristic of Subjects
Non T2D (n=11) T2D (n=11)
Subject Characteristics p-value
Mean ± SD Mean ± SD
Age (years) 36.27 ± 5.69 43.82 ± 4.17 0.0021*
Weight (kg) 55.71 ± 5.76 5.68 ± 8.30 0.7531
Height (cm) 152.60 ± 4.77 150.62 ± 6.40 0.4201
Body Mass Index/ BMI (kg/m2) 23.94 ± 2.33 24.99 ±3.26 0.3931
Normal 7 (63.6 %) 6 (54.5 %)
Overweight 4 (36.4%) 5 (45.5 %)
Waist Circumference/WC (cm) 82.03 ± 5.70 85.96 ± 7.46 0.1801
Hip Circumference/HC (cm) 96.20 ± 4.67 95.03 ± 6,26 0.6241
WHR (Waist Hip Ratio) 0.85 ± 0.04 0.90 ± 0.05 0.0111*
Systolic 115.09 ± 10.80 124.45 ± 16.90 0.0612
Diastolic 77.73 ± 3.38 81.09 ± 9.69 0.4892
Fasting Blood Sugar /FBS (mg/dL) 81.09 ± 6.28 149.55 ± 53.58 0.0002*
HbA1c (%) 5.42 ± 0.39 8.19 ± 1.84 0.0002*
Total cholesterol (mg/dL) 175.64 ± 32.09 193.45 ± 39.81 0.2611
Duration of T2D (year) - 2.5 ± 1.55
< 1 year 2 (18.2%)
1 - 3 years 6 (54.5%
> 3 years 3 (27.3%)
Type of drugs
Metformin 2 (18.2%)
Metformin & Glimepiride 8 (72.7%)
Metformin & Gliabetes 1 (9.1%)
1 independent t-test; 2 Mann Whitney

Subject characteristics in T2D group had significantly higher age, FBS, HbA1c, and waist-to-hip ratio
(WHR) than non-T2D group. Body mass index (BMI), cholesterol total, systolic, and diastolic were not
significantly different between the two groups. The mean WHR in the T2D group (0.90 ± 0.05 cm) was higher
than in the non-T2D group (0.85 ± 0.04 cm).

Food Intake and Physical Activity

The results of food intake and physical activity can be seen in Table 3. Food intakes per day, including
calories, protein, fat, and dietary fiber, were not significantly different in the two groups. In both groups, only
protein relative intake was in the range of WHO recommendations (protein 10% to 15%). Meanwhile,
carbohydrate relative intake was lower (carbohydrate, 55% to 75%) and the fat relative ratio was higher than
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the WHO recommendation (fat, 15 to 30%). However, the T2D group had a significantly higher total water
daily intake (2073.36 ± 374.89 ml) than the non-T2D group (1401.13 ± 477.57 ml).

Table 3. Macronutrient Intake and Physical Activity Per Day in Two Groups

Macronutrient Intake Non-T2D(n=11) T2D (n=11)

p-value
and Physical activity Mean ± SD (% Energy) Mean ± SD (% Energy)

Energy (kcal) 1395.66 ± 220.61 1453.55 ± 491.70 0.4502

Carbohydrate (g) 186.80 ± 45.94 (53.0) 186.66 ± 62.24 (51.6) 0.3702
Protein (g) 52.66 ± 13.87 (15.1) 46.75 ± 19.34 (12.9) 0.2002
Fat (g) 48.65 ± 12.15 (31.7) 61.46 ± 25.74 (37.5) 0.5992
Dietary Fiber (g) 13.22 ± 3.61 10.41 ± 6.87 0.0612
Total water (ml) 1401.13± 477.57 2073.36 ± 374.89 0.0021*
Physical activity (MET) 7354.6 ± 2618.8 6872.5 ± 3709.2 0.5981
1
independent t-test; 2 Mann Whitney

Fecal pH and Short-Chain Fatty Acids (SCFA)

In the feces of the non-T2D and T2D groups, there was no significant variation in pH, total SCFA,
acetic, propionic, and butyric acid (Table 4). Acetic acid was the most predominant SCFA. The acidity of the
gut environment as a result of microbial metabolites was measured using fecal pH.
Table 4. The short chain fatty acid (SCFA) and fecal stool pH profile in two group
Mean ± SD
Characteristics p-value
Non T2D (n=11) T2D (n=11)
pH 6.17 ± 0.43 6.11 ± 0.45 0.7781
a
Total SCFA (mmol/g) 21.04 ± 8.70 22.46 ± 10.42 0.7321
Acetic acid (mmol/g) 12.97 ± 5.36 12.89 ± 5.76 0.9731
Propionic acid (mmol/g) 3.87 ± 2.21 5.53 ± 4.26 0.5622
Butyric acid (mmol/g) 2.75 ± 1.21 2.75 ± 1.49 0.9961
a
Total SCFA was the sum of acetic, propionic, iso-butyric, butyric, iso-valeric, valeric, and iso-caproic acid.
1
independent t-test; 2 Mann Whitney

Gut Microbiota
Table 5. Gut Microbiota between Two Treatment Groups
Log10 bacterial cell/g (detection rate)
Type of microbial p value
Non T2D (n=11) T2D (n=11)
L. plantarum 4.41 ± 0.29 (100) 4.72 ± 0.54 (100) 0.1071
Bifidobacterium 7.11 ± 0.47 (100) 6.72 ± 0.82 (100) 0.1811
Prevotella 7.85 ± 0.97 (100) 7.32 ± 1.35 (100) 0.3021
1
independent t-test; 2 Mann Whitney

As the bacteria of interest, qPCR analysis was used to determine the counts of L. plantarum,
Bifidobacterium, and Prevotella (Table 5). Compared to the T2D group, L. plantarum in the non-T2D group
tended to be lower, but Bifidobacterium and Prevotella tended to be greater. There was no significant
difference in L. plantarum, Bifidobacterium, and Prevotella in the T2D and non-T2D groups.

Correlations Between Metabolic Markers, Food Intake, SCFA, and Gut Microbiota
The correlation of the two investigated parameters using corrplot was seen in Figure 2. Both FBS and
HbA1c showed a positive correlation with age (r: 0.498, p: 0.018 and r: 0.591, p: 0.004, respectively), duration
of T2D (r: 0.838, p: 0.000 and r: 0.819, p: 0.000, respectively), total water (r: 0.456, p: 0.033 and r: 0.445, p:
0.038, respectively). Meanwhile they showed slightly negatively with dietary fiber intake (r: -0.397, p: 0.067
and r: 0.-363, p: 0.097, respectively). FBS had negative correlate with Prevotella (r: -0.400, p: 0.065), but
HbA1c with Bifidobacterium (r: -0.417, p: 0.054). FBS positively correlate with HbA1c (r: 0.828, p: 0.033).
HbA1c was positive correlation with WC and fat ratio (r: 0.442, p: 0.039 and r: 0.401, p: 0.065, respectively).
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There was a positive correlation between total cholesterol and fat intake ratio (r: 0.502, p: 0.017), but
a slightly negative correlation with carbohydrate ratio (r: -0.408, p: 0.060). Systolic displayed a positive
correlation with total water (r: 0.467, p: 0.029) and a slightly positive correlation with duration of T2D, calorie
and carbohydrates intake (r: 0.373, p: 0.082; r: 0.381, p: 0.080 and r: 0.387, p: 0.075, respectively).
Fecal pH displayed a negative correlation with WC (r: -0.460, p: 0.031), WHR (r: -0.430, p: 0.046),
acetic acid (r: -0.501, p: 0.018), propionic acid (r: -0.574, p: 0.005), total SCFA (r: -0.493, p: 0.020). Acetic,
propionic, and butyric acid acid was positively correlated with carbohydrate intake (r: 0.478, p: 0.024; r: 0.535,
p: 0.010; r: 0.417, p: 0.053 respectively). Acetic acid had slightly negatively correlation with Bifidobacterium
(r: -0.400, p: 0.065), meanwhile butyric acid had slightly positively correlation with L. plantarum (r: 0.363, p:
0.097) and calorie intake (r: 0.363, p: 0.097).

Figure 2. Corrplot Showing the Correlation between Two Parameters Analyzed using The Spearman Method. A
Bigger Circle shows A Higher Correlation Coefficient. A Blue Circle Means Positive Correlation, While A Red
Circle Indicates Negative Correlation.

DISCUSSION
There was no significant difference in L. plantarum, Bifidobacterium, and Prevotella in the T2D and
non-T2D groups. Meanwhile, the relative abundance of Bifidobacterium, in the DM2 group was lower than
the non-DM2 group. Bifidobacterium has a negative correlation with fasting blood glucose [22].
Bifidobacterium is often associated with protective properties in DM2 [10]. However, metformin significantly
increased Bifidobacterium to improve glucose tolerance [23]. Prevotella levels tends to decreased in the T2D
group. Because T2D was shown to have more Bacteroides, which has an antagonistic relationship with
Prevotella, as seen in enterotypes, according to several studies [24]. Several studies have shown that there is
a positive effect of metformin on the Bacteroidetes phylum, in particular increasing the abundance of
Bacteroides, one of the genera within the Bacteroidetes phylum [25]. Another study shows that Prevotella
level in lean T2D Indonesian subjects was lower than in the non-T2D subjects. In this study all T2D patients
used metformin. some T2D patients in the overweight BMI category.
However, Lactobacillus plantarum, Bifidobacterium, and Prevotella in two groups were lower than L.
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plantarum, Bifidobacterium, and Prevotella in the young healthy subject in Yogyakarta by about 5.0 ± 1.0 ;
9.4 ± 0.6 and 10.0 ± 1.2 log10 bacterial cell/g, respectively [16]. In the T2D group, Lactobacillus plantarum
tended to be more prevalent than in the non-T2D group. Lactobacillus plantarum was shown to be the most
prevalent among the higher-level Lactobacillus species detected in the gut.
Food intakes per day, including calories, protein, fat, and dietary fiber, were not significantly different
in the two groups. Similar to another study, there were no significant differences in intake of total calories,
carbohydrates, protein, or fat between the diabetes group and the control group [24]. The T2D group had a
significantly higher total water daily intake (2073.36 ± 374.89 ml) than the non-T2D group (1401.13 ± 477.57
ml). Diabetic patients have symptoms including polyuria and polydipsia because of glucose homeostasis. For
diabetics, drinking water can help to reduce your blood sugar (glucose) levels by diluting the amount of sugar
in the bloodstream. Adequate intake of water also helps to alleviate the dehydration that comes with excess
urination caused by high glucose levels[1,26]. In this study, water intake correlated positively with L.
plantarum, but the mechanisms are still unclear.
In the feces of the non-T2D and T2D groups, there was no significant variation in pH, total SCFA,
acetic, propionic, and butyric acid. These results are the same as a study in Japan which showed no significant
difference in stool pH between the control group, namely 6.85 ± 0.85 and DM2, namely 6.74 ± 0.75 [27]. Stool
pH under normal conditions ranges from 6.0 to 7.2 [28]. The pH of the stool indicates the acidity of the
intestinal environment related to organic acids which are the result of commensal microbial fermentation in
the colon such as acetic, propionic, and butyric acids [29,30]. It was proven in this study that the results of
Spearman's correlation analysis showed a negative correlation between fecal pH and acetic acid (r: -0.501, p:
0.018), propionic acid (r: -0.574, p: 0.005) and total SCFA (r: -0.501, p: 0.018).
In DM2 patients who used metformin, SCFA increased again after decreasing in prediabetic and DM2
patients who did not use metformin [31,32].Propionic acid in the DM2 group, namely 5.53 ± 4.26 mmol/g,
tended to be higher than non-DM2, namely 3.87 ± 2.21 mmol/g. This is because the relative abundance of
Bacteroidetes which produce acetate and propionate in the DM2 group is significantly higher than the non-
DM2 group. Firmicutes produces butyrate as a metabolic end product [8,29]. Acetic acid is the most dominant
SCFA. The molar ratio of acetate, propionate and butyrate is approximately 60:20:20 [29]. Propionate has
been demonstrated to cause the gut peptides glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) to
secrete, which are implicated in hunger regulation, glucose metabolism, and inflammation [33]. The SCFA
formed in the intestine is influenced by diet, the number and type of microbiota, and the transit time in the
intestine. SCFA is mostly produced from anaerobic fermentation of dietary fiber and carbohydrates, especially
resistant starch. r: 0.438, p: 0.042). Meanwhile, butyric acid has a positive correlation with dietary fiber (r:
0.456, p: 0.033)
A diet high in complex carbs and fiber can improve HbA1c and FBS because they can produce amount
of producing bacteria short-chain fatty acids and produce mucus. The non-T2D group had a diverse gut
microbiota that was enriched with short-chain fatty acid-producing bacteria and mucus-producing bacteria like
Faecalibacterium, Akkermansia, Lachnospira, and Roseburia, which inhibited Collinsella and Streptococcus
with proinflammatory effects and reduced inflammation in T2D. The presence of SCFA-producing bacteria
can increased levels of intestinal SCFAs, reduced proliferation of dangerous bacteria, activated intestinal cells
to release GLP-1, and increased insulin and HbA1c levels in patients [34].
This study has some limitations, which are listed below. The sample size was insufficient to provide
significant statistical power, implying that further samples would be necessary to validate the study's findings.

CONCLUSIONS
The two groups did not differ significantly in terms of their macronutrient intake (calories, carbs,
protein, and fat), total water, and dietary fiber. Fecal pH and GM did not statistically differ between the control
and T2D groups. FBS and HbA1c were positively associated with age, duration of T2D, WHR, and total water
consumption, but slightly negatively associated with dietary fiber intake. FBS was also slightly negatively
associated with Prevotella, meanwhile HbA1c with Bifidobacterium. Carbohydrate intake were positively
correlated with acetic, propionic, and butyric acid levels.

ACKNOWLEDGMENT
Thank for supported financially by the Ministry of Research and Technology (No. 3083/UN1.DITLIT/DIT-
LIT/PT/2020), Grant for Dissertation of Doctorate Candidate (No. 27/E1/KPT/2020), and the Center of
Excellence in Science and Technology (No. 6648/UN1/DITLIT/DIT-LIT/PT/2021).

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