Journal of the American College of Nutrition

ISSN: 0731-5724 (Print) 1541-1087 (Online) Journal homepage: https://www.tandfonline.com/loi/uacn20

Antiobesity and Uric Acid-Lowering Effect of

Lactobacillus plantarum GKM3 in High-Fat-Diet-
Induced Obese Rats

Chin-Lin Hsu, Yu-Hsin Hou, Ci-Sian Wang, Shih-Wei Lin, Bo-Yi Jhou, Chin-Chu
Chen & Yen-Lien Chen

To cite this article: Chin-Lin Hsu, Yu-Hsin Hou, Ci-Sian Wang, Shih-Wei Lin, Bo-Yi Jhou, Chin-
Chu Chen & Yen-Lien Chen (2019): Antiobesity and Uric Acid-Lowering Effect of Lactobacillus
plantarum GKM3 in High-Fat-Diet-Induced Obese Rats, Journal of the American College of
Nutrition, DOI: 10.1080/07315724.2019.1571454

To link to this article: https://doi.org/10.1080/07315724.2019.1571454

Published online: 22 Feb 2019.

Submit your article to this journal

View Crossmark data

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=uacn20
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION
https://doi.org/10.1080/07315724.2019.1571454

Antiobesity and Uric Acid-Lowering Effect of Lactobacillus plantarum GKM3 in

High-Fat-Diet-Induced Obese Rats
Chin-Lin Hsua,b, Yu-Hsin Houc, Ci-Sian Wangc, Shih-Wei Linc, Bo-Yi Jhouc, Chin-Chu Chenc,d,e,f, and
Yen-Lien Chenc
a
School of Nutrition, Chung Shan Medical University, Taichung City, Taiwan; bDepartment of Nutrition, Chung Shan Medical University
Hospital, Taichung City, Taiwan; cGrape King Bio Ltd, Taoyuan City, Taiwan; dDepartment of Food Science, Nutrition, and Nutraceutical
Biotechnology, Shih Chien University, Taipei City, Taiwan; eInstitute of Food Science and Technology, National Taiwan University, Taipei
City, Taiwan; fDepartment of Bioscience Technology, Chung Yuan Christian University, Taoyuan City, Taiwan

ABSTRACT ARTICLE HISTORY

Objective: Obesity has become one of the world’s biggest issues. This condition has a great Received 16 October 2018
impact on several metabolic and chronic diseases. For example, obesity is often accompanied by Accepted 15 January 2019
hyperuricemia or gout. However, few drugs are available for the treatment of obesity. The present
KEYWORDS
study is to evaluate the antiobesity effect of Lactobacillus plantarum GKM3 in high-fat-diet-induced
Lactobacillus plantarum;
obese rats and whether taking L plantarum GKM3 can effectively reduce uric acid accumulation obesity; high-fat diet; uric
caused by obesity and ameliorate other harmful factors. acid; lactic acid bacteria
Method: Sixty male Wistar rats were divided into five groups as follows: (1) ND group, fed normal
diet; (2) HFC group, fed AIN93G-based high-fat diet containing 65% solids, 7% soybean oil, and
25% lard; (3) HFL group, fed AIN93G-based high-fat diet supplemented with 102.7 mg/kg/d L plan-
tarum GKM3; (4) HFM group, fed AIN93G-based high-fat diet supplemented with 205.4 mg/kg/d L
plantarum GKM3; and (5) HFH group, fed AIN93G-based high-fat diet supplemented with
513.5 mg/kg/d L plantarum GKM3. After 6 weeks, the body, organ, and fat weights; food intake;
blood serum levels; and adipocyte size were measured.
Results: Results showed that rats fed on the high-fat diet showed more body weight, increased
feed efficiency, higher fat deposition, higher total liver weight, elevated serum lipid levels, and
increased adipocyte size compared with those on the normal diet. All these effects were reversed
by supplementation of L plantarum GKM3.
Conclusions: In conclusion, we suggest that the L plantarum GKM3 supplement may have benefi-
cial antiobesity and uric acid–lowering effects.

Abbreviations: ALT: alanine aminotransferase; AST: aspartate aminotransferase; BCRC: Bioresource

Collection and Research Center; CGMCC: China General Microbiological Culture Collection Center;
Cl-: chlorine; HDL-C: high-density lipoprotein cholesterol; HFC: high-fat-diet group; HFD: high-fat
diet; HFH: high-fat-diet group supplemented with Lactobacillus plantarum GKM3-high dose; HFL:
high-fat-diet group supplemented with L plantarum GKM3-low dose; HFM: high-fat-diet group sup-
plemented with L plantarum GKM3-medium dose; Kþ: potassium; LDL-C: low-density lipoprotein
cholesterol; ND: normal diet; Naþ: sodium

Introduction their effectiveness is limited to production and maintenance

Obesity is one of the biggest problems in modern society.
The rapid increase in world obesity prevalence points to life-
style changes as the main cause, especially in eating habits.
The World Health Organization recognizes obesity as the
greatest public health challenges of the 21st century. Obesity
is the result of sustained disequilibrium between energy
intake and energy expenditure (1), and this condition has a
great impact on several metabolic and chronic diseases
including type 2 diabetes associated with insulin resistance,
atherosclerosis, cardiovascular disease, nonalcoholic fatty liver
disease, hypertension, and hyperlipidemia (2–4). In addition,
obesity is associated with hyperuricemia and gout (5). To
date, few drugs are available for the treatment of obesity, and
of weight loss (6). Therefore, further research is needed to
discover new therapies to reduce the prevalence of obesity.
Hyperuricemia occurs when there is too much uric acid in
the blood. The main causes for higher plasma uric acid are
either lower excretion, higher synthesis, or both. Some studies
showed that hyperuricemia is closely related to body fat accu-
mulation (7, 8). Obesity is associated with higher insulin
resistance and leptin production, and both reduce uric acid
excretion (9). Previous studies have also shown that hyperuri-
cemia increases the risk for gout, cardiovascular diseases,
hypertension, hepatic disease, diabetes, obesity, renal failure,
and stroke (10–15). As a result, it is necessary to prevent
hyperuricemia-related diseases by reducing uric acid levels.

CONTACT Yen-Lien Chen, PhD lan.chen@grapeking.com.tw No.60, Sec. 3, Longgang Road, Zhongli District, Taoyuan City 320, Taiwan.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/icnv.
ß 2019 American College of Nutrition
2 C.-L. HSU ET AL.
Probiotics are live microbial dietary supplements that
exert beneficial effects on host health (16). Probiotics attract
public attention because of their potential effectiveness for
both prevention and treatment of immune diseases (17).
Recent studies have also demonstrated the preventive effects
of some bacterial strains on obesity (4, 18). Lactobacillus are
the most common probiotics in the stomach, duodenum,
and jejunum of humans and are a type of lactic acid bacteria
(19). Lactobacillus plantarum exhibits a variety of potential
beneficial effects on human health, such as improvement of
microbiota, immunomodulation, suppression of fat accumula-
tion, and reduction of serum triglyceride and serum choles-
terol (20–25). In one study, L plantarum administration
significantly reduced the mean adipocyte size in female
C57BL/6 mice fed a high-fat diet (HFD) (22). Another study
found that a strain of L plantarum exhibited a significant
effect in lowering serum cholesterol and triglycerides in
hypercholesterolemic mice (24). These data suggest that some
specific strains of L plantarum related to lipid metabolism
may be a potential new candidate in the treatment of obesity.
The aim of this study was to evaluate the antiobesity
effect of L plantarum GKM3 in HFD-induced obese rats
and whether taking L plantarum GKM3 can effectively
reduce uric acid accumulation caused by obesity and ameli-
orate other harmful factors.
Materials and methods
Preparation of L plantarum GKM3
L plantarum GKM3 was isolated by Grape King Bio Ltd,
Taoyuan, Taiwan. L plantarum GKM3 is preserved at the
Bioresource Collection and Research Center (BCRC) of the
Food Industry Research and Development Institute
(Hsinchu, Taiwan), with the preservation numbers of BCRC
910787, and preserved at the China General Microbiological
Culture Collection Center (CGMCC, Beijing, China) with
the preservation numbers of CGMCC 14565.
For functional assessment, L plantarum GKM3 grown in MRS
broth (DIFCO) under anaerobic conditions was transferred to a
2.0-L flask containing 1.2 L of MRS medium. The whole medium
was cultivated at 37  C for 1 day on a rotary shaker (10–20 rpm)
for seed culturing prior to its scale-up production step. The fer-
mented broth (1.2 L) was inoculated into a 5-ton fermentor with
80% working volume (MRS medium; pH 6.0), and agitated at 10
to 20 rpm at 37  C for 1 day. The obtained L plantarum GKM3
powder was ground after a freeze-drying process, with counts of
greater than or equal to 2  1010 CFU/g.
Animals
Sixty 6-week-old male Wistar rats, weighing 201 to 225 g, were
obtained from BioLASCO Taiwan Co., Ltd. After quarantine
and accommodation for one week, the rats were randomized
into experimental and control groups. The rats had free access
to commercial rodent diet and sterile reverse osmosis water.
They were housed in stainless steel cages and maintained at
controlled temperature (22 ± 2  C), relative humidity
(60%–80%), and light cycle (12 hours light/12 hours dark).
Study design
The protocol was approved by Chung Shan Medical
University Animal Care Committee (IACUC AHPP-Groval
NO: 1199) before the beginning of the study. The rats were
randomly divided into five groups, each comprising 12 rats.
The experiment was conducted for 6 weeks. One group (the
control group) continuously received the normal diet (ND),
and four groups received an HFD supplemented with different
doses of L plantarum GKM3—group 1 ND: rats were fed a
normal diet; group 2 HFC: rats were fed AIN93G-based HFD
containing 65% solids, 7% soybean oil, and 25% lard; group 3
HFL: rats were fed AIN93G-based HFD supplemented with
gavaged 102.7 mg/kg/d L plantarum GKM3; group 4 HFM:
rats were fed AIN93G-based HFD supplemented with gavaged
205.4 mg/kg/d L plantarum GKM3; and group 5 HFH: rats
were fed AIN93G-based HFD supplemented with gavaged
513.5 mg/kg/d L plantarum GKM3. The body weights of rats
were measured prior to the study and every two days during
the study. Measurement of feed and water intake for all rats
was conducted daily during the study.
Serum biochemistry
After 12 hours of overnight fasting, all rats were euthanized with
CO2 asphyxiation and blood samples were collected. The follow-
ing serum biochemistry parameters were analyzed by using com-
mercial kits (Diasys Co Ltd.): triglycerides, high-density
lipoprotein cholesterol (HDL-C), low-density lipoprotein choles-
terol (LDL-C), aspartate aminotransferase (AST), alanine amino-
transferase (ALT), cholesterol, creatinine, glucose, uric acid,
sodium (Naþ), potassium (Kþ), and chlorine (Cl-). We used
commercial kits (Denka Seiken Co Ltd. to analyze ketone bodies.
Total body fat and body fat percentage
Visceral adipose tissue (epididymal, perirenal, and mesenteric
fats) and subcutaneous tissue (retroperitoneal and inguinal
fats) were collected, washed, and weighed. Total body fat (mg/
g rat) = [visceral adipose tissue (mg) þ subcutaneous adipose
tissue (mg)]  final body weight (g). Body fat percentage (%)
= Total body fat (g)  final body weight (g)  100%. Visceral
adipose tissue (mg/g rat) = [perirenal adipose tissue (mg) þ
epididymal adipose tissue (mg) þ mesenteric adipose tissue
(mg)]  final body weight (g). Subcutaneous adipose tissue
(mg/g rat) = [retroperitoneal adipose tissue (mg) þ inguinal
adipose tissue (mg)]  final body weight (g).
Determination of liver and fecal lipids
The total lipids from liver homogenates and fecal matters
were extracted according to Tzang et al. method (26). Each
0.3 g sample of liver tissue and oven-dried fecal matters was
homogenized with chloroform/methanol (2/1) to a final vol-
ume 23 times the volume of the tissue sample (0.3 g in 6.9 mL

of solvent mixture). The homogenate was evaporated to con-
stant weight by a nitrogen-blowing concentrator. The total
lipid from liver or fecal matters was calculated according to
the formula: The total lipid from liver or fecal matters (mg/g)
= constant dry weight of homogenate  0.3 g sample.
The dried extract was then dissolved in isopropanol for
following determination. We used commercial kits (TG
assay kit, Teco Diagnosis) to detect hepatic triglycerides and
fecal triglycerides. Liver total cholesterol and fecal total chol-
esterol were analyzed by commercial kits (Cholesterol assay
kit, Teco Diagnostics, Rendox Laboratories Co Ltd.).
Pathology
A histopathological test was performed to examine liver and
perirenal adipose tissues. The collected organ and tissues were
dehydrated, clarified, infiltrated with paraffin, and embedded
after trimming, forming paraffin tissue blocks, and cut into tis-
sue slices. Serial sections were stained with hematoxylin and
eosin. The histopathological changes were evaluated using
optical microscope and recorded by Charge Coupled Device
and Carl Zeiss Axio Vision microscopy software 4.9.
In vitro experiment method on the uric acid–reducing
effect of lactic acid bacteria
The following method was used to examine the purine-
decomposing ability in each type of lactic acid bacteria (27).
Using a BD Difco Lactobacilli MRS Broth medium, L planta-
rum GKM3 was anaerobically cultured overnight at 37  C.
Bacterial cells were harvested by centrifugation at 3000 rpm
for 10 minutes (4  C) followed by resuspension in phosphate-
buffered saline containing 1.25 mM inosine and 1.25 mM
guanosine. These 1  109 CFU/mL bacterial cell suspension
solutions were shake-cultured for 2 hours at 120 rpm, 37  C.
The consumption level of nucleosides were measured by high
performance liquid chromatography (HPLC).
The specific operational conditions of the HPLC are as fol-
lows. Guanosine and inosine were determined by HPLC
equipped with a reverse-phase column (CAPCELL PAK C18
SG120, 2504.6 mm) and Waters 996 photodiode array
detector. The mobile phase used included 25 mM KH2PO4
(0.1% methanol) (A) and 25 mM KH2PO4 (0.1% methanol)/
methanol (75:25) (B) as the gradient. The gradient began at
100% A. Composition was remained constant for 20 minutes,
then reversed to 20% A over 5 minutes, and finally 100% A
over 15 minutes. The flow rate was 1.0 mL/min, and the col-
umn was kept at room temperature. Absorption spectra of
eluted compounds were recorded at 254 nm. The degradation
rates were calculated according to the following equation:
Degradation rate = 100 – (amount of inosine or guanosine/
amount of inosine or guanosine in the blank)  100.
Statistical analysis
Statistical analysis was performed with SPSS statistic software
package. Values are expressed as mean ± standard error of the
mean and analyzed using PROC analysis of variance followed
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 3
by Duncan’s multiple range test for comparisons of group
means. A p value < 0.05 is considered statistically significant.
Results
Effect of L plantarum GKM3 on body weights, weight
change, food intake, feed efficiency, energy intake, and
water intake
The body weight of all groups was increased every week,
especially that of the HFD-fed groups compared to that of
the ND group (Table 1). The HFD promoted a significant
increase in body weight over the 6-week period. At the end
of the experiment, the gain in body weight was higher in
the HFC group than in the HFD supplemented with L plan-
tarum GKM3 groups. The mean body weight increased by
266.62 ± 8.31 g in the ND group, 318.54 ± 11.49 g in the HFC
group, 266.89 ± 10.13 g in the HFL group, 276.41 ± 8.95 g in
the HFM group, and 261.20 ± 12.48 g in the HFH group.
The daily food intake of groups fed an HFD was lower
(p < 0.05) than that of the control group but did not differ
among the HFD-fed groups (Table 2). Feed efficiency was
significantly higher in all HFD groups than in the ND group,
but feed efficiency in HFD supplemented with L plantarum
GKM3 groups was significantly lower than in the HFC group.
There was no significant difference in energy intake and
water intake among the obese and the non-obese groups.
Effect of L plantarum GKM3 on the weights of organs
Except for the liver of the HFC group, no significant differ-
ences in organ weights was observed of all rats among
HFD-fed groups and the ND group (Table 3).
Effect of L plantarum GKM3 on the weights of total
body fat, body fat percentage, visceral adipose tissue,
and subcutaneous adipose tissue
Throughout the 6-week experimental period, total body fat,
body fat percentage, and visceral adipose tissue were signifi-
cantly lower (p < 0.05) in the L plantarum GKM3 low-,
medium-, and high-dose groups compared with those in the
HFC group (Table 4). There were no significant differences
in analysis of subcutaneous adipose tissue for all rats
between the obese and the non-obese groups. Visceral
Table 1. Effect of Lactobacillus plantarum GKM3 on body weights and weight
change in high-fat-diet-induced obese rats.
Groups Initial body weight (g) Final body weight (g) Weight change (g)
ND 231.55 ± 3.01a 498.17 ± 7.96b 266.62 ± 8.31b
HFC 234.24 ± 3.47a 552.78 ± 10.21a 318.54 ± 11.49a
HFL 230.98 ± 3.52a 497.88 ± 10.68b 266.89 ± 10.13b
HFM 231.80 ± 3.36a 508.21 ± 10.71b 276.41 ± 8.95b
HFH 231.38 ± 3.43a 492.58 ± 13.98b 261.20 ± 12.48b
The reported values are the mean ± standard error of the mean (n ¼ 12).
Mean values with different letters were significantly different (p < 0.05).
ND ¼ normal diet; HFC ¼ high-fat diet; HFL ¼ high-fat diet supplemented
with Lactobacillus plantarum GKM3-low dose; HFM ¼ high-fat diet supple-
mented with L plantarum GKM3-medium dose; HFH ¼ high-fat diet supple-
mented with L plantarum GKM3-high dose. Weight change (g) ¼ final body
weight (g)  initial body weight (g). Results of ND, HFL, HFM, and HFH
were compared with HFC.
4 C.-L. HSU ET AL.

Table 2. Effect of Lactobacillus plantarum GKM3 on food intake, energy intake, water intake, and feed efficiency in high-fat-diet-induced obese rats.
Groups Food intake (g/rat/d) Feed efficiency (%) Energy intake (kcal/rat/d) Water intake (mL/rat/d)
ND 25.75 ± 0.26a 19.89 ± 0.51c 101.95 ± 1.04a 41.78 ± 1.47a
HFC 20.51 ± 0.28b 38.76 ± 1.10a 106.88 ± 1.44a 46.12 ± 1.87a
HFL 19.45 ± 0.36b 34.29 ± 1.14b 101.35 ± 1.89a 42.35 ± 1.23a
HFM 19.76 ± 0.28b 34.94 ± 0.93b 102.95 ± 1.47a 43.08 ± 2.20a
HFH 19.54 ± 0.49b 33.22 ± 0.91b 101.80 ± 2.56a 43.15 ± 1.44a
The reported values are the mean ± standard error of the mean (n ¼ 12). Mean values with different letters were significantly different (p < 0.05). ND ¼ normal
diet; HFC ¼ high-fat diet; HFL ¼ high-fat diet supplemented with Lactobacillus plantarum GKM3-low dose; HFM ¼ high-fat diet supplemented with L plantarum
GKM3-medium dose; HFH ¼ high-fat diet supplemented with L plantarum GKM3-high dose. Results of ND, HFL, HFM, and HFH were compared with HFC.

Table 3. Effect of Lactobacillus plantarum GKM3 on the weights of organs in high-fat-diet-induced obese rats.
Groups Heart (g/rat) Liver (g/rat) Spleen (g/rat) Lung (g/rat) Kidney (g/rat)
ND 1.49 ± 0.03a 15.92 ± 0.31b 0.92 ± 0.04a 2.33 ± 0.10a 3.51 ± 0.05a
HFC 1.51 ± 0.03a 17.65 ± 0.47a 0.91 ± 0.03a 2.31 ± 0.12a 3.47 ± 0.08a
HFL 1.49 ± 0.03a 15.13 ± 0.44b 0.87 ± 0.03a 3.57 ± 1.32a 3.30 ± 0.06a
HFM 1.48 ± 0.03a 15.98 ± 0.50b 0.86 ± 0.03a 2.07 ± 0.09a 3.33 ± 0.07a
HFH 1.47 ± 0.03a 15.05 ± 0.44b 0.87 ± 0.04a 2.15 ± 0.07a 3.33 ± 0.09a
The reported values are the mean ± standard error of the mean (n ¼ 12). Mean values with different letters were significantly different (p < 0.05). ND ¼ normal
diet; HFC ¼ high-fat diet; HFL ¼ high-fat diet supplemented with Lactobacillus plantarum GKM3-low dose; HFM ¼ high-fat diet supplemented with L plantarum
GKM3-medium dose; HFH ¼ high-fat diet supplemented with L plantarum GKM3-high dose. Results of ND, HFL, HFM, and HFH were compared with HFC.

Table 4. Effect of Lactobacillus plantarum GKM3 on the weights of total body fat, body fat percentage, visceral adipose tissue, and subcutaneous adipose tissue
in high-fat-diet-induced obese rats.
Groups Total body fat (mg/g rat) Body fat percentage (%) Visceral adipose tissue (mg/g rat) Subcutaneous adipose tissue (mg/g rat)
ND 107.94 ± 8.45b 10.79 ± 0.84b 73.49 ± 5.20b 34.45 ± 3.43a
HFC 136.66 ± 3.83a 13.67 ± 0.38a 94.30 ± 2.28a 42.35 ± 2.21a
HFL 117.69 ± 5.88b 11.77 ± 0.59b 80.41 ± 3.77b 37.28 ± 2.70a
HFM 113.72 ± 7.12b 11.37 ± 0.71b 79.73 ± 4.14b 33.98 ± 3.30a
HFH 113.64 ± 6.96b 11.36 ± 0.70b 80.20 ± 4.50b 33.44 ± 2.92a
The reported values are the mean ± standard error of the mean (n ¼ 12). Mean values with different letters were significantly different (p < 0.05). ND ¼ normal
diet; HFC ¼ high-fat diet; HFL ¼ high-fat diet supplemented with Lactobacillus plantarum GKM3-low dose; HFM ¼ high-fat diet supplemented with L plantarum
GKM3-medium dose; HFH ¼ high-fat diet supplemented with L plantarum GKM3-high dose. Total body fat (mg/g rat) ¼ [visceral adipose tissue (mg) þ sub-
cutaneous adipose tissue (mg)]  final body weight (g). Body fat percentage (%) ¼ Total body fat (g)  final body weight (g)  100%. Visceral adipose tissue
(mg/g rat) ¼ [perirenal adipose tissue (mg) þ epididymal adipose tissue (mg) þ mesenteric adipose tissue (mg)]  final body weight (g). Subcutaneous adi-
pose tissue (mg/g rat) ¼ [retroperitoneal adipose tissue (mg) þ inguinal adipose tissue (mg)]  final body weight (g). Results of ND, HFL, HFM, and HFH were
compared with HFC.

fat mass was assessed by weighing the total perirenal, epi-
didymal, and mesenteric adipose tissues after dissection
(Table 5). Supplementation of L plantarum GKM3 to the
HFD led to a significant reduction (p < 0.05) in the perirenal
and epididymal adipose tissue weights.
Effect of L plantarum GKM3 on serum
biochemical parameters
It was observed that rats supplemented with L plantarum
GKM3 (the HFL, HFM, and HFH groups) for 6 consecutive
weeks showed a significant decrease in the levels of serum tri-
glycerides and LDL-C/HDL-C and a significant increase in
the levels of serum HDL-C compared to the HFC group
(p < 0.05; Table 6). Triglyceride, total cholesterol, and LDL-C
levels and LDL-C/HDL-C in these L plantarum GKM3
groups were even significantly lower than those of the ND
group (p < 0.05). No significant differences in the glucose lev-
els among the five groups were observed in this study.
The AST, ALT, uric acid, creatinine, ketone body, Naþ,
þ triglyceride, and cholesterol
K , and Cl- levels in serum in each group are summarized
in Table 7. Compared with the ND group, the HFC group
had significantly increased uric acid and ketone body levels
and decreased Kþ levels. AST, ALT, creatinine, Naþ, and
Cl- levels were not different. HFL, HFM, and HFH groups
had significantly reduced uric acid levels compared to the
HFC group. Mean uric acid levels were 5.73 ± 0.05 mg/dL
in the ND group, 7.53 ± 0.10 mg/dL in the HFC group,
4.53 ± 0.03 mg/dL in the HFL group, 6.48 ± 0.05 mg/dL in
the HFM group, and 5.58 ± 0.09 mg/dL in the HFH group.
The low-, medium-, and high-dose L plantarum GKM3
groups significantly and dose-dependently reversed the
HFD-induced elevation of ketone body level. No significant
differences in the serum Kþ levels among the ND, HFL,
HFM, and HFH groups were observed in this study.
Effect of L plantarum GKM3 on the hepatic total lipid,
triglyceride, and cholesterol levels
Hepatic lipid contents of the animals as shown in Table 8
indicate that consumption of the HFD resulted in a significant
increase in hepatic total lipids, triglycerides, and cholesterol.
Consumption of L plantarum GKM3 significantly and dose-
dependently reversed all these lipid perturbations.
Effect of L plantarum GKM3 on the fecal total lipid,
The effect of L plantarum GKM3 on the fecal total lipids,
triglycerides, and total cholesterol excretions are shown in
Table 9. Fecal total lipids, triglycerides, and cholesterol were
significantly higher in the HFD-fed groups compared with
the ND group, and the HFD-fed groups supplemented with
 JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 5

Table 5. Effect of Lactobacillus plantarum GKM3 on the weights of adipose tissue in high-fat-diet-induced obese rats.
Retroperitoneal adipose
Perirenal adipose Epididymal adipose Mesenteric adipose tissue (mg/g Inguinal adipose
Groups tissue (mg/g rat) tissue (mg/g rat) tissue (mg/g rat) rat) tissue (mg/g rat)
ND 30.95 ± 2.56b 22.59 ± 1.41b 19.94 ± 1.51a 20.92 ± 2.02a 13.53 ± 1.72a
HFC 39.82 ± 1.31a 30.99 ± 0.67a 23.49 ± 1.06a 26.53 ± 1.76a 15.82 ± 1.41a
HFL 32.72 ± 1.80b 25.96 ± 1.46b 21.73 ± 1.16a 22.88 ± 1.54a 14.41 ± 1.56a
HFM 32.54 ± 2.23b 26.42 ± 1.28b 20.78 ± 1.12a 21.23 ± 1.83a 12.75 ± 2.02a
HFH 33.32 ± 1.71b 26.24 ± 2.13b 20.64 ± 1.48a 21.32 ± 2.32a 12.12 ± 1.74a
The reported values are the mean ± standard error of the mean (n ¼ 12). Mean values with different letters were significantly different (p < 0.05). ND ¼ normal
diet; HFC ¼ high-fat diet; HFL ¼ high-fat diet supplemented with Lactobacillus plantarum GKM3-low dose; HFM ¼ high-fat diet supplemented with L plantarum
GKM3-medium dose; HFH ¼ high-fat diet supplemented with L plantarum GKM3-high dose. Results of ND, HFL, HFM, and HFH were compared with HFC.

Table 6. Effect of Lactobacillus plantarum GKM3 on serum biochemical parameters (serum lipid profile and glucose) in high-fat-diet-induced obese rats.
Groups Triglyceride (mg/dL) Total cholesterol (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL) LDL-C/HDL-C (ratio) Glucose (mg/dL)
ND 118.58 ± 12.44b 107.86 ± 4.69a 69.00 ± 2.00a 36.67 ± 1.81a 0.53 ± 0.02a 249.20 ± 4.95a
HFC 236.92 ± 7.89a 104.55 ± 2.26a,b 57.92 ± 3.65b 28.33 ± 1.63a 0.50 ± 0.03a 251.15 ± 5.49a
HFL 85.17 ± 8.43c 98.19 ± 2.55b 72.58 ± 2.71a 27.17 ± 1.78b 0.37 ± 0.02b 235.15 ± 9.47a
HFM 88.83 ± 4.61c 97.73 ± 1.98b 66.33 ± 3.14a 25.25 ± 0.92b 0.39 ± 0.02b 242.96 ± 4.24a
HFH 82.83 ± 8.72c 97.87 ± 2.47b 67.42 ± 2.73a 26.42 ± 0.78b 0.40 ± 0.01b 239.98 ± 11.41a
The reported values are the mean ± standard error of the mean (n ¼ 12). Mean values with different letters were significantly different (p < 0.05). HDL-C ¼ high-
density lipoprotein cholesterol; LDL-C ¼ low-density lipoprotein cholesterol; ND ¼ normal diet; HFC ¼ high-fat diet; HFL ¼ high-fat diet supplemented with
Lactobacillus plantarum GKM3-low dose; HFM ¼ high-fat diet supplemented with L plantarum GKM3-medium dose; HFH ¼ high-fat diet supplemented with L
plantarum GKM3-high dose. Results of ND, HFL, HFM, and HFH were compared with HFC.

Table 7. Effect of Lactobacillus plantarum GKM3 on serum biochemical parameters (safety) in high-fat-diet-induced obese rats.
Groups AST (U/L) ALT (U/L) Uric acid (mg/dL) Creatinine (mg/dL) Ketone body (mmol/L) Naþ (mmol/L) Kþ (mmol/L) Cl- (mmol/L)
a a c a b a a
ND 77.75 ± 6.32 32.42 ± 2.65 5.73 ± 0.05 0.58 ± 0.01 6.01 ± 0.16 144.75 ± 0.51 4.57 ± 0.04 105.67 ± 0.19a
HFC 72.75 ± 8.02a 38.00 ± 2.66a 7.53 ± 0.10a 0.57 ± 0.02a 7.32 ± 0.27a 145.25 ± 0.35a 4.43 ± 0.04b 105.50 ± 0.23a
HFL 76.25 ± 6.98a 40.58 ± 3.69a 4.53 ± 0.03d 0.58 ± 0.01a 6.13 ± 0.21b 145.25 ± 0.25a 4.53 ± 0.03a 105.25 ± 0.28a
HFM 73.50 ± 7.87a 35.75 ± 4.45a 6.48 ± 0.05b 0.54 ± 0.01a 5.81 ± 0.14b 144.25 ± 0.45a 4.50 ± 0.03a,b 105.25 ± 0.25a
HFH 70.58 ± 4.53a 33.92 ± 2.06a 5.58 ± 0.09c 0.56 ± 0.01a 5.07 ± 0.09c 145.08 ± 0.26a 4.52 ± 0.03a,b 105.25 ± 0.28a
The reported values are the mean ± standard error of the mean (n ¼ 12). Mean values with different letters were significantly different (p < 0.05). AST ¼ aspartate
aminotransferase; ALT ¼ alanine aminotransferase; ND ¼ normal diet; HFC ¼ high-fat diet; HFL ¼ high-fat diet supplemented with Lactobacillus plantarum GKM3-
low dose; HFM ¼ high-fat diet supplemented with L plantarum GKM3-medium dose; HFH ¼ high-fat diet supplemented with L plantarum GKM3-high dose.
Results of ND, HFL, HFM, and HFH were compared with HFC.

Table 8. Effect of Lactobacillus plantarum GKM3 on the hepatic total lipid, triglyceride, and cholesterol in high-fat-diet-induced obese rats.
Groups Hepatic total lipid (mg/g tissue) Hepatic triglyceride (mg/g tissue) Hepatic cholesterol (mg/g tissue)
ND 66.13 ± 2.91c 22.79 ± 0.61c 10.56 ± 0.35b
HFC 91.19 ± 5.70a 30.04 ± 1.21a 12.36 ± 0.33a
HFL 82.05 ± 2.13a,b 25.89 ± 1.22b 10.41 ± 0.35b
HFM 80.76 ± 2.99b 21.29 ± 1.36c 9.86 ± 0.18b
HFH 77.32 ± 2.11b 19.73 ± 0.68c 9.64 ± 0.25b
The reported values are the mean ± standard error of the mean (n ¼ 12). Mean values with different letters were significantly different (p < 0.05). ND ¼ normal
diet; HFC ¼ high-fat diet; HFL ¼ high-fat diet supplemented with Lactobacillus plantarum GKM3-low dose; HFM ¼ high-fat diet supplemented with L plantarum
GKM3-medium dose; HFH ¼ high-fat diet supplemented with L plantarum GKM3-high dose. Results of ND, HFL, HFM, and HFH were compared with HFC.

Table 9. Effect of Lactobacillus plantarum GKM3 on the fecal total lipid, triglyceride, and cholesterol in high-fat-diet-induced obese rats.
Groups Fecal total lipid (mg/g dried feces) Fecal triglyceride (mg/g dried feces) Fecal cholesterol (mg/g dried feces)
ND 22.67 ± 1.88c 3.96 ± 0.04c 5.25 ± 0.24c
HFC 35.08 ± 1.53b 6.51 ± 0.33b 7.57 ± 0.12b
HFL 46.96 ± 4.19a 7.07 ± 0.11a 8.21 ± 0.23a
HFM 49.19 ± 5.91a 7.13 ± 0.12a 8.24 ± 0.04a
HFH 52.18 ± 2.40a 7.23 ± 0.14a 8.43 ± 0.13a
The reported values are the mean ± standard error of the mean (n ¼ 12). Mean values with different letters were significantly different (p < 0.05). ND ¼ normal
diet; HFC ¼ high-fat diet; HFL ¼ high-fat diet supplemented with Lactobacillus plantarum GKM3-low dose; HFM ¼ high-fat diet supplemented with L plantarum
GKM3-medium dose; HFH ¼ high-fat diet supplemented with L plantarum GKM3-high dose. Results of ND, HFL, HFM, and HFH were compared with HFC.

L plantarum GKM3 were significantly and dose-dependently
higher than the HFC group.
Effect of L plantarum GKM3 on hepatosteatosis and the
size of perirenal adipocyte
The hepatocytes of the rats in the HFC group showed severe
macrovascular accumulation of lipid droplets within the
cytoplasm, and ballooning injury was observed around the
central vein (Figure 1). In contrast, the hepatocytes of L
plantarum GKM3-fed rats showed only mild accumulation
of lipid droplets and ballooning injury.
Histologic examination of perirenal adipose tissue
demonstrates that HFD progressively increased the size of
adipocytes (Figures 2 and 3). On the other hand,
6 C.-L. HSU ET AL.

hematoxylin and eosin staining of perirenal adipose tissue L plantarum GKM3 has a stronger uric acid–lowering
revealed that supplementation with L plantarum GKM3 was effect than other lactic acid bacterias
associated with a significant reduction in average adipocyte
Figures 4 and 5 shows the purine-decomposing ability of each
size in perirenal adipose tissue, as compared with the
of the lactic acid bacteria when they were cultured in the
HFC group.

Figure 1. Effect of Lactobacillus plantarum GKM3 on hepatosteatosis in high-fat-diet-induced obese rats. Liver was stained with hematoxylin and eosin. Original
magnification: 200. The oil drop marked by an arrow.

presence of inosine or guanosine. Among the lactic acid bac-
teria, L plantarum GKM3 had the highest purine-degrad-
ation rate.
Discussion
Various materials have been purported to have antiobesity
activity, and there is sufficient scientific evidence that lactic
Figure 2. Effect of Lactobacillus plantarum GKM3 on the size of perirenal adipocyte in high-fat-diet-induced obese rats. Perirenal adipose tissues were stained with
hematoxylin and eosin. Original magnification: 200.
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 7
acid bacteria possess this activity via inhibiting the activity
of intestinal pathogens (28), suppressing the absorption of
excess lipids (29), decreasing the level of LDL-C (30), and
inhibiting the differentiation of 3T3-L1 adipocytes (29, 30).
After 6 weeks of feeding, supplementation with L planta-
rum GKM3 reversed HFD-induced body weight gain and
the increase in total body fat and body fat percentage.
8 C.-L. HSU ET AL.
However, there was no significant differences in these
parameters in rats among the HFL, HFM, and HFH groups.
This can be explained on the basis that the low
Figure 3. Effect of Lactobacillus plantarum GKM3 on the size of perirenal adipo-
cyte in high-fat-diet-induced obese rats. The reported values are the mean-
± standard error of the mean (n ¼ 3). Mean values with different letters were
significantly different (p < 0.05).
Figure 4. Rate of guanosine decomposition (%).
Figure 5. Rate of inosine decomposition (%).
concentration of L plantarum GKM3 was sufficient for col-
onization in the intestines. The food intake of the HFD
group was lower than that of the control group. The explan-
ation for this may be the higher calorie intake of the HFD
group, which in turn reduced food intake. In addition, the
feeding efficiency of the HFD group was significantly
improved compared with that of the ND group, suggesting
that the intake of an HFD resulted in greater body weight
gain. Treatment with L plantarum GKM3 significantly
decreased the feeding efficiency compared with the HFC
group, suggesting that L plantarum GKM3 may affect hosts’
ability to harvest energy from the diet and produce a differ-
ence in energy utilization and storage.
An HFD increased liver weight and serum lipid levels.
The reduction of triglycerides, total cholesterol, or LDL-C in
serum is reported to lower the risk of coronary heart disease
(31, 32). Recent studies showed that probiotics, including
Bifidobacterium longum (33) and Lactobacillus acidophilus
(34), possess significant hypolipidemic activity in both rats
and humans. Notably, it was observed that probiotic supple-
mentation may promote perirenal and epididymal fat loss
and decrease serum lipid levels in rats with HFD-induced

obesity. This result demonstrates that L plantarum GKM3
supplementation has a protective effect against obesity
induced by an HFD in rats.
Interactions between the intestinal microbiota and miner-
als and the effect of this interaction on the health of the
host were shown (35). Elevation of blood ketone bodies by
HFD resulted in elevation of blood uric acid, which can lead
to gout. This can be prevented by oral potassium citrate
supplementation (36). The L plantarum GKM3-fed groups
significantly and dose-dependently reversed the HFD-
induced elevation of ketone bodies. In addition, L plantarum
GKM3 treatment significantly reversed HFD-induced uric
acid levels. According to the results, we suggest that L plan-
tarum GKM3 feeding resulted in increased potassium levels,
allowing rapid excretion of ketones and uric acid. Previous
studies screened serum uric acid–lowering lactic acid bac-
teria. Among these probiotics, Lactobacillus gasseri PA-3
exhibits excellent uric acid–lowering activity (37). Compared
with L gasseri PA-3 and other commercial lactic acid bac-
teria, L plantarum GKM3 still has the highest purine-deg-
radation rate. According to these results, L plantarum
GKM3 is a promising therapy in obese individuals with
hyperuricemia or gout.
Total lipid, cholesterol, and triglyceride levels were lower
in the livers of L plantarum GKM3-fed groups compared
with the HFC group after 6 weeks. Total fecal lipid, choles-
terol, and triglyceride excretion were in contrast increased
in L plantarum GKM3-fed groups compared with the HFC
group. A possible mechanism is that L plantarum GKM3
changed the intestinal microbiota composition and then
reduced intestinal lipid absorption, leading to decreased hep-
atic lipid accumulation and increased fecal fat loss. On the
other hand, we observed a decreased number of lipid drop-
lets in the hepatic tissue when the HFD-fed rats were treated
with L plantarum GKM3. Results also showed the differen-
ces in the cell size in the perirenal fat pads of the groups.
Taken together, these results clearly demonstrated that L
plantarum GKM3 reduces fat accumulation in rats with
HFD-induced obesity.
Study limitations
The greatest limitation of this study was the relatively short
time of the intervention. However, the study managed to
show significant results in a period as short as 6 weeks.
Moreover, healthy rats without hyperuricemia and associated
diseases were used.
Study strong points
The greatest strong point of the study is the comparative
mode of the trial in a wide dose range of the probiotic.
Conclusion
We suggest that the lactic acid bacteria supplement (L plan-
tarum GKM3) used in this study may have beneficial antio-
besity and uric acid–lowering effects. L plantarum GKM3
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 9
may therefore protect obese individuals against elevated
serum uric acid levels and resulting hyperuricemia and gout.
Further clinical trials should therefore be conducted to con-
firm these effects.
References
1. Woods SC, Seeley RJ, Porte D, Schwartz MW. Signals that regu-
late food intake and energy homeostasis. Science. 1998;280(5368):
1378–1383. doi:10.1126/science.280.5368.1378.
2. Kopelman PG. Obesity as a medical problem. Nature. 2000;
404(6778):635–643. doi:10.1038/35007508.
3. Skrypnik K, Bogda
nski P, Łoniewski I, Reguła J, Suliburska J.
Effect of probiotic supplementation on liver function and lipid
status in rats. Acta Scientiarum Polonorum. Technologia
Alimentaria. 2018;17(2):185–192
4. Szuli
nska M, Łoniewski I, Skrypnik K, Sobieska M, Korybalska
K, Suliburska J, Bogda
nski P. Multispecies probiotic supplemen-
tation favorably affects vascular function and reduces arterial
stiffness in obese postmenopausal women—a 12-week placebo-
controlled and randomized clinical study. Nutrients. 2018;10(11):
1672. doi:10.3390/nu10111672.
5. Hikita M, Ohno I, Mori Y, Ichida K, Yokose T, Hosoya T.
Relationship between hyperuricemia and body fat distribution. Intern
Med. 2007;46(17):1353–1358. doi:10.2169/internalmedicine.46.0045.
6. Glenny AM, O’Meara S, Melville A, Sheldon TA, Wilson C. The
treatment and prevention of obesity: a systematic review of the lit-
erature. Int J Obes. 1997;21(9):715–737. doi:10.1038/sj.ijo.0800495.
7. Hosoya T, Hikita M, Okabe H. Obesity and hyperuricemia.
Himan Kenkyu. 1998;4:79–85.
8. Tamba S, Nishizawa H, Funahashi T, Okauchi Y, Ogawa T,
Noguchi M, Fujita K, Ryo M, Kihara S, Iwahashi H, et al.
Relationship between the serum uric acid level, visceral fat accu-
mulation and serum adiponectin concentration in Japanese men.
Intern Med. 2008;47(13):1175–1180. doi:10.2169/internalmedi-
cine.47.0603.
9. de Oliveira EP, Burini RC. High plasma uric acid concentration:
causes and consequences. Diabetol Metab Syndr. 2012;4(1):12.
doi:10.1186/1758-5996-4-12.
10. Lin KC, Lin HY, Chou P. The interaction between uric acid level
and other risk factors on the development of gout among
asymptomatic hyperuricemic men in a prospective study. J
Rheumatol. 2000;271501–1505.
11. Verdecchia P, Schillaci G, Reboldi G, Santeusanio F, Porcellati C,
Brunetti P. Relation between serum uric acid and risk of cardiovas-
cular disease in essential hypertension. The PIUMA Study.
Hypertension. 2000;36(6):1072–1078. doi:10.1161/01.HYP.36.6.1072.
12. Taniguchi Y, Hayashi T, Tsumura K, Endo G, Fujii S, Okada K.
Serum uric acid and the risk for hypertension and type 2 dia-
betes in Japanese men: the osaka health survey. J Hypertens.
2001;19(7):1209–1215.
13. Sundstro€M J, Sullivan L, D’Agostino RB, Levy D, Kannel WB,
Vasan RS. Relations of serum uric acid to longitudinal blood
pressure tracking and hypertension incidence. Hypertension.
2005;45(1):28–33. doi:10.1161/01.HYP.0000150784.92944.9a.
14. Masuo K, Kawaguchi H, Mikami H, Ogihara T, Tuck ML. Serum
uric acid and plasma norepinephrine concentrations predict subse-
quent weight gain and blood pressure elevation. Hypertension.
2003;42(4):474–480. doi:10.1161/01.HYP.0000091371.53502.D3.
15. Tomita M, Mizuno S, Yamanaka H, Hosoda Y, Sakuma K,
Matuoka Y, Odaka M, Yamaguchi M, Yosida H, Morisawa H,
Murayama T. Does hyperuricemia affect mortality? A prospective
cohort study of Japanese male workers. J Epidemiol. 2000;10(6):
403–409. doi:10.2188/jea.10.403.
16. Fuller R. Probiotics in man and animals. J Appl Bacteriol. 1989;
66(5):365–378.
10 C.-L. HSU ET AL.
17. Borchers AT, Selmi C, Meyers FJ, Keen CL, Gershwin ME.
Probiotics and immunity. J Gastroenterol. 2009;44(1):26–46. doi:
10.1007/s00535-008-2296-0.
18. An H, Park S, Lee D, Kim J, Cha M, Lee S, Lim H, Kim K, Ha
N. Antiobesity and lipid-lowering effects of bifidobacterium spp.
in high fat diet-induced obese rats. Lipids Health Dis. 2011;10(1):
116. doi:10.1186/1476-511X-10-116.
19. Mitsuoka T, Emeritus P. The human gastrointestinal tract. The
Lactic Acid Bacteria. 1992;1:69–114. doi:10.1007/978-1-4615-
3522-5.
20. Axling U, Olsson C, Xu J, Fernandez C, Larsson S, Str€ om K.
Green tea powder and Lactobacillus plantarum affect gut micro-
biota, lipid metabolism and inflammation in high-fat fed C57BL/
6J mice. Nutr Metab (Lond). 2012;9:105. doi:10.1186/1743-7075-
9-105.
21. Naruszewicz M, Johansson ML, Zapolska-Downar D, Bukowska
H. Effect of Lactobacillus plantarum 299v on cardiovascular dis-
ease risk factors in smokers. Am J Clin Nutr. 2002;76(6):
1249–1255. doi:10.1093/ajcn/76.6.1249.
22. Takemura N, Okubo T, Sonoyama K. Lactobacillus plantarum
strain No. 14 reduces adipocyte size in mice fed high-fat diet.
Exp Biol Med (Maywood). 2010;235(7):849–856. doi:10.1258/
ebm.2010.009377.
23. Murosaki S, Yamamoto Y, Ito K, Inokuchi T, Kusaka H, Ikeda
H, Yoshikai Y. Heat-killed Lactobacillus plantarum L-137 sup-
presses naturally fed antigen–specific IgE production by stimula-
tion of IL-12 production in mice. J Allergy Clin Immunol. 1998;
102(1):57–64. doi:10.1016/S0091-6749(98)70055-7.
24. Nguyen TD, Kang JH, Lee MS. Characterization of Lactobacillus
plantarum PH04, a potential probiotic bacterium with choles-
terol-lowering effects. Int J Food Microbiol. 2007;113(3):358–361.
doi:10.1016/j.ijfoodmicro.2006.08.015.
25. Duary RK, Bhausaheb MA, Batish VK, Grover S. Anti-inflamma-
tory and immunomodulatory efficacy of indigenous probiotic
Lactobacillus plantarum Lp91 in colitis mouse model. Mol Biol
Rep. 2012;39(4):4765–4775. doi:10.1007/s11033-011-1269-1.
26. . Tzang BS, Yang SF, SG, Yang HC, Sun HL, Chen YC. Effects
of dietary flaxseed oil on cholesterol metabolism of hamsters.
Food Chem. 2009;144:1450–1455.
26. Tsuboi H, Kaneko N, Satou A, Tsuchiya Y. inventors; Meiji
(Japan), assignees. Lactic acid bacteria having action of lowering
blood uric acid level. Worldwide patent WO/2009/069219. 2009
June 4.
27. Tsai YT, Cheng PC, Pan TM. Immunomodulating activity of
Lactobacillus paracasei subsp. paracasei NTU 101 in enterohe-
morrhagic Escherichia coli O157H7-infected mice. J Agric Food
Chem. 2010;58(21):11265–11272. doi:10.1021/jf103011z.
28. Choi I, Kim Y, Park Y, Seog H, Choi H. Anti-obesity activities
of fermented soygerm isoflavones by Bifidobacterium breve.
Biofactors. 2007;29(2-3):105–112. doi:10.1002/biof.552029201.
29. Kim N-H, Kim H-M, An H-J, Um J-Y, Moon P-D, Kim S-J,
Choi I-Y, Hong S-H, Myung N-Y, Jeong H-J. Lipid profile low-
ering effect of Soypro fermented with lactic acid bacteria isolated
from Kimchi in high-fat diet-induced obese rats. Biofactors.
2008;33(1):49–60. doi:10.1002/biof.5520330105.
30. McBride P. Triglycerides and risk for coronary artery disease.
Curr Atheroscler Rep. 2008;10(5):386–390.
31. Taylor GR, Williams CM. Effects of probiotics and prebiotics on
blood lipids. Br J Nutr. 1998;80(4):S225–S230.
32. Xiao JZ, Kondo S, Takahashi N, Miyaji K, Oshida K, Hiramatsu
A, Iwatsuki K, Kokubo S, Hosono A. Effects of milk products
fermented by Bifidobacterium longum on blood lipids in rats and
healthy adult male volunteers. J Dairy Sci. 2003;86(7):2452–2461.
doi:10.3168/jds.S0022-0302(03)73839-9.
33. Park YH, Kim JG, Shin YW, Kim SH, Whang KY. Effect of diet-
ary inclusion of Lactobacillus acidophilus ATCC 43121 on chol-
esterol metabolism in rats. J Microbiol Biotechnol. 2007;
17655–662.
34. Skrypnik K, Suliburska J. Association between the gut microbiota
and mineral metabolism. J Sci Food Agric. 2018;98(7):2449–2460.
35. Sampath A, Kossoff EH, Furth SL, Pyzik PL, Vining EP. Kidney
stones and the ketogenic diet: risk factors and prevention. J Child
Neurol. 2007;22(4):375–378. doi:10.1177/0883073807301926.
36. Yamada N, Saito-Iwamoto C, Nakamura M, Soeda M, Chiba Y,
Kano H, Asami Y. Lactobacillus gasseri PA-3 uses the purines IMP,
inosine and hypoxanthine and reduces their absorption in rats.
Microorganisms. 2017;5(1):10. doi:10.3390/microorganisms5010010.