Acute and sub-chronic (28-day) oral toxicity profiles of newly synthesized prebiotic butyl-fructooligosaccharide in ICR mouse and Wistar rat models

Abstract

B-FOS (butyl-fructooligosaccharide) is a newly synthesized prebiotic molecule, formed by the combination of FOS and butyrate by ester bonds. B-FOS has been reported to have the potential prebiotic effect of promoting intestinal flora diversity and enhancing butyrate production. The aim of this study was to investigate the potential acute and sub-chronic toxicity of B-FOS in Institute of Cancer Research (ICR) mice and Wistar rats to verify its biosafety. ICR mice were administered a single oral gavage of B-FOS at doses of 0, 500, 1000, and 2000 mg/kg body weight and observed for signs of acute toxicity for 14 days. Sub-chronic toxicity was evaluated by repeated oral administration of B-FOS at 2000 mg/kg for 28 days, in accordance with Organization for Economic Co-operation and Development (OECD) protocol test numbers 420 and 407. No mortality or abnormal clinical signs were observed during the experimental periods after B-FOS administration. Furthermore, no significant changes in body weight, organ weight, serum biochemical parameters, or tissue histology were observed after animal sacrifice. These in vivo results indicate that B-FOS does not exert any acute or sub-chronic toxicity at a dose of 2000 mg/kg, and this novel molecule can be regarded as a safe prebiotic substance for use in the food and nutraceutical industries.

Introduction

Butyl-fructooligosaccharide (B-FOS) is newly synthesized prebiotic molecule, combining fructooligosaccharide (FOS) with one or two butyrate molecules by ester bonds [1]. The two major B-FOS compounds are GF3-1B [O-(1-buty-β-D-fru-(2 → 1)-O-β-D-fru-(2 → 1)-O-β-D-fru-O-α-D-glu] and GF3-2B [O-(1-buty)-β-D-fru-(2 → 1)-O-β-D-fru-(2 → 1)-O-(4-buty)-β-D-fru-O-α-D-glu], which are synthesized in the ratio of approximately 5:3 [1]. B-FOS enables butyrate to be delivered to the distal colon; it also promotes colonic health and remodels gut microbiota. Based on our previous study, B-FOS selectively promotes the growth of probiotics (i.e. Bifidobacterium and Lactobacillus) and butyrate-producing bacteria (i.e. Faecalibacterium) and increases butyrate production during fecal batch fermentation in vitro [1]. Based on in vivo study results, B-FOS alters the composition of intestinal bacteria, stimulates the growth of Bifidobaterium, and increases cecal butyrate production, accompanied by an increase in cecal weight in healthy ICR mice. Moreover, B-FOS reduces colitis symptoms and colonic inflammation and damage. B-FOS may become an alternative to butyrate, alleviating human irritable bowel disease (IBD) in the future (unpublished). Although the biological activities of B-FOS have been investigated to some degree, related toxicological information is unknown. As B-FOS is a newly synthesized substance, it is important to evaluate and predict its toxicity in experimental animals to determine safe dosages in humans [2]. The objective of this study was, therefore, to evaluate the acute and sub-chronic oral toxicity of B-FOS in Institute of Cancer Research (ICR) mice and Wistar rats based on single dose acute oral toxicity and repeated dose 28-day oral toxicity studies, as described in OECD guidelines 407 and 420 [3, 4].

Materials and Methods

Animals and treatment

The acute toxicity experiment was performed on 6-week old, healthy ICR mice (20 females and 20 males), and the sub-chronic toxicity experiment was conducted on 5-week-old healthy Wistar rats (10 females and 10 males). All animals were obtained from Central Lab Animal Inc. (Seoul, Korea). A standard diet (AIN-93G) was purchased from Doo Yeol Biotech, Korea, and fed to all animals. Its composition is shown in Table 1. Animals were grouped by sex and housed in polycarbonate cages with stainless steel mesh lids in a ventilated animal room. Room temperature was maintained at 23 ± 2°C and 60 ± 10% relative humidity, and a 12 hours light/dark cycle was employed. Distilled water and sterilized food were available ad libitum, except during the fasting period. Animals were acclimated to this environment for 2 weeks prior to dosing. B-FOS was provided by Bifido Inc. (Gangwon-do, Korea). The Institutional Animal Care and Use Committee of Seoul National University approved the facilities and protocols used in this study.

Table 1.

Diet composition (g/kg)

Diet ingredients	g/kg
Casein	200
Corn starch	397.486
L-Cystine	3
Cellulose	50
Sucrose	100
Dextrose	132
AIN-93G mineral mix	35
AIN-93G vitamin mix	10
Choline bitartrate	2.5
Soybean oil	70
t-Butylhydroquinone	0.014

Acute oral toxicity study

The acute oral toxicity of B-FOS in mice was conducted based on the fixed dose procedure described by the Organization for Economic Cooperation and Development (OECD, 2001). ICR mice were randomly divided into four groups (five females and five males each): control, 500, 1000, and 2000 mg/kg body weight of B-FOS. B-FOS was orally administered by sonde into the stomach (gastric gavage) with respective doses in treatment groups or with sterile water in the control group. The mice were fasted approximately 4 hours prior to dosing, but they had free access to drinking water during this time. After B-FOS was administered, food was withheld for a further 1–2 hours. The mice were observed carefully for any signs of toxicity in the first 3 hours after the treatment period and daily thereafter for a period of 14 days. Observations for mortality, signs of illness, injury, pain, distress, allergic reactions, changes of outer appearance, behavioral alterations (i.e. ataxia, hyperactivity, hypoactivity), and general stimulation or sedation were conducted twice daily. All observations were systematically recorded. After 2 weeks of observation, all mice were sacrificed by CO₂ inhalation, and body weights were recorded. Blood samples were collected from the heart by cardiac puncture, and the blood was centrifuged at 2500 rpm for 10 minutes to harvest serum samples. The organs (liver, spleen, kidneys, heart, lung, brain, and testicle/ovary) were excised. Liver, spleen, and kidneys were weighed accurately, and all organs were stripped and immediately fixed in a 10% formalin solution for further histopathological diagnosis.

Sub-chronic oral toxicity study

The sub-chronic oral toxicity of B-FOS in Wistar rats was performed based on the limit test of repeated dose 28-day oral toxicity study in rodents described by the OECD (2008). The Wistar rats were randomly divided into control and B-FOS groups, consisting of five females and five males in each group. A total of 2000 mg/kg body weight of B-FOS was orally administered to the B-FOS rats daily for 4 weeks. The rats were observed carefully for any signs of toxicity daily. After 28 days of administration, the rats were sacrificed by CO₂ inhalation, and body weights were recorded. Blood samples were collected in heparin tubes, and EDTA-treated tubes were used to collect blood from the heart by cardiac puncture technique. The organs (liver, spleen, kidneys, heart, lung, brain, and testicle/ovary) were excised, stripped, and immediately fixed in a 10% formalin solution for further histopathological diagnosis.

Observations

The clinical observations included obvious symptoms of toxicity: changes in the skin, fur, eyes, mucous membranes, and autonomic activity; changes in gait, posture, and response to handling; the presence of clonic/tonic movement; and stereotypical or bizarre behaviors.

Coefficients of the liver, kidney and spleen

Coefficients of the liver, kidneys, and spleen to body weight were calculated as the ratio of tissues (wet weight, mg) to body weight (g).

Hematological and biochemical analysis

Blood samples collected in the heparin tubes were centrifuged at 3000 rpm for 10 minutes at 4°C. The separated serum samples were used to measure serum levels of albumin, total bilirubin levels (TBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (Cr), and lactate dehydrogenase (LDH) enzyme using an AU680 Biochemical Autoanalyzer (Beckman coulter, US).

Hematological analysis was conducted with the whole blood collected in the EDTA-treated tubes. Erythrocyte (RBC), total and differential leukocyte (WBC), hematocrit, hemoglobin, platelet count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean platelet volume (MPV), platelet distribution width (PDW), and red distribution width (RDW) of blood samples were determined using an ADVIA2120i hematology analyzer (Siemens, German).

Histopathological examination

Histopathology of the heart, liver, kidney, lung, spleen, stomach, colon, and testicle/ovary tissues was performed. All tissues fixed in 10% formalin were immersed in paraffin blocks, sliced into 5-mm-thin sections, mounted onto slides, and stained with hematoxylin–eosin (HE). The tissue slices on the slides were observed and photographed using an optical microscope (Nikon U-III Multipoint Sensor System, USA). The source and identity of the pathology slides were blind to the pathologist.

Statistical analysis

Results are expressed as the mean ± standard deviation. Statistical analysis was performed by Dunnett test in a one-way analysis of variance (ANOVA) test in the acute toxicity study and a t-test in the 28-day repeated toxicity study. The level of significance was set at P < 0.05 to evaluate the control and treatment group(s).

Results

Vital signs, body weights, and coefficients of the liver, spleen, and kidneys of ICR mice in the acute study

After oral administration, no unusual behavior was observed in the first 3 hours and daily thereafter over 2 weeks. No mortality occurred during the study period. The mice were weighed on day 0 and day 14 after exposure to B-FOS. On day 14, all mice were sacrificed, and organs/tissues were harvested. The weight of the livers, spleens, and kidneys was accurately measured. Table 2 shows the coefficients of the liver, spleen, and kidney to body weight. There were no significant differences in body weight within the male and female groups or the coefficients of the liver, spleen, and kidney within either group. Any significant increase in these coefficients would have suggested that inflammation had been induced.

Table 2.

Body weight changes and coefficients of the liver, spleen, and kidneys after single oral exposure to B-FOS

	Groups	Body weight (g)		Liver (mg/g)	Spleen (mg/g)	Kidney (mg/g)
		Before	After
Male	Control	35.04 ± 4.17	41.06 ± 6.55	41.06 ± 9.51	3.84 ± 1.02	7.18 ± 1.54
	500 mg/kg	34.80 ± 0.67	39.58 ± 2.32	39.58 ± 7.09	3.57 ± 1.48	7.55 ± 1.55
	1000 mg/kg	34.04 ± 1.25	38.72 ± 1.61	38.72 ± 5.12	4.66 ± 1.20	7.76 ± 0.33
	2000 mg/kg	36.28 ± 4.65	42.14 ± 5.98	42.14 ± 4.13	4.25 ± 0.90	7.52 ± 1.32
Female	Control	27.96 ± 1.47	31.56 ± 0.79	45.67 ± 3.34	3.79 ± 1.33	7.59 ± 1.66
	500 mg/kg	27.86 ± 2.67	32.18 ± 3.00	47.90 ± 2.53	4.32 ± 1.52	6.86 ± 1.30
	1000 mg/kg	26.94 ± 1.78	30.20 ± 1.48	47.07 ± 4.54	4.00 ± 1.60	6.65 ± 2.46
	2000 mg/kg	26.60 ± 1.76	30.82 ± 1.65	46.34 ± 7.89	3.91 ± 1.48	7.16 ± 1.52

Data are expressed as mean ± SD (n = 5). ^*P < 0.05 compared with control group using ANOVA (Dunnett).

Acute study biochemical parameters in ICR mouse serum

Indications of functional changes in the whole body, such as infection or disease, can be identified by biochemical analysis of blood serum. Figure 1 displays the changes in biochemical parameters in the serum of female and male mice after exposure to B-FOS at doses of 0, 500, 1000, and 2000 mg/kg. No significant changes were observed in serum albumin, TBIL, and the enzymes AST and ALT (P > 0.05) (indicators of liver function) after oral administration [5–7]. No elevation of blood urea nitrogen (BUN) or creatinine (Cr) was observed in this study, which suggested no kidney damage [8]. Cell membrane injury and tissue damage were evaluated by lactate dehydrogenase testing (LDH) [9]. There was no significant change in LDH levels in either the female or male groups.

Figure 1.

Biochemical parameters in blood serum after single-dosage oral exposure to B-FOS (n = 5). ^*P < 0.05 compared with control group using ANOVA (Dunnett).

Acute study histological evaluation of ICR mouse

The index of necrosis with inflammatory cell infiltration can be assessed by visual evaluation of histological changes, because toxic compounds induce neutrophilic infiltration and cell death [8, 10]. Histopathology of ICR mouse heart, liver, kidney, lung, spleen, stomach, colon, and testicle/ovary tissues was performed. There were no abnormal pathological changes in the tissues between control and treatment groups at any dosage.

Sub-chronic study vital signs, body weights, and coefficients of the liver, spleen, and kidneys of Wistar rats

No mortality or physical/behavioral changes were observed in the male and female Wistar rats treated with B-FOS at 2000 mg/kg of body weight daily for 28 days. There were no significant differences in body weights of male Wistar rats between the control and the treatment group, while the body weights of female Wistar rats in the B-FOS group were significantly heavier than the control (P < 0.05) after 28 days of oral administration (Table 3). This was probably due to differences in food intake between the groups (P < 0.05, Fig. 2). In addition, no significant differences were found in the coefficients of the liver, spleen, and kidney between the groups (Table 3).

Table 3.

Coefficients of the liver, spleen and kidney of Wistar rat after 28-day repeated oral exposure to B-FOS

	Groups	Body weight (g)		Liver (mg/g)	Spleen (mg/g)	Kidney (mg/g)
		Before	After
Male	Control	206.6 ± 4.5	390.8 ± 25.5	39.90 ± 8.02	1.48 ± 0.22	7.77 ± 0.98
	2000 mg/kg	207.8 ± 7.2	376.8 ± 12.9	37.14 ± 2.39	1.75 ± 0.13	7.37 ± 0.25
Female	Control	148.0 ± 3.9	234.0 ± 7.0	39.95 ± 3.36	1.97 ± 0.27	8.02 ± 0.87
	2000 mg/kg	151.8 ± 5.2	246.4 ± 6.4*	34.97 ± 4.56	2.03 ± 0.05	7.79 ± 0.73

Data are expressed as mean ± SD (n = 5). ^*P < 0.05 compared with control group using unpaired t-test.

Figure 2.

Food intake changes during 28-day repeated dose administration in male (A) and female (B) Wistar rats. Data are expressed as mean ± SD (n = 5). ^*P < 0.05 compared with the control group using an unpaired t-test.

Sub-chronic study biochemical parameters in the serum of Wistar rats

The results of biochemical analysis of blood serum are shown in Fig. 3. There were no significant differences observed in the plasma levels of albumin, total-Bil, AST, ALT, BUN, Cr, or LDH.

Figure 3.

Biochemical parameters in blood serum after 28-day repeated oral exposure to B-FOS (n = 5). ^*P < 0.05 compared with the control group using an unpaired t-test.

Sub-chronic study hematological analysis of Wistar rats

Alteration of hematological parameters is highly predictive of human toxicity [11]. Based on the hematological analysis results presented in Table 4, B-FOS did not induce significant changes in hemoglobin, hematocrit, red blood cell (RBC) count, RBC indices (MCV, MCH, MCHC), platelet count, and MPV and WBC counts when compared with the control. This indicates that B-FOS did not exert any harmful effects on blood cell counts or hemoglobin content, which means that B-FOS did not have toxic effects on blood.

Table 4.

Hematological parameters in whole blood after 28-day repeated oral exposure to B-FOS

	Groups	Hemoglobin (g/dl)	Hematocrit (%)	RDW (%)	MPV (fl)	Platelets (×10 3 cells/μl)	WBC (×10 6 cells/μl)	RBC (×10 6 cells/μl)
Male	Control	14.96 ± 1.05	53.48 ± 3.88	11.26 ± 0.40	9.22 ± 0.66	601.6 ± 37.6	9.51 ± 4.38	8.46 ± 0.40
	2000 mg/kg	15.2 ± 0.57	53.96 ± 2.50	10.96 ± 0.36	8.32 ± 0.42	717.4 ± 49.8	10.14 ± 4.7	8.59 ± 0.35
Female	Control	15.06 ± 0.31	53.18 ± 1.59	11.04 ± 0.27	8.86 ± 0.48	688.0 ± 121.6	6.91 ± 1.65	8.25 ± 0.21
	2000 mg/kg	14.80 ± 1.07	50.70 ± 4.15	11.58 ± 1.16	9.7 ± 1.61	601.8 ± 137.4	7.99 ± 3.82	8.16 ± 0.52
WBC differential counting (%)					RBC indices
Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils	MCV (fL)	MCH (pg)	MCHC (g/dL)
15.62 ± 7.67	78.84 ± 9.22	1.8 ± 0.49	2.1 ± 1.19	1.06 ± 0.38	63.18 ± 2.75	17.68 ± 0.60	27.98 ± 0.61
11.16 ± 3.79	83.14 ± 4.73	1.58 ± 0.63	1.72 ± 0.67	0.72 ± 0.26	62.76 ± 0.63	17.7 ± 0.28	28.18 ± 0.61
8.06 ± 1.54	86.62 ± 1.55	1.48 ± 0.26	1.90 ± 0.99	0.70 ± 0.10	64.48 ± 1.28	18.26 ± 0.21	28.32 ± 0.67
12.74 ± 7.47	82.70 ± 9.01	1.58 ± 0.80	1.70 ± 1.03	0.64 ± 0.40	62.12 ± 2.83	18.16 ± 1.03	29.26 ± 0.68

Data are expressed as mean ± SD (n = 5). ^*P < 0.05 compared with the control group using an unpaired t-test.

Sub-chronic study histological evaluation of Wistar rats

Necropsy and histopathological findings shown in Figs 4 and 5 are summarized in Table 5, including focal degeneration and fibrosis of the heart and focal necrosis, focal fibrosis, and local microvesicular steatosis of the liver in male and female Wistar rats. All male and female rats in the control group and the B-FOS group displayed focal muscle degeneration, appearing around the blood vessels of the interventricular septum muscle in the heart, but differences between the groups was not observed within gender. In addition, focal fibrosis of the heart was also found in both the control and B-FOS groups of male rats. Three female rats showed local microvesicular steatosis, and one female rat showed focal hepatic cell necrosis in the B-FOS group, while all the females maintained normal liver histologic structure in the control group. This indicates that hepatocellular fat degeneration was significantly increased in the B-FOS treatment versus the control group. Meanwhile, most of the male rats had focal fibrosis and fatty hepatocellular necrosis in both the control and B-FOS treatment groups, but no difference in degree of damage was found between the two groups. There were no abnormal pathological changes observed in the other tissues in either group.

Figure 4.

Heart tissue stained hematoxylin and eosin (H&E, 100× & 200×) showing the effect of B-FOS in Wistar rats. (A) Control (B) B-FOS were from male samples, and (C) control (D) B-FOS were from female samples. FD, focal degeneration; FF, focal fibrosis.

Figure 5.

Liver tissue stained hematoxylin and eosin (H&E, 100× & 200×) showing the effect of B-FOS in Wistar rats. (A) Control (B) B-FOS were from male samples, and (C) control (D) B-FOS were from female samples. FN, focal necrosis; FF, focal fibrosis; MS, microvesicular steatosis.

Table 5.

Histological findings in the 28-day repeated oral dose toxicity study

Dose (mg/kg)	Male		Female
	0	2000	0	2000
No. of animals	5	5	5	5
Heart
Focal degeneration	5	4	5	5
Focal fibrosis	1	1	0	0
Liver
Focal necrosis, focal fibrosis	3	4	0	1
Local microvesicular steatosis	5	4	0	3*

Data are expressed as mean ± SD (n = 5). ^*P < 0.05 compared with the control group using an unpaired t-test.

Discussion

B-FOS is a newly synthesized compound, formed by the combination of FOS and butyrate, which can be considered as a prebiotic. This study was conducted to evaluate the biosafety of B-FOS by acute and 28-day repeated sub-chronic oral toxicity tests. Toxicological evaluations in animal models are usually performed to evaluate the potential health risk of a new substance to humans [12].

The amount of an orally administered test substance which causes the death of 50% of a group of test animals is known as the LD₅₀ value [13]. No clinical symptoms or motility were observed after B-FOS single oral administration in the acute toxicity study, so the LD₅₀ of orally administered B-FOS was higher than 2000 mg/kg in both sexes. In addition, no statistically significant differences were observed between the body weights, organ coefficients, chemical parameters, and histopathology diagnoses of the control and treated groups.

Sub-chronic toxicity testing was performed to examine the possible adverse effects of B-FOS after repeated exposure over a period of time. Neither abnormal animal behavior nor mortality was observed in the B-FOS treatment group at 2000 mg/kg in this limit test. Although no significant changes in body weight gain or organ weight were observed in male Wistar rats, female Wistar rats gained significantly greater body weight in the B-FOS treatment group. Changes in body weight can be attributed to adverse drug effects [14]. For instance, the reduction of caloric intake or increased body fat accumulation might be due to stress or physiological adaptation to the drug intake [15]. In this study, the significantly higher body weight gain in the female rats could be associated with greater food intake by the B-FOS group compared with the control.

In addition, blood parameter assessment was used to evaluate the risk of alterations to the hematological system in humans [16]. No alterations of the hematological and biochemical parameters in the blood of male or female rats treated with or without B-FOS were observed. The liver is an imperative organ that contributes to drug biotransformation, and its normal function is monitored by serum biomarker enzymes [17]. In this study, the levels of AST, ALT, total-Bil enzymes, and albumin were investigated to assess possible hepatocellular damage caused by B-FOS. ALT, AST, and total-Bil are specific indicators of hepatocellular necrosis/degeneration [8, 18]. ALT and AST mainly exist in the hepatocytes and their increases in blood serum imply hepatic cell damage [19]. Albumin is usually considered as a binding and transport protein, which is only synthesized in the liver [5, 20]. Low albumin levels are relevant to cirrhosis and ascites [7]. The results of this study showed no significant alteration in the AST, ALT, total-Bil, and albumin levels due to B-FOS treatment. Moreover, kidney dysfunction was assessed by assaying Cr and BUN. Creatinine is formed by the hydrolysis of creatine and phosphocreatine in muscles. Its level goes up when renal function is impaired [8]. BUN, an end product of protein metabolism, is mainly synthesized in the liver. Its high level indicates extensive impairment of kidney function [21]. In this study, the levels of creatinine and BUN were not affected by treatment with B-FOS. Therefore, B-FOS did not cause any impairment of the liver or kidneys.

Furthermore, MCH, MCHC, and MCV are associated with individual red blood cells, whereas hemoglobin and RBC are related with the total population of red blood cells in blood [22]. B-FOS did not affect the incorporation of hemoglobin into red blood cells or the morphology and osmotic fragility of red blood cells generated, which means that B-FOS did not exert toxic effects on the blood system and its oxygen-carrying capacity. In addition, WSC and its contents were not affected by B-FOS treatment, which suggests that B-FOS did not trigger immune responses associated with the destruction or impaired production of white blood cells.

Although focal degeneration of the heart and focal necrosis and focal fibrosis of the liver were observed via necropsy, there was no significant difference in the severity between the control and B-FOS treatment groups, which suggests that the alteration was probably caused by individual health status rather than external factors.

The liver is the major site in the body for drug metabolism [23]. Liver microvesicular steatosis is characterized by the excessive and pathologic intrahepatocellular accumulation of small fat vacuoles [24]. In this study, the occurrence of hepatic microvesicular steatosis was significantly increased in female rats in the B-FOS group compared to the control. Though microvesicular steatosis is considered a potentially severe liver lesion possibly associated with impaired fatty acid metabolism [25, 26], solely fat stored in small vesicles were witnessed, and the levels of ALT, AST, albumin, and total-Bil (indicators of liver functions) were not elevated in the female B-FOS group compared with the control. Besides, the microvesicular steatosis appeared in all but one male rat. Consequently, the increased occurrence of the microvesicular steatosis was probably due to significantly larger food intake and body weight in the female B-FOS treatment group.

This study provides valuable information on the acute and sub-chronic oral toxicity profile of B-FOS, which might be useful for B-FOS clinical studies in the future. Nonetheless, this study also has limitations. The limited number of Wistar rats used in the sub-chronic study is insufficient to completely validate the safety of B-FOS, and food intake was not controlled, introducing an unaccounted-for factor. The low number of animals used in the study could also have been the cause of relatively high standard deviations of some parameters. Further studies are necessary to confirm the safety of this newly synthesized compound in humans.

In summary, the results of body/organ weights, chemical/hematological analysis, and histopathology evaluations indicate that B-FOS administered in doses of 2000 mg/kg daily for 28 days did not cause any mortality or evidence of severe toxicity. As all data were within normal limits in acute and sub-chronic toxicity studies, oral administration of B-FOS was considered to be low or non-toxic in this study. It is expected that B-FOS will become a key compound in functional foods and drugs designed to promote human intestinal health.

Author Contributions

Experimental design, Sini Kang, Seockmo Ku, and Geun Eog Ji; animal experiments, Sini Kang; data analysis, Sini Kang and Seockmo Ku; writing (original draft preparation), Sini Kang and Seockmo Ku; writing (review and editing), Tony V. Johnston, Seockmo Ku, and Geun Eog; supervision, Seockmo Ku and Geun Eog Ji; funding acquisition, Tony V. Johnston, Seockmo Ku, and Geun Eog Ji. Tony V. Johnston and Seockmo Ku participated in this work based on a non-disclosure research agreement between Middle Tennessee State University and BIFIDO Co., Ltd. All authors have read and agreed to the published version of the manuscript.

Funding

This work was carried out with support from the Ministry of Small and Medium-sized Enterprises (SMEs) and Startups(MSS), Korea, under the “Regional Specialized Industry Development Program (R&D, Project number S2848321)” supervised by the Korea Institute for Advancement of Technology (KIAT) and the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-341-2017M3A9F3041747). This work was also supported by a Faculty Research and Creative Activity Committee (FRCAC) grant (No. 221745) funded by Middle Tennessee State University in the USA.

Conflicts of interest statement

Geun Eog Ji holds BIFIDO Ltd. stocks. Other authors declare no conflict of interests.