Effects of 3 months continuous intake of supplement containing Pantoea agglomerans LPS to maintain normal bloodstream in adults: Parallel double‐blind randomized controlled study

Abstract In this study, the effects on the maintenance of normal bloodstream by lipopolysaccharide (LPS) were investigated in the parallel‐group randomized double‐blind study using supplement containing Pantoea agglomerans LPS (201.5 μg/tablet as LPS). Screening was previously performed in the implementation of the study. Adult males and females with normal value to borderline (healthy subjects) in the hematologic parameters, for which reference values were given, were chosen in this study. The period of ingestion of the supplement was 3 months. As the result, a significant decrease in the rate of change (the ratio when the baseline was 1) of HbA1c, which is a glycative stress marker, was found in the group which ingested LPS supplement after 3 months. Also, a significant increase in the number of fingertip capillary vessels was found compared with the control group. From these results, the effects of the maintenance of bloodstream by ingestion of LPS were shown.

(2) In 47 males and females with high blood glucose levels and serum lipid levels aged from 40 years or older to 70 years or younger, it was confirmed that HbA1c levels and LDL marker were decreased by the ingestion of LPSp Salacia tea for 2 months; 500 μg LPSp/day (double-blind study) (Nakata et al., 2011). (3) In 52 females aged from 40 to 79 years, it was confirmed that a decrease in the bone mineral density was suppressed by the ingestion of an LPSp soymilk for 3 months; 600 μg LPSp/day (double-blind study) (Nakata et al., 2014).
(4) In 20 males and females aged from 20 to 65 years, it was confirmed that triglyceride markers were decreased and malaise, lassitude, stiff neck, asthenopia, and constipation tended to improve by the ingestion of LPS beverage contained another Pantoea sp. for 1 month; 1200 μg LPS/day (open study).
Since these study results showed the safety of the oral ingestion of LPS and the usefulness in health maintenance from the improvement of stiff neck and asthenopia seen in (1) or (4), it is suggested that LPSp is more likely to promote improvement of the bloodstream.
LPS controls macrophages. Macrophages are known to produce nitric oxide (NO) with vasodilatory effects. Although the contribution of NO production by macrophages on vasodilatation has not been reported, there may be that possibility.
Also, macrophages function to eliminate unwanted molecules in the body by phagocytosis. From this point of view, there is the possibility that LPS eliminates intravascular degenerative foreign matter, for example, one of the AGEs, HbA1c, oxidized LDL, or triglycerides, by enhancing the phagocytosis of macrophages to contribute to improvement of the blood stream.
In addition, the bloodstream is associated with the number of capillary vessels, and it is known that macrophages produce vascular endothelial growth factor (VEGF) by stimulating with LPS (Cattin et al., 2015) (Spiller et al., 2014). The enhancement of VEGF production from macrophage and an increase in the number of capillary vessels by the ingestion of LPSp may lead to the improvement of the bloodstream.
In this study, in order to verify the functions in the maintenance of normal bloodstream of LPSp, HbA1c, which is one of the glycative stress markers, and oxidized LDL, which is one of the oxidative stress marker, the number of fingertip capillary vessels as well as safety markers were investigated by using the supplement containing LPSp alone and controls.

| Study design
This study was conducted as a randomized, parallel-group, doubleblind study using reference products, and each parameter before, during, and after the ingestion of this test food was compared. Also, the LPSp supplement group and control group were compared. The study was conducted in accordance with the Declaration of Helsinki (1964)  The study was conducted at the Miyake Medical Institute Group Central Park Clinic from 13 October 2015 to 5 February 2016, and the period of ingestion of the test food was 3 consecutive months. The grouping of the group which ingested the LPSp supplement and the group which ingested the controls was performed according to the envelope method, and this study was conducted according to the doubleblind approach. The study design is shown in Figure 1. Examinations were held four times at Central Park Clinic: at the screening, before the start of ingestion, after 1.5 months of ingestion, and after the completion of ingestion (after 3 months). The subjects were fasted after 9:00 p.m. on the day before examination.

| Subjects
The subjects in this study included males and females aged 20 years or older and 74 years or younger, and subjects who were within the range of the hematological values before the start of the study excluding oxidized LDL, IgA, and CRP as shown in Table 1 (Mild abnormalities in the "Japan Society of Ningen Dock Criteria [Revised on 1 April 2014]").
In this blood test, the screening was performed prior to the start of the study. Other exclusion criteria were the following four items. (1) Subjects who are using the pharmaceuticals (pharmaceuticals used for the improvement of lipid metabolism or bloodstream) or foods (foods for specified health use or foods with function claims in which the improvement of lipid metabolism or bloodstream is clearly documented) that the investigator or the subinvestigator judged to have an impact on participation in the study. (2) Subjects who are participating or will be participating in another clinical study. (3) Subjects who have donated more than 400 ml of blood 3 months before the date of informed consent or more than 200 ml of blood 1 month before the date of informed consent. (4) Other subjects who are judged not to be eligible for participation in the study by the investigator or the subinvestigator.

| Test food
One tablet of this LPSp supplement compounds 25 mg of fermented wheat extract "Somacy-FP100" produced by MACROPHI Inc. (Kagawa, Japan) and Somacy-FP100 contains 10 mg/g of LPSp (measured value was 201.5 ± 22.8 μg/tablet by ELISA). For the control, the supplement replaced the LPSp of the LPSp supplement with dextrin. The name and mixing amount of the components of the test product are shown in Table 2.

| Fermented wheat extract Somacy-FP100
Somacy-FP 100 is a raw material containing LPSp of P. agglomerans, a wheat symbiotic bacterium, as an active ingredient. Briefly, it is manufactured as follows. After P. agglomerans was cultured in a medium containing wheat flour as the carbon source, LPSp was extracted by heating and then purified by activated charcoal treatment, celite filtration, and membrane filtration. Finally, dextrin was added and spray dried so that the LPSp content was 10 mg/g to 15 mg/g. The components other than LPS contained in the raw material were as follows: moisture 4.7%, dextrin 89.9%, fiber 2.7%, protein 0.9%, lipid F I G U R E 1 Study design and screening of subjects. Among the members enrolled in the LSIN, enrollment consent forms were obtained from persons who wished to participate in this study or persons who were newly recruited and consented to the enrollment. The content of the study was explained to 107 volunteers, the consent of the study purpose and the content was obtained, and the enrollment of this study was completed by obtaining the study consent forms at the same time (the informed consent was obtained). The screening was performed 2 weeks before the start of ingestion. Blood was collected from 104 subjects in the screening and a blood test was performed. The subinvestigator selected 57 subjects who met the inclusion criteria and did not conflict with the exclusion criteria based on the background of the subjects and the results of the blood test. Among these subjects, two subjects were declined to participate in the study before the start of the study and 55 subjects initiated the study Two tablets of LPSp supplement (403.0 μg LPSp/day) or control were ingested once daily before dinner in general. The supplement was dissolved by chewing or licking in the mouth and ingested in an unchanged form or with water. 0.5%, and ash content 0.3%. In addition, the wheat protein residue of Somacy-FP 100 is "not detected." On the other hand, it has been reported that various physiological activities, such as hyperlipidemia and allergy improvement, can be seen by ingesting LPS at 10 μg kg −1 day −1 (1 mg kg −1 per −1 day in terms of this raw material). The functional involved component is considered to be only LPSp.

| Blood test
As the parameter that has an impact on the bloodstream, HbA1c was measured as a glycative stress marker. The lipid metabolism markers, such as LDL, HDL, triglyceride, and oxidized LDL, were measured at a time. The white blood cell count (WBC), red blood cell count (RBC), AST (GOT), ALT (GPT), creatinine, CRP, and IgA were measured as the safety markers. Measurements were held three times: before the start of ingestion, after 1.5 months of ingestion, and at the completion of ingestion (after 3 months). This blood test was analyzed by Shikoku Chuken Inc. (Kawaga, Japan).

| Vascular investigations
As direct investigations on the bloodstream and vessels, the inves-

| Others
As a general physical examination, the body weight and percent of body fat were measured. In addition, as the evaluation for improvement in QOL, questionnaire surveys were administered on stiff neck, For items other than shoulder stiffness, it was chosen from a given choice in a question paper for the presence and extent of subjective symptoms within the past week from the examination date.

| Statistical analyses
By using statistical software (Excel Statistics 2012), the data quantified from the results of the blood test, investigation of arteriosclerosis, and vascular images were analyzed by the Steel-Dwass test for intragroup comparisons and analyzed by the Mann-Whitney U test for intergroup comparisons. The statistical processing was carried out by excluding the data of subjects who were withdrawn or dropped out and missing (two-sided significance level of 5%).

| Subject
The study was started in 55 subjects through the screening. After the start of the study, a total of three subjects withdrew due to the following reasons: (1) the condition that should be excluded from the study was found after the start of the study, (2) subject wanted to withdraw due to the inconvenience of the examination schedule, and (3) the subinvestigator judged that it was difficult to continue the study through the interview. As a result, 52 subjects (26 in the LPSp supplement group and 26 in the control group) were eventually analyzed.
The ratio of male to female in each group was 9 males to 17 females in both groups. The mean age was 37.8 ± 7.2 years in the LPSp supplement group and 34.6 ± 9.9 years in the control group (Table 3).
The body weight and percent of body fat at the start of ingestion were 56.6 ± 10.4 kg and 22.9 ± 5.4% in the LPSp supplement group and 56.8 ± 11.8 kg and 22.9 ± 5.3% in the control group, respectively.
No significant changes in both body weight and percent of body fat were seen within and between groups (Table 4)

| Blood test
As the results of this study, according to the criteria as provided under the Japan Society of Ningen Dock Criteria (revised on 1 April 2014), the number of subjects with borderline or higher of each hematologic parameter was 1 or 2 subjects in the parameters of LDL, TG, WBC, ALT, creatinine, and CRP (Table 5).
According to the JDS criteria, 24 subjects in the LPSp supplement group and 20 subjects in the control group were within the borderline at the baseline in HbA1c which is a glycative stress marker. Including the subjects with borderline, a significant decrease was found in the LPSp supplement group after 3 months of ingestion (Tables 5-7, Figure 2).
No significant changes in LDL, HDL, triglyceride, WBC, RBC, AST, ALT, creatinine, and CRP were found within and between groups in both the LPSp supplement and control groups, and these parameters were changed within the normal ranges (Tables 5-7).
The normal/borderline ranges were not specified in oxidized LDL and IgA, and no significant changes were found within and between groups in the measurement results (Tables 6 and 7).
No significant changes in the measured values of WBC, RBC, AST (GOT), ALT (GPT), creatinine, CRP, and IgA measured as safety markers in this study were found during the 3 months ingestion period, therefore it was confirmed that LPSp supplement was of no safety concern.

| Bloodstream
No significant change in CAVI was found within and between groups in both the LPSp supplement and control groups, and this parameter was changed within the normal range. CAVI was significantly low in the control group at the baseline between groups, but no significant difference was found after 3 months (Table 8).
By using the capillary bloodstream assessment system, when the number of fingertip capillary vessels that can be confirmed within the specified field was assessed as an indicator, a significant increase in the number of fingertip capillary vessels was found in the LPSp supplement group after 3 months compared with the control group (Table 9, Figure 3).

| QOL
No significant difference in QOL was found in all parameters within the study period and between groups. However, in the LPSp supplement group, the subjects who developed stiff neck at the start of ingestion tended to improve after 3 months (Table 10). Also, the number of subjects who "did not feel coldness" was five at the start of ingestion, and the number of subjects doubled after 3 months for a total of 10 subjects (no data available).

| DISCUSSION
In this study, in order to verify the efficacy of the ingestion of LPS in the maintenance of normal bloodstream, the double-blind study was conducted by the ingestion of LPSp supplement. Since this study assessed the functionality as the foods, it was conducted in healthy subjects and subjects with borderline.
LPS is known as an endotoxin that causes serious inflammation when it is released in the blood during the infection of Gram-negative bacteria. On the other hand, LPS is a substance that is universally present in the environment. It is reported that it is not toxic by orally and transdermally ingested LPS, and it contributes to the activation of innate immunity.
LPSp used in this study is derived from wheat symbiotic bacteria P. agglomerans. This LPSp consists of rhamnose and glucose in the O-antigen part, and is characterized by a shorter sugar chain length than that of Escherichia coli (Inagawa et al., 1992). The chromosomal aberration assay (1.5 mg LPSp/plate), bacterial reverse mutation test (1.5 mg LPSp/ml), single-dose toxicity (600 mg LPSp/kg/day), and 28-day repeated-dose toxicity (300 mg LPSp/kg/day) have confirmed the safety of this LPSp ). In the results of this study, it was also confirmed that no adverse events were found in the Body fat (%) 22.9 ± 5.3 23.7 ± 5.8 24.9 ± 5.5 22.9 ± 5.4 23.5 ± 5.0 23.9 ± 5.4 Body weight and body fat were measured by ingested LPSp supplement or control. Subjects ingested test product for 3 months. Medical assessments were held three times; 0 m is baseline, +1.5 m is products ingested after 1.5 months, and +3 m is products ingested after 3 months. All numerical values are M ± SD.  Subjects ingested test product for 3 months. Medical assessments were held three times; 0 m is baseline, +1.5 m is products ingested after 1.5 months, and +3 m is products ingested after 3 months. a One volunteer could not be measured after 3 months.
healthy subjects and subjects with borderline with the oral ingestion of 500 μg/day LPSp.
In the bloodstream, Wiesner et al. (2010) reported that macrophage and intraperitoneally administered LPS, which is an activator of macrophage, may exacerbate arteriosclerosis. However, in this study, CAVI used as an indicator of arteriosclerosis was not changed in either group. Since the present study was performed on healthy volunteers, we cannot deny Wiesner's results, at least by oral administration; consequently, LPS may not be a factor in arteriosclerosis. No significant differences in other body markers and hematologic markers were found.
Here, we focused on the improved function of the bloodstream by the oral ingestion of LPSp. HbA1c is called advanced glycation end products (AGEs) in which hemoglobin and glucose are bound.
In this study, 18.2% of subjects were included in the subjects with the borderline of HbA1c in both groups, respectively. A significant decrease (p = .007) in the rate of change in HbA1c was found in the LPSp supplement group after 3 months. This improvement was more significant for the female segment by gender. This may be because the number in (borderline for HbA1c in) the female segment was larger than that of the male segment. The decrease in HbA1c was consistent with the results of Nakata et al. (2011). Since macrophage distinguish denatured protein, such as AGEs, as a foreign matter and eliminate (Ott et al., 2014), the decrease in HbA1c may be related with phagocytic elimination by macrophages activated through oral administration of LPS. On the other hand, no change in oxidized LDL as an oxide marker was found in either group. It was considered that LDL in the subjects was within the normal range in both groups, in this study.

F I G U R E 2
The changes in relative HbA1c levels in the LPSp supplement and control groups. HbA1c was measured before the start of ingestion (0), at 1.5 months, and 3 months after the start of ingestion. The graphic chart is expressed by M ± SE of the relative value in which the measured value before the start of ingestion is 1. P: Relative risk by Steel-Dwass test (before the start vs. after 3 months of ingestion) In association with the bloodstream, the number of fingertip capillary vessels was investigated in this study. Hypertensive patients showed lower capillary density, and capillary density was used for the assessment of antihypertensive drugs, etc. (Penna et al., 2008). Also, diabetic patients showed lower capillary density (Spiller et al., 2014).
Since an increase in capillary density is associated with improvement of diseases (the effect of the uptake of glucose to tissues by insulin stimulation is dependent on the capillary vessel or the ability of oxygen diffusion to skeletal muscle tissue is enhanced), the capillary density is often used as an indicator of assessment. In everyday life excluding diseases, since the number of capillary vessels is decreased by stress or aging, antistress and the improvement of bloodstream can be assessed by investigating the number and status of capillary vessels.
In this study, an increase in the number of fingertip capillary vessels was found in the LPSp supplement group. As a result, it was newly suggested that LPSp promoted angiogenesis by stimulating the production of VEGF from macrophage and was likely to contribute to the improvement of bloodstream.
Moreover, IgA produced at mucosa was also measured as a secondary parameter in this study. This is because it has been reported that LPSp enhances the effect of the influenza vaccine when administered sublingually and promotes the systemic induction of IgA which is the mucosal immunity (Fukasaka et al., 2015). In this study, no increase in IgA was observed probably because there was no exposure to antigens during the study period.
Recently, it has been reported that orally and transdermally ingested LPS have a variety of effects on health maintenance and disease prevention. It has been reported that the oral ingestion of LPSp improved the alopecia status in mice that developed atopic dermatitis (Wakame, Komatsu, Inagawa, & Nishizawa, 2015), and it was also suggested that the gene expression of VEGF, when stimulated by LPSp, was increased in an LPSp dose-dependent manner in human dermal papilla cell (Wakame et al., 2016). Also, it was confirmed that 62.5% (10 of 16 patients who were in remission or improved) of cancer patients were improved by the oral ingestion of LPSp (Morishima & Inagawa, 2016). As described in these reports, LPSp was found to exhibit effects on remission and the improvement of symptoms according to diseases.
From the results of this study, an increase in the number of capillary vessels and antiglycative stress effects by the oral ingestion of LPSp were found in the healthy subjects. Thus, it was suggested that orally ingested LPSp contributed to the maintenance of normal bloodstream. However, more study will be needed to determine the optimal dose of LPSp for the bloodstream.

F I G U R E 3
The changes in relative number of vessels per field in the LPSp supplement and control groups. The number of vessels was measured before the start of ingestion (0) and at 3 months after ingestion. The graphic chart is expressed by M ± SE of the relative value in which the measured value before the start of ingestion is 1. P: Relative risk by Mann-Whitney U test (test product group vs. control group) VAS of stiff neck was researched by ingested LPSp supplement or control. Subjects ingested test product for 3 months. Medical assessments were held three times; 0 m is baseline, +1.5 m is products ingested after 1.5 months, and +3 m is products ingested after 3 months. All numerical values are M ± SE.