Safety Evaluation of the Excessive Intake of Ceramide-Containing Acetic Acid Bacteria - A Randomized, Double-Blind, Placebo-Controlled Study Over a 4-week Period.

Ceramide plays an important role in maintaining the skin barrier function. Aging and atopic dermatitis are known to reduce the levels of ceramide. Application of exogenous ceramide to the skin can restore the barrier function. In recent years, the effect of oral intake of ceramide has been demonstrated to improve the skin barrier function, and it has been marketed as a food supplement. Therefore, it is important to provide information on the safety of unintentional overdose of ceramide. This randomized, double-blind, placebo-controlled study was conducted in 30 healthy adults, aged between 20 and 60 years of age (both female and male). The subjects consumed either dietary supplement, comprising 1197 mg of acetic acid bacteria containing 9.06 mg of ceramide, or placebo for four consecutive weeks. Safety was evaluated based on physical measurements, blood test, urinalysis, adverse events, and side effects. The results showed several significant differences in physical measures and blood tests between the two groups. However, these differences were considered to be unrelated to the intake of the ceramide-containing acetic acid bacteria or placebo. Thus, no adverse effects or clinically concerning changes in physical, blood, and urine parameters were observed due to the excessive intake of the ceramide-containing acetic acid bacteria in the present study.TRIAL REGISTRATION: UMIN000035481.


Introduction
The skin barrier function not only prevents the evaporation of water from inside the stratum corneum, but also plays an important role in protecting the living body from external stressors such as ultraviolet rays, infiltration of chemical substances, and microbial infection. The barrier function of the epidermis is strongly related to the intercellular lipid layer structure of the stratum corneum. Intercellular lipids are mainly composed of ceramide, cholesterol, and free fatty acids, and among these components, ceramide is the most important molecule for maintaining skin barrier homeostasis 1 . Reports indicating decreased levels of ceramide 2 , increased transepidermal water loss TEWL , and decreased water content in the stratum corneum of skin lesions in patients with atopic dermatitis all support this theory 3 . Ceramide levels are also known to brewed vinegar, Caspian Sea yogurt, and Nata de coco 10 . Acetic acid bacteria are able to tolerate osmotic stress and low pH caused by the acetic acid, produced by the bacteria themselves, and it has been reported that the molecule that confers this resistance is ceramide accumulated in the cells 11 . Acetobacter malorum NCI 1683 S24 , derived from fermented milk, is known to accumulate a large amount of ceramide 12 , and the oral intake of this bacteria has been reported to improve the skin barrier function 13 . The ceramide used in this study refers to the dihydroceramide produced by hydroxylation of the sphingosine base, unless otherwise stated. In detail, the ceramide molecule is 2-hydroxypalmitoyl-sphinganine dihydroceramide , which has no double bond, and its chemical formula is C 34 H 69 NO 4 .
There has been progress in the development of cosmetics and foods to improve skin function, and these products are currently marketed as food supplements. Although previous human clinical trials have not yet identified any problematic events, in terms of safety, for ceramide-containing acetic acid bacteria 13 , food supplements pose the risk of unintentional overdose because these products are easy to take. Thus, it is important to evaluate safety of food supplements with ceramide-containing acetic acid bacteria, in case of overdose. Therefore, we conducted a randomized, double-blind, placebo-controlled, parallel-group comparison study in healthy adults, aged 20 to 60 years, to evaluate the safety of excessive intake of ceramide-containing acetic acid bacteria. The dose used as an excessive intake was 1197 mg per day as acetic acid bacteria, 9.06 mg as ceramide , which was more than five times the recommended daily intake, as specified in the precautions stipulated during preparation of the application for food for specified health uses, and safety of this dose has been confirmed in animal experiments In-house examinations: data not shown .

Study implementation system
This study was reviewed and approved by the Ethics Review Committee of the Oriental Occupational Health Association Tokyo Branch, Oriental Ueno Medical Center approval date: December 6, 2018 , which comprised of a third party not involved in the study, to protect the human rights and safety of the subjects and to ensure the reliability of the test data. The study was then implemented from January to February 2019, based on the study protocol of the Ueno Asagao Clinic Investigator: Takahiro Ono UMIN study ID: UMIN000035481 . This study complied with ethical principles and was conducted in accordance with the tenets of the Declaration of Helsinki amended at the 2013 WMA Fortaleza General Assembly Brazil and the Ethical Guidelines for Medical and Health Research Involv-ing Human Subjects Notice from the Ministry of Education, Culture, Sports, Science and Technology and Ministry of Health, Labour and Welfare .

Study participants
The study details were explained to the subjects at recruitment, and a preliminary examination SCR: Screening was conducted for 68 subjects who were deemed suitable as test subjects. The selection criteria for this study were 1 men and women, aged between 20 and 60 years inclusive at the time of consent for participation in the study, 2 healthy individuals with no chronic physical disease, including skin disease, 3 individuals who had received sufficient explanation about the purpose and content of this study, had the ability to consent, had agreed to participate voluntarily with full understanding of the content of the study, and were able to consent to participate in this study in writing, 4 individuals who were able to visit the clinic on designated examination days, and able to undergo examination, and 5 individuals deemed to be suitable for participation in the study by the principal investigator. The exclusion criteria for this study were 1 individuals currently receiving drug treatment for some kind of disease, 2 individuals with a medical history or current mental disease, sleep disorder, hypertension, diabetes, dyslipidemia, and/or serious disease, 3 individuals with a habit of taking medication to treat disease in the past month excluding single dose medication for headache, menstrual pain, common cold, etc. , 4 individuals with serious medical history or current condition of the liver, kidneys, heart, lungs or blood, 5 individuals with serious medical history or comorbidity of the digestive system, 6 individuals with body mass index BMI 18.5 kg/m 2 or ≥ 30 kg/m 2 , 7 individuals with a systolic blood pressure of ≥ 140 mmHg, a diastolic blood pressure of ≥ 90 mmHg, and/or a pulse rate of ≥ 100 bpm, 8 individuals who had donated more than 200 mL of blood within the last month or more than 400 mL of blood within the last 3 months, 9 individuals who felt unwell after previous blood collection, 10 individuals who may develop allergy symptoms to the test food, or who may develop serious allergy symptoms to other foods or drugs, 11 individuals who regularly took food supplements for specified health uses, foods with functional claims, and/or health foods however, this did not apply to individuals who were able to stop taking the foods in question for the duration of the study after consent was obtained . 12 individuals whose mean daily alcohol consumption normally exceeded 60 g/day, 13 individuals whose daily life may change during the study period going on extended holidays, etc. , 14 women who were pregnant, breastfeeding, or may be pregnant, 15 individuals who were currently participating in another human clinical trial, or who participated in another human clinical trial less than 3 months ago, 16 individuals or their family members who worked at companies that develop, manufacture or sell health food, foods with functional claims and/or cosmetics, and 17 other individuals who were deemed unsuitable for participation in this study by the principal investigator. Based on the selection criteria, 30 subjects were selected for participation in this study.
As the basis for setting the number of subjects considered the importance of incorporating a broad age range of the population expected to take the test food, as the purpose of this study was to ascertain the safety of the test food. Therefore, it was considered necessary to incorporate equal ratio of male and female subjects, across a wide age range aged 20-60 years . Considering withdrawals and the short study period 4 weeks , the number of subjects was set to 15 per group.

Selection, randomization, and blinding
The subjects gender, age, and BMI at preliminary examination SCR were used as stratification factors, and after randomly assigning the subjects to two groups, using the stratified block randomization method, it was confirmed that there was no significant difference between the allocation groups.
Two types of food supplements were investigated: test food food containing acetic acid bacteria and placebo food food not containing acetic acid bacteria . The study contractor ensured that each type of test food could not be identified based on their appearance, by printing the relevant identification codes on the test food and delivering it to the study outsource provider. The identification codes were sealed in an envelope and sent to the person-in-charge of allocation, who confirmed that each test food sent from the study contractor could not be distinguished by its appearance and/or smell. Next, the person-in-charge of allocation replaced the test food food containing acetic acid bacteria and placebo food food not containing acetic acid bacteria codes with another test product control code that was not easy identifiable, thereby blinding the study. The products were then handed over to the test product manager. The test item control codes were tightly sealed in an envelope together with the correspondence table and kept by the person-in-charge of allocation, until the study was unblinded.

Test food
Dried A. malorum NCI 1683 acetic acid bacteria strain and A. malorum NCI 1683 acetic acid bacteria strain-derived ceramides were used as the test food.
The ingredients and nutritional compositions of the test food and placebo food used in the study are shown in Tables 1a and 1b. Hard gelatin capsules, containing dried A. malorum NCI 1683 acetic acid bacteria strain 1197 mg containing 9.06 mg of dihydroceramides as mentioned in the introduction. Since the industrial know-how regarding the optimal fermentation of A. malorum NCI 1683 S24 found from fermented milk was included, the culture conditions, preparations for the bacteria were not described. In briefly, the ceramide was prepared by solvent extraction using chloroform/methanol. 7.5 mL of chloroform and 7.5 mL of methanol were added to 80 mg of lyophilized bacterial cells. The mixture was extracted effectively under sonication. 6 mL of distilled water was added to chloroform/ methanol mixture to extract effectively under sonication. 15 mL of chloroform and 7.5 mL of methanol were added and mixed by convective vortex, and centrifuged at 3000 rpm for 10 minutes to remove the upper layer and insoluble bacterial cells. 20 mL of distilled water was added and mixed by convective vortex, and centrifuged at 3000 rpm for 10 minutes to remove the upper layer and insoluble bacterial cells. The lower layer was transferred to a glass tube and dried using a rotary evaporator at 3000 rpm and 42 . The dried residue highly including the ceramide was reconstituted by chloroform: methanol 2:1 solution and subjected to quantification analysis. The absorbance could not be measured, because there is no double bond. Therefore the ceramide molecule was quantified using a combination of an evaporative light scattering detector ELSD and HPLC using a separation column Shim-Pack HRC-SIL 4.6 250 mm; Shimadzu Corporation, Japan .
The placebo capsules contained dextrin only. It was confirmed that that test food could not identified based on its appearance or smell.
The test food was taken as 9 capsules per day. All the capsules were taken once a day water or lukewarm water.
In the nutritional components shown in Table 1b, calories were calculated assuming that protein was 4 kcal/g, fat was 9 kcal/g, and carbohydrate was 4 kcal/g. Protein was measured by the combustion method, lipid by the acid decomposition method, and sodium by the atomic absorption spectrophotometry. Carbohydrates were calculated by subtracting protein, fat, ash and water content from the total weight. The ash content was measured by the direct ashing method. Sodium chloride equivalent was calculated as sodium equivalent g/100 g sodium content in food mg/ 100 g 2.54/1,000.

Study design
This was a placebo-controlled, randomized, double-blind, parallel-group study. The main endpoints were the presence or absence of side effects, and the number and incidence of side effects. Secondary endpoints included the number and incidence of adverse events, blood pressure systolic/diastolic /pulse, body weight/body fat percentage/ BMI, hematology tests, biochemical examination of blood, and general urinalysis. During the study period, the subjects were instructed to avoid an irregular lifestyle lack of sleep, excessive drinking and eating, etc. . They were instructed to refrain from drinking excessive alcohol 500 mL of beer per day: within the equivalent of 20 g based on alcohol conversion . They were also prohibited from suddenly changing the amount of alcohol consumption, such as suddenly stopping alcohol. The subjects were instructed to maintain the same quantity and quality of diet, exercise, and sleep as before the start of this study. In particular, excessive exercise and lack of sleep on the day before the tests was prohibited. The use of pharmaceuticals including external preparations , quasi-drugs, and herbal medicines was generally prohibited. If pharmaceuticals were used or the use was unavoidable, the subjects were asked to contact the consultation counter and to record the name of the product used, the manufacturer, and the reason for use in a diary. Intake of food for specified health uses, foods with functional claims, and/or health foods was prohibited. When intake was unavoidable, the name of the product, composition, and the amount taken were recorded in the diary. There were no particular restrictions on smoking during the study period. However, the subject was asked to maintain their usual smoking habits frequency, brand, etc. as much as possible, and sudden changes in smoking, such as suddenly stopping smoking, were prohibited. Blood collection other than those performed in this study and blood donation were prohibited from the time of becoming a subject to the end of the study blood collection for health checkups etc. was allowed . The subjects were instructed to complete the diary every day and to submit it on the specified day. They were asked to visit the hospital on the designated examination day and obliged to follow the precautions at the time of the examination. Participation in other human clinical trials was prohibited during the trial period. Other matters that may affect the test results were also prohibited. Consumption of alcohol was prohibited from the day before the test until completion of the test on the test day. The subjects fasted for 12 hours before the scheduled blood sampling time until the completion of the test. However, the minimum required amount of water was allowed.
All procedures followed were in accordance with the Ethics Committee of the Oriental Ueno Detection Center, General Incorporated Association Oriental Occupational Health Association Tokyo Branch Tokyo, Japan; Study ID: HR-2018-MKH03 and with the Helsinki Declaration of 1964 and later versions.

Statistical analysis
Statistical analysis was performed using appropriate statistical analysis software, such as SAS SAS 9.4 or SPSS Statistics 25 , and the significance level for all tests was 5 on both sides and the trends at 10 level. Calculated data shown in the table and graphs, was presented as the mean standard deviation. Fisher s exact test was used for intergroup comparison of the number and incidence of side effects, and the number and incidence of adverse events. The paired t-test was used for intergroup comparison of blood pressure systolic/diastolic /pulse, weight/body fat ratio/BMI, hematology tests, and biochemical examination of blood. The Wilcoxon signed rank test was used for semi-quantitative urinalysis. The unpaired t-test was used for intergroup comparison.

Patient disposition
This study conducted a preliminary examination on 68 people, amongst which 30 were selected as subjects for this study based on the selection criteria. One subject could not attend the clinic for the examination before starting the intake of the test food due to contracting influenza, hence the subject withdrew from the study. Therefore, the study was initiated with 29 subjects: test food group: n 15 men: n 7, women: n 8 and placebo food group: n 14 men: n 7, women: n 7 . The study was completed with one subject withdrawing during the study due to personal circumstances. Subject background intent to treat analysis set included age of the test food group which was 34.  Table 2 .

Adverse drug reactions and adverse events
There were no side effects thought to be caused by the test food in any of the subjects.
Adverse events occurred in both the groups: test food group n 3: 4 events: elevated CK, knee burn, pharyngitis, influenza A , and placebo food group n 2: 2 events: common cold cough, pharyngitis, nasal discharge , boil left foot Tables 3a and 3b .

Blood pressure systolic/diastolic , pulse
For blood pressure and pulse, the systolic blood pressure significantly decreased in the test food group at four weeks after starting the intake compared to that before the intake Table 4 . Conversely, in the placebo food group, systolic blood pressure significantly increased at two weeks and four weeks after starting the intake compared to that before the intake. Similar result was seen in the amount of change value , and especially with the amount of change between groups, the test food group was significantly lower than the placebo food group at two weeks and four weeks after starting the intake. The diastolic blood pressure significantly decreased in the test food group at two weeks after starting the intake compared to that   before the intake. In the placebo food group, diastolic blood pressure significantly increased at four weeks after starting the intake compared with before the intake, while a decreasing tendency was noted at two weeks after starting the intake. In terms of the amount of change, the test food group had a significant decrease at two weeks after starting the intake compared to that before the intake. While the placebo food group had a significant increase at four weeks after starting the intake compared to that before the intake, and a decreasing tendency was noted at two weeks after starting the intake. The test food group had a significantly lower amount of change than the placebo food group at two weeks and four weeks after starting the intake, compared to that in the placebo food group. There was no significant variation in either the measured value or the amount of change in the pulse throughout the study period, in both the test food and placebo food groups.

Body weight, body fat ratio, BMI
Amongst weight, body fat ratio, and BMI, both the measured value and the amount of change were significantly decreased for body fat ratio, only in the test food group at 4 weeks after the intake compared to that before the intake p 0.0475 . No significant change was observed for weight and BMI throughout the study period in both measured values and amount of change for both the groups data not shown .

Hematology tests
In the hematology tests, no significant change was observed in measured values and amount of change for WBC, MCHC, PLT, and WBC profile, in both the test food group and placebo food group throughout the study period. No significant change was observed in RBC in the test food group throughout the study period. However, in the placebo food group, a significant decline was noted at 2 weeks and 4 weeks after starting the intake, compared to that before the intake. There was no significant difference between the groups in RBC. Similarly, there was no significant variation in the amount of change in the test food group throughout the study period. In contrast, in the placebo food group a significant decline was noted at 2 weeks and 4 weeks after starting the intake, as a result, a statistically significant difference was found between the groups, but no significant change was observed in the test food, in RBC. No significant change was observed in Hb levels in the test food group throughout the study period, while in the placebo food group a significant decline was noted at 2 weeks and 4 weeks after starting the intake when compared to that before the intake. There was no significant difference between the groups. Similarly, while there was no significant variation in the amount of change in the test food group throughout the study period, in the placebo food group a significant decline was noted at 2 weeks and 4 weeks after starting the intake, when compared to that before the intake, which this resulted in a significant increase in the test food group at 2 weeks after starting intake, and an increasing trend at 4 weeks compared to that in the placebo food group. No significant change was observed in Ht in the test food group throughout the study period. However, in the placebo food group, a significant decline was noted at 2 weeks and 4 weeks after starting the intake, compared to that before the intake. There was no significant difference between the groups. Similarly, there was no significant variation in the amount of change in the test food group throughout the study period, but in the placebo food group a significant decline was noted at 2 weeks and 4 weeks after starting the intake, when compared to that before the intake. This resulted in a significant increase in the test food group at 2 weeks after starting the intake, and an increasing trend at 4 weeks compared to that in the placebo food group. There was a significant decline in the MCV in the test food group at 4 weeks after starting the intake, compared to that before the intake. No significant variation was noted in the placebo food group throughout the study period. The results were similar for the amount of change. A decreasing trend in the MCH levels was noted in the test food group at 4 weeks after starting the intake, compared to that before the intake. No significant variation was noted in the placebo food group throughout the study period. The results were similar for the amount of change. There was a significant decline in basophil count in the test food group at 2 weeks and 4 weeks after starting the intake, compared to that before the intake. No significant variation was noted in the placebo food group throughout the study period. There was no significant difference between the groups. Similarly, there was a significant decline in the amount of change in the test food group at 2 weeks and 4 weeks after starting the intake, compared to that before the intake. No significant variation was noted in the placebo food group throughout the study period. A decreasing trend was noted in the test food group at 2 weeks after starting the intake compared to that in the placebo food group. There was no significant variation in eosinophil count in the test food group throughout the study period. A decreasing trend was noted in the placebo food group at 4 weeks after starting the intake compared to that before the intake; however, there was no difference between the groups. The results were similar for the amount of change. There was a significant increase in neutrophil count in the test food group at 2 weeks after starting the intake compared to that before the intake. There was no significant variation in eosinophil count in the placebo food group throughout the study period. There was an increasing trend in the test food group at 2 weeks and 4 weeks after starting the intake compared to that in the placebo food group. There was no difference in the amount of change between the groups. There was a significant decrease in the lymphocyte count in the test food group at 2 weeks after starting the intake compared to that before the intake. There was no significant variation in the placebo food group throughout the study period. There was a decreasing trend in the test food group at 2 weeks and 4 weeks after starting the intake compared to that in the placebo food group. The results were similar for the amount of change; however, there was no difference between the groups. There was an increasing trend in monocyte count in the test food group at 2 weeks after starting the intake compared to that before the intake, but there was no significant variation in the placebo food group throughout the study period. There was no difference between the groups. There was an increasing trend in the amount of change in the test food group at 2 weeks after starting the intake compared to that before the intake, but there was no significant variation in the placebo food group throughout the study period. However, there was an increasing trend in test food group at 4 weeks after the intake compared to that in the placebo food group Table 5 .

Biochemical examination of blood
In the biochemical examination of blood, there were no significant changes in the levels of ALB, UN, CRE, UA, CK, TG, T-BIL, Na, K, Cl, and Fe in either the test food group or the placebo food group throughout the study period. The results were similar for the amount of change. There was a significant decline in TP content in the test food group at 4 weeks after starting the intake compared to that before the intake, and a significant decline in the placebo food group at 2 weeks and 4 weeks after starting the intake. The results were similar for the amount of change. There was no significant variation in AST in the test food group throughout the study period, but a decreasing trend was noted in the placebo food group at 2 weeks after starting the intake compared to that before the intake. The results were similar for the amount of change. There was a decreasing trend in ALT concentration in the test food group at 2 weeks after starting intake compared to before intake. There was no significant variation in the placebo food group throughout the study period. The results were similar for the amount of change. There was a significant decline in γ-GT in the test food group at 2 weeks after starting the intake compared to that before the intake. There was a significant decline in the placebo food group at 2 weeks and 4 weeks after starting the intake compared to that before the intake. The results were similar for the amount of change. There was no significant variation in the ALP in both the test food group and the placebo food group throughout the study period. However, the test food group had an increasing trend in actual measurements compared to that in the placebo food group at 4 weeks after starting the intake. There was a significant decline in LDH levels in the test food group at 2 weeks after starting the intake compared to that before the intake. There was a significant decline in the placebo food group at 2 weeks and a declining trend at 4 weeks after starting the intake compared to that before the intake. The results were similar for the amount of change. There was a significant decline in T-cho in the test food group at 2 weeks and 4 weeks after starting the intake compared to that before the intake. There was a significant decline in the placebo food group at 4 weeks after starting the intake compared to that before the intake. The results were similar for the amount of change. Analysis of the amount of change revealed a declining trend in the test food group compared to that in the placebo food group at 2 after starting the intake. There was a significant decline in the levels of LDL-C in both the test food group and the placebo food group at 2 weeks and 4 weeks after starting the intake compared to that before the intake. There was a decreasing trend in HDL-C in the test food group at 2 weeks after the starting intake compared to that before the intake, which progressed to a significant decline at 4 weeks. There was no significant variation in the placebo food group throughout the study period. The results were similar for the amount of change. There was a decreasing trend in Ca in the test food group at 2 weeks after starting the intake compared to that before the intake, which progressed to a significant decline at 4 weeks. There was a significant decline in the placebo food group at 2 weeks and 4 weeks after starting the intake compared to that before the intake. The results were similar for the amount of change. There was a significant increase in GLU in the test food group at 2 weeks and 4 weeks after starting the intake compared to that before the intake. There was no significant variation in the placebo food group throughout the study period. The results were similar for the amount of change. Analysis of the amount of change revealed an increasing trend in the test food group compared to that in the placebo food group at 4 weeks after starting the intake. There was a significant increase in HbA1c in both the test food group and the placebo food group at 2 weeks and 4 weeks after starting the intake compared to that before the intake. The results were similar for the amount of change. Analysis of the actual measurements revealed an increasing trend in the test food group compared to that in the placebo food group at 2 weeks and 4 weeks after starting the intake. On the other hand, there was a significant increase in CRP quantification in the test food group compared to that in the placebo food group at 2 weeks after starting the intake. However, no significant variations were seen in either the test food group or the placebo food group throughout the study period Table 6 .

General urinalysis
In general urinalysis, there were no significant changes in urobilinogen, bilirubin, ketone body qualitative measurements, glucose qualitative measurements, protein, pH, and specific gravity in either the test food group or placebo food group throughout the study period Table 7 . There was no significant change in occult blood reaction in either the test food group or the placebo food group throughout the study period. In the test food group, occult blood reaction was significantly increased compared to the placebo food group at 4 weeks after starting the intake. However, there was no difference between the groups with respect to the amount of change.

Discussion
There were no side effects attributable to the test product observed in any subjects throughout the study period. Adverse events, such as the common cold and influenza, which occurred during the cold and influenza season, as well as other events, disappeared in a short period of time. Moreover, since these adverse events were temporary, the study was continued at the doctor s discretion. No adverse events that may have been attributable to the test product were observed in any of the subjects.
This study also confirmed a significant decrease in blood pressure and body fat ratio due to intake of the test food. However, the scope of decrease in measured values was within the physiological range.
Although there were significant fluctuations in the hematology tests, the fluctuation range was slight and within the physiological range. CRP quantification in biochemical examination of blood confirmed an upward swing in the mean value of the test food group due to the presence of 3 cases, 2 weeks after intake that exceeded the standard value 0.3 mg/dL . The spikes were transient in all cases, and the results were within the normal range when examined 4 weeks later. It was assumed that these spikes may have been due to inflammation associated with some kind of infection because it is not considered to be an effect that occurs with continuous ingestion of the test food. As described above, although significant changes were observed in several parameters in the biochemical examination of blood, all were slight changes, which were considered to be physiological fluctuations within the normal range.
The occult blood reaction test in urinalysis revealed one case with a score of 3 and three cases with a score of 1 in the test food group 4 weeks after intake. All the cases were reported in women. The case with a score of 3 also had a score of 3 before intake 4 weeks before , and the cases with a score of 1 had a score of 2 before intake 4 weeks before , so the involvement of the sexual cycle was suggested. Therefore, it was strongly suspected that menstrual blood had contaminated the urine. As described above, the intake of the test food is thought to have had no effect on general urinalysis.

Conclusion
A safety study on excessive intake of acetic acid bacteria containing ceramide found no clinically significant events for blood pressure systolic/diastolic /pulse, weight/body fat ratio/BMI, hematology tests, biochemical examination of blood, general urinalysis, adverse events, and side effects. Therefore, the study confirmed the safety of 4 consecutive weeks intake of 1197 mg of acetic acid bacteria containing 9.06 mg of ceramide.