Takahiro Oda,Central Research Institute, Mizkan Group Corporation,

2-6 Nakamura, Handa-city, Aichi 475-8585 JapanTel: +81-569-24-5128 / Fax: +81-569-24-5028 / E-mail: 3ne@mizkan.co.jp

Anti-Aging Medicine 7 (5) : 50-54, 2010(c) Japanese Society of Anti-Aging Medicine

Original Article

Effect of Oral Intake of Ceramide-Containing Acetic Acid Bcteria on Skin Barrier Function

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The lipid matrix in the stratum corneum is important to the barrier function of mammalian skin. Ceramides are main components of intercellular lipids in the stratum corneum and play an essential role in skin barrier function. Moreover, recent investigations have demonstrated improvement in skin barrier properties after oral intake of ceramides of plant origin in mice and healthy adults. However, beneficial effects on skin barrier function by oral intake of ceramides other than those of plant origin have not been reported. It is known that acetic acid bacteria accumulate intracellular ceramides. Therefore, we examined the dietary effect of ceramide-containing acetic acid bacteria on skin barrier function. Studied were 3 groups, each comprised of 20 healthy adults with awareness of dry skin. The control group was given a placebo, the low acetic acid bacteria group [low-dose group] was given 55.6 mg dry acetic acid bacteria containing 400 μg ceramide, and the high acetic acid bacteria group [high-dose group] was administered 111.1 mg dry acetic acid bacteria containing 800 μg ceramide per day for 8 weeks. Transepidermal water loss (TEWL) and stratum corneum hydration (SCH) were measured as indexes of skin barrier function on the cheek, upper inner arm and back of the neck of all study subjects. The most marked effect was on the cheek, the area most exposed. On the cheek, the TEWL value increased significantly on week 4 after starting administration in the control group, but remained at the baseline level in both the low-dose and high-dose groups. In both the low-dose and high-dose groups, TEWL values decreased significantly on week 6. The SCH value increased significantly after 6 weeks administration in the control group, whereas in both the low-dose and high-dose groups the SCH value increased significantly after just 4 weeks of intake. This increase occurred within a shorter period than in the control group. These results suggest that oral intake of ceramide-containing acetic acid bacteria effects to maintain skin barrier function in healthy adults with awareness of dry skin.

Abstract

Takahiro Oda 1), Hideki Tachimoto 1), Mikiya Kishi 1), Takayuki Kaga 1), Masamitsu Ichihashi 2)

1) Central Research Institute, Mizkan Group Corporation

2) Sun Care Institute, Sun Clinic

KEY WORDS: acetic acid bacteria, ceramide, oral intake, stratum corneum hydration, transepidermal water loss

Received: Jan. 7, 2010Accepted: Mar. 3, 2010Published online: Mar. 25, 2010

Introduction The skin barrier function prevents dryness and dehydration of the skin and protects against infection. The barrier function of the epidermis is strongly related to the intercellular lipid lamellar structure of the stratum corneum. Intercellular lipids mainly consist of ceramides, cholesterol and free fatty acids. Among these components, ceramides are the most important for maintaining skin barrier homeostasis 1). The lesional skin of patients with atopic dermatitis has decreased ceramide levels 2), increased transepidermal water loss (TEWL), and decreased stratum corneum hydration (SCH) 3). The level of ceramides in the stratum corneum also decreases in aged skin 2). Supplying ceramides to the skin may contribute to maintaining or improving skin barrier function. Actually, it has been reported that application of a cream containing ceramides ameliorated skin barrier destruction induced by tape-stripping or by sodium lauryl sulfate treatment 4). Also, recent investigations have demonstrated that oral intake of ceramides derived from plants decreased TEWL in mice 5,6) and in healthy adults 6,7). However, reports are still scant on the effects of orally administered ceramides on skin barrier function.

Although ceramides are principally found in animals and plants, they are also found in an extremely limited number of species of gram negative bacteria. Acetic acid bacteria are gram negative and obligate aerobes with a strong ability to oxidize ethanol into acetic acid and are used industrially to produce vinegar. These bacteria are capable of growing in an acidic medium and are tolerant of high osmotic stress and low pH caused by the acetic acid. A unique membrane lipid that protects the organism from the outside environment was shown to allow acetic acid bacteria to grow in extreme environments and that intracellular accumulated ceramide was a lipid component 8). In addition to vinegar manufacture, acetic acid bacteria are used to produce fermented foods, such as tea fungus beverages and nata de coco. Acetobacter malorum NCI 1683 (S24) derived from fermented milk, accumulated the high amount of ceramide 9). Moreover, ingestion of Acetobacter malorum NCI 1683 (S24) is effective at improving cognitive function 10). In this study we investigated whether oral administration of Acetobacter malorum NCI 1683 (S24) could affect skin barrier function.

Methods

Measurement of Ceramide Content in Acetic Acid Bacteria Cells

Total lipids were extracted by the method of Bligh and Dyer 11) from 30 mg of dry acetic acid bacteria. The total lipids were saponified with 3 ml 0.4 N KOH in methanol at 37 °C for 2 hours to decompose glyceryl phospholipids. The resultant alkali-stable lipid was extracted from the reaction mixture by Folch’s method 12). Benzoylation of ceramide was achieved by treating the dried alkali-stable lipid with 0.5 ml of benzoyl chloride/anhydrous pyridine (1:9, v/v) for 15 minutes at 70 °C followed by addition of 0.5 ml of methanol/water (8:2, v/v). The benzoylated lipids were applied to a Sep-PakC18 column (Waters Corp., Milford, MA, USA) equilibrated with methanol/water (8:2, v/v). After elution with methanol, the eluates were dried and dissolved in 1ml of hexane/isopropanol (100:0.8, v/v). The benzoylated ceramide was separated and quantified by HPLC on a Lichrospher 100 CN, 5 μm, 250×4 mm (Merck KGaA, Darmstadt, Germany), eluted with hexane/isopropanol (100:0.8, v/v) at a flow rate of 1.0 ml/min and monitored by UV absorption at 230 nm.

Study Design This was a randomized, double-blinded and placebo-controlled study performed at a single center. The SCH value is used as a screening test, with 50 arbitrary units (AU). Eligible subjects were men and women aged 35 to 60 years who had mild dry skin and were free of skin diseases such as atopic dermatitis and psoriasis and other diseases with severe xeroderma. Also, persons with systemic diseases that might contribute to skin conditions were not eligible. The final study group consisted of 65 healthy adult volunteers with awareness of dry skin who were aged 35–56 years (29 men, 36 women). The study period was 8 weeks from 12 February 2007 to 11 April 2007. This investigation conforms with the principles outlined in the Declaration of Helsinki, and the ethics committee at our institution approved this study. Subjects gave written, informed consent to participate. After confirmation that the subjects fulfilled the defined eligibility criteria, they were randomly assigned into 3 groups according to whether they received capsules having either 120.0 mg dextrin (placebo, control group); 55.6 mg dry Acetobacter malorum NCI 1683 (S24) (ceramide concentration of 7.2 mg/g dry cell weight), which contained 400 μg ceramide and 94.4 mg dextrin (low-dose group); or 111.1 mg dry Acetobacter malorum NCI 1683 (S24), which contained 800 μg ceramide and 48.9 mg dextrin (high-dose group). The doses were based on the previous reports which showed the effect of ingested ceramides on skin barrier function 6,7) and the safety of Acetobacter malorum NCI 1683 (S24) 10). Subjects took 1 capsule per day at any time during the 8 weeks of the trial, regardless of the level of symptoms. Compliance was evaluated by the number of capsules remaining at each visit.

Results

Sixty-five subjects were initially enrolled in the study. Five subjects withdrew from the study for personal reasons; thus, the final study population was 60 (27 males, age 43.9±4.7 years; 33 females, age 39.8±4.2 years). There were no significant differences in gender distribution between groups (Table 1). During the study period, none of the subjects, including 5 dropouts, experienced any adverse effect based on the results of blood analysis and clinical interviews. Therefore, oral intake of acetic acid bacteria was considered safe.

Transepidermal Water Loss and Stratum Corneum Hydration TEWL and SCH were measured every 2 weeks (±1 day). TEWL was determined using the Vapometer (Delfin Technologies, Ltd., Kuopio, Finland) and SCH was determined using the Corneometer CM825 (Courage+Khazaka electronic GmbH, Cologne, Germany). Both TEWL and SCH were measured on the cheek, upper inner arm and back of the neck. These measurements were performed after a 30-minute acclimation in a room controlled to specified conditions (temperature: 21±1°C, humidity: 45±5%).

Safety Evaluation Blood sampling and clinical interview were performed every 4 weeks. Blood samples were analyzed for creatinine, fasting blood glucose, uric acid, blood urea nitrogen, albumin, globulins, alkaline phosphatase, alanine transaminase, aspartate transaminase, gamma-glutamyl transferase, total protein, total bilirubin, direct bilirubin, c-reactive protein, lactate dehydrogenase, triglycerides, total cholesterol, red blood cell count, mean cell haemoglobin, mean corpuscular hemoglobin concentration, mean cell volume, haematocrit, glycosylated hemoglobin, haemoglobin, white blood cell count, basophils, eosinophils, lymphocytes, monocytes, neutrophils, fibrinogen and platelet count.

Statistical Analysis Data analyses and statistical analyses were done by a computer program (Excel Statistics 2006, Social Survey Research Information Co., Ltd., Shinjuku-ku, Tokyo). Data are expressed as means ±SE. Intra-group comparisons using raw data between values at baseline and those on week 2, 4, 6 and 8 were analyzed using one-way repeated measures ANOVA with the Tukey post-hoc test. All data were transformed to relative values and expressed as percentages of the 100% baseline value. Inter-group comparisons using transformed data were made by one-way factorial ANOVA with the Williams multiple comparison post-hoc test. p < 0.05 was considered significant.

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Table 1 Demographic characteristics of study subjects

Group n Mean age

20

20

20

41.5

41.6

41.8

Range

35 - 48

35 - 56

35 - 52

Control group

Low-dose group

High-dose group

n

9

8

10

Mean age

43.4

43.8

44.3

Range

38 - 48

37 - 53

35 - 52

Men

n

11

12

10

Mean age

39.9

40.1

39.3

Range

35 - 47

35 - 56

35 - 45

Women

52

Acetic Acid Bacteria and Skin Barrier Function

41.7

43.3

40.9

3.4

3.1

3.0

±

±

±

104.4

112.6

113.4

3.8

5.5

5.3

±

±

±

Significantly different (*p < 0.05, **p < 0.01, one-way repeated measures ANOVA with Tukey post-hoc test) from the corresponding value at baseline. Not significantly different from the corresponding value for the control group.

Table 3 A SCH on cheek

Table 3 Stratum corneum hydration (SCH) values measured on the cheek, upper inner arm and back of the neck of study subjects pre and post administration

Group Baseline

AU week 2

113.8

119.3

121.4

3.7

6.9

7.5

*

*

±

±

±

week 4

115.9

130.3

133.0

4.3

7.8

9.4

*

**

**

±

±

±

week 6

126.3

133.7

140.7

7.0

8.9

9.3

**

**

**

±

±

±

week 8

Control group

Low-dose group

High-dose group

% changes related to baseline

34.8

33.6

34.1

1.6

1.3

1.5

±

±

±

97.2

95.0

99.9

5.0

2.8

2.8

±

±

±

Table 3 B SCH on upper inner arm

Group Baseline

AU week 2

103.5

105.8

105.4

3.8

3.4

2.5

±

±

±

week 4

97.8

99.7

97.1

3.5

4.3

2.8

±

±

±

week 6

110.1

110.8

113.1

4.4

3.5

3.1 **

±

±

±

week 8

Control group

Low-dose group

High-dose group

% changes related to baseline

47.0

51.5

52.1

±

±

±

1.6

2.1

1.9

96.8

91.5

94.9

3.1

2.9

3.2

±

±

±

Table 3 C SCH on back of the neck

Group Baseline

AU week 2

105.6

100.9

102.7

3.9

3.2

2.6

±

±

±

week 4

103.3

98.0

101.9

3.6

3.1

1.9

±

±

±

week 6

114.0

108.5

110.3

4.0

3.3

2.3

**

**

*

±

±

±

week 8

Control group

Low-dose group

High-dose group

% changes related to baseline

19.8

23.1

21.4

1.3

1.6

1.8

±

±

±

101.5

97.3

100.3

5.9

3.5

4.2

±

±

±

Significantly different (*p < 0.05, **p < 0.01, one-way repeated measures ANOVA with Tukey post-hoc test) from the corresponding value at baseline. Significantly different (†p < 0.05, one-way factorial ANOVA with Williams multiple comparison post-hoc test) from the corresponding value for the control group.

Table 2 A TEWL on cheek

Table 2 Transepidermal water loss (TEWL) values measured on the cheek, upper inner arm and back of the neck of study subjects pre and post administration

Group Baseline

g/m2/h week 2

118.0

105.7

104.0

4.6

5.4

4.3

**

†

†

±

±

±

week 4

89.5

85.6

85.6

4.1

4.0

4.4

**

**

±

±

±

week 6

102.7

96.5

95.6

3.9

4.3

4.4

±

±

±

week 8

Control group

Low-dose group

High-dose group

% changes related to baseline

8.3

8.4

8.3

0.3

0.2

0.3

±

±

±

110.2

102.6

107.8

1.9

2.6

3.6

±

±

±

Table 2 B TEWL on upper inner arm

Group Baseline

g/m2/h week 2

* 103.2

98.2

100.7

3.3

2.8

3.6

±

±

±

week 4

111.2

103.2

103.5

4.7

3.2

2.8

*±

±

±

week 6

97.1

93.9

93.0

3.1

2.8

2.8

±

±

±

week 8

Control group

Low-dose group

High-dose group

% changes related to baseline

10.2

10.6

10.6

±

±

±

0.5

0.8

0.8

110.3

108.2

102.0

3.5

4.6

4.6

±

±

±

Table 2 C TEWL on back of the neck

Group Baseline

g/m2/h week 2

98.7

103.4

93.9

3.5

4.4

3.6

±

±

±

week 4

110.2

100.9

99.9

6.0

4.1

4.3

±

±

±

week 6

100.7

90.4

92.5

5.3

4.2

4.0

*

±

±

±

week 8

Control group

Low-dose group

High-dose group

% changes related to baseline

in hairless mice. It was therefore suggested that oral intake of ceramide might reduce damage from the external environment, as Uchiyama et al. also discussed 6). Accordingly, the TEWL value for the cheek would be effective in comparison with the non-exposed regions (upper arm and back of the neck) in this study. Unlike the TEWL, the SCH was improved in all regions and groups by week 8, a result that might be caused by an increase in temperature. But, SCH is not influenced by many skin barrier dysfunction methods compared with TEWL 15). This may be a reason why inverse correlation could not been observed between TEWL values and SCH values on the cheek. Ceramides consist of a sphingoid base linked to a fatty acid via an amide bond. Ceramide derived from plants, which reportedly improves skin barrier function by oral intake, is a complex of a sphingoid base with two trans-double bonds called sphingadienine, whereas ceramide derived from acetic acid bacteria is a complex of a sphingoid base without the double bond called sphinganine, which is a dihydroceramide 8). It is reported that dietary ceramides are hydrolyzed to a sphingoid base and fatty acids by ceramidase in the intestinal tract, and are absorbed in the small intestine in mice 16). Sphingadienines are not well absorbed in Caco-2 in comparison with sphingosine 17). However, sphinganine is well absorbed in comparison with sphingosine in examinations using rats 18). Consequently, ceramide derived from acetic acid bacteria, which is a complex of sphinganine, is expected to be more readily absorbed after oral administration than ceramide derived from plants. In addition, it has been shown that dihydroceramide is used in mammals as a precursor of mammalian-type ceramide 19). Therefore, ceramide derived from acetic acid bacteria can be expected to be more easily used by mammals when reconstructed as dihydroceramide after degradation and absorption as sphinganine or to be absorbed if it remains as dihydroceramide. However, there are no reports of absorption of ceramides from acetic acid bacteria. Therefore, we need to investigate absorption in the intestine and accumulation to the skin. In this study, on a molar basis, oral intake of dihydroceramide contained in acetic acid bacteria effected on skin barrier function less than that reported for ceramide derived from plants 6,7). In the future, dihydroceramide and dihydroceramide-containing acetic acid bacteria are expected to become promising agents for relieving dry skin conditions.

Tables 2 and 3 summarize statistical data on TEWL and SCH. Tables 2 shows the TEWL values for each region measured. As for the cheek, TEWL values in the control group on week 4 was significantly increased from baseline, and TEWL in the low-dose and high-dose groups were significantly decreased on week 6 (Table.2A). On week 4, TEWL values in the low-dose and high-dose groups were significantly lower than that in the control group. TEWL on the upper arm in the control group was significantly increased over baseline on week 2 and 6 (Table.2B). With regard to the back of the neck, the TWEL in the low-dose group was decreased significantly on week 8 compared with baseline (Table.2C). The SCH value of each region measured is shown in Tables 3. The SCH value on the cheek significantly increased from baseline in all groups. The value in the low-dose and high-dose groups were higher than that in the control group, and statistically significant differences between baseline were observed week 4, 6,and 8 in the low-dose and high-dose groups whereas the differences from baseline in the control group were statistically significant only on week 6 and 8 (Table.3A). The SCH of the upper inner arm in the high-dose group increased significantly on week 8 in comparison with the baseline value (Table.3B). On the back of the neck, SCH significantly decreased from baseline on week 2 in the low-dose group, and significantly increased from baseline on week 8 in the control and high-dose groups (Table.3C).

Discussion

To our knowledge, this is the first report about the oral intake of acetic acid bacteria containing ceramide on skin barrier function. Especially on cheek, although TEWL values in the control group increased as a result of a decline in skin barrier function, high- and low-dose groups were significantly lower than that in the control groups. Moreover, significantly higher SCH over baseline in high- and low-dose groups were observed earlier than that in the control group. These results indicated that oral intake of acetic acid bacteria, even the low-dose, showed useful effect to maintain skin barrier function in healthy adults with awareness of dry skin. Orally administered acetic acid bacteria may be expected to preserve a better skin condition and relieve itchiness derived from dry skin. In this study, the effect of acetic acid bacteria administration were remarkably observed on cheek compared with other regions studied. The number of cell layers of the stratum corneum in the cheek is similar to that of the upper arm and the neck 13). However, the exposed cheek is more likely than non-exposed regions to be damaged by the environment. Therefore, the skin barrier function on the cheek is impaired in winter, as evidenced by a higher TEWL value in winter than in summer 14). In this study, when the TEWLs of each studied part were compared before administration of the capsules, the cheek had a higher TEWL value than the upper arm and back of the neck (Table 2). Moreover, in the control group, TEWL values for the cheek were increased on week 4, as a sign of a decline in skin barrier functions possibly caused by environmental factors. These factors might have been a rapid decrease in humidity and temperature around week 4. In addition, subjects in the control group had decreased TEWL values on week 6 (Table.2A), due to rapid increases in humidity and temperature after week 4. Moreover, oral administration of ceramide extracted from plants has been shown to improve skin barrier dysfunction induced by tape-stripping 5) or by sodium lauryl sulfate treatment 6)

Acknowledgments

We would like to thank Dr. Atsushi Ishikawa, Dr. Takashi Fushimi, Mr. Hiroyuki Fukami and Mr. Kenichi Sugiyama for useful discussions, Mr. Shin Ogawa for supplying the dry Acetobacter malorum NCI 1683 (S24) we used in this study, and Ms. Tomoko Hayashi for technical assistance.

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References

Acetic Acid Bacteria and Skin Barrier Function