The effect of Lactobacillus helveticus fermented milk on sleep ... - Nature

European Journal of Clinical Nutrition (2009) 63, 100–105

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ORIGINAL ARTICLE

The effect of Lactobacillus helveticus fermented milk on sleep and health perception in elderly subjects S Yamamura1, H Morishima1, T Kumano-go1,2, N Suganuma1,2, H Matsumoto1, H Adachi1,2, Y Sigedo1, A Mikami1,3, T Kai4, A Masuyama4, T Takano4, Y Sugita1,2 and M Takeda1 1

Psychiatry, Department of Integrated Medicine, Division of Internal Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; 2Osaka University Health Care Center, Toyonaka, Osaka, Japan; 3Osaka Prefectural Mental Health Center, Osaka, Japan and 4Research and Development Center, CALPIS Co., Ltd., Sagamihara, Kanagawa, Japan

Objective: To study the effect of Lactobacillus helveticus fermented milk on sleep and health perception in elderly healthy subjects. Subjects: The study included 29 healthy elderly subjects aged 60–81 years. Methods: Prospective, randomized, double-blind and placebo-controlled, with a crossover design. The study included two intervention periods of 3 weeks each, separated by a 3-week washout period. Subjects took 100 g of fermented milk drink or a placebo drink (artificially acidified milk) daily in the first supplementary period and the other drink in the second supplementary period. For each period, we measured sleep quality by means of actigraphy and a sleep questionnaire, and assessed the quality of life (QOL) by SF-36 health survey. Results: There was a significant improvement in sleep efficiency (P ¼ 0.03) and number of wakening episodes (P ¼ 0.007) in actigraph data after intake of fermented milk, whereas no significant changes were observed for the placebo. Fermented milk did not improve the SF-36 scores significantly from the baseline period. In the GH domain (general health perception) of the SF36, however, there was marginal improvement as compared to the baseline period. Although the difference between fermented milk and placebo was not statistically significant for any of the sleep or QOL parameters, fermented milk produced slightly greater mean values for many parameters. Conclusion: This short-term (3-week) intervention study indicates that Lactobacillus helveticus fermented milk may have a more favorable effect on improving sleep in healthy elderly people as compared with placebo.

European Journal of Clinical Nutrition (2009) 63, 100–105; doi:10.1038/sj.ejcn.1602898; published online 12 September 2007 Keywords: sleep; actigraph; SF-36; fermented milk; elderly

Introduction In an aging society like in Japan, dealing with insomnia among the elderly has become a major social issue. According to Schubert et al. (2002), symptoms of insomnia are common among older adults and are associated with a Correspondence: Dr S Yamamura, Psychiatry, Department of Integrated Medicine, Division of Internal Medicine, Osaka University Graduate School of Medicine, D3 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: [email protected] Guarantor: M Takeda. Contributors: SY contributed to the study plan, subject recruitment, data analysis and interpretation, and was the principal author of the manuscript. HMo, TKu, NS, HMa, HA, TKa and AMa recruited the subjects, collected the data and assisted the data analysis. TKa, AMa and TT developed and distributed the test products. TKu, YSi, AMi, AMa, TT, YSu and MT contributed to the study plan and data interpretation. Received 23 October 2006; revised 8 July 2007; accepted 18 July 2007; published online 12 September 2007

decrease in health-related quality of life. Almost all epidemiological studies have reported that the prevalence of insomnia symptoms increases with age. A study in Japan found that the overall prevalence of insomnia increased with age, from 18.1% for the younger group to 29.5% for the older group (Kim et al., 2000). In the elderly, about 20% of older adults who responded to the National Sleep Foundation (2003) poll indicated that they took something to aid sleep at least a few nights per week. Eleven percent reported taking a prescription medication, 6% used over-the-counter medications and 6% used alcohol to help them sleep. Many individuals affected with insomnia, especially elderly people, do not wish to use conventional hypnotic drugs because of concerns about side effects and the risks of tolerance and dependence (Attele et al., 2000). In turn, there is an increasing interest in the use of herbal and dietary supplements, partly because of their natural properties and perceived absence of residual effects.

The effect of fermented milk on sleep and health perception S Yamamura et al

101 Milk and fermented milk have been widely consumed all over the world. Milk protein-derived bioactive peptides are claimed to be health-enhancing components that can be used to reduce the risk of disease or to enhance a certain physiological function (Meisel, 2004). Studies have reported in recent years that bioactive peptides have antihypertensive effects, antioxidative effects, hypocholesterolemic effects, opioid activity and anxiolytic-like stress-relieving properties (Korhonen and Pihlanto, 2003). Lactobacillus helveticus CM4 (CM4), selected from a starter culture of Calpis sour milk (Calpis, Tokyo, Japan), has very potent protease activity and can efficiently produce milk protein-derived bioactive peptides (Yamamoto et al., 2003). Because it has been reported that bioactive peptides can improve physical and mental well-being, we expected that bioactive peptides present in CM4 fermented milk might show a beneficial effect not only on quality of life but also on sleep. But research into the effect of fermented milk on sleep has not been done until now. In a pilot study carried out on healthy student volunteers by our group at Osaka University, CM4 fermented milk significantly improved sleep latency as compared with placebo (Kumano-go et al., 2006). The variability of social life conditions (for example, staying up all-night and reduced sleeping hours for club activities, examine revision and report writing, and so on) of the study population, however, posed a considerable problem. Therefore, here we have examined the beneficial effect on sleep and health perception of fermented milk ingestion in healthy elderly people, whose life rhythm is comparatively stable.

Subjects and methods Subjects A total of 30 elderly subjects participated in the study. They all attend lectures of the lifelong learning course of the Osaka Prefectural Senior Citizens Center, and are living a healthy and regular life. Subjects were screened by means of a medical questionnaire to ensure the absence of current, or past history of, serious, severe, or unstable physical disorders (for example, arthritis, asthma, chronic obstructive pulmonary disease, heart disease) or psychiatric illness (for example, major depression, generalized anxiety disorder) that could significantly affect sleep states. In addition, it was checked at the interview stage that each subject had no history of milk allergy or lactose intolerance and that there was no use of substances that influence sleep including hypnotic treatment or over-the-counter sleep aids. All female subjects had reached menopause. The purpose and the protocol were explained fully to the subjects, who all provided written informed consent to participation. This study was approved by Osaka University Healthcare Center Ethics Committee.

Trial design The trial was double-blind, randomized and placebocontrolled, with a crossover design. The trial was performed within the autumn season from September 4, to November 27, 2003. This time period in Japan is not associated with large changes in photoperiod or temperature; therefore, it is thought that seasonal changes should have little influence on human circadian rhythms. The trial was done in four periods, each lasting 3 weeks. The four periods were the baseline period, first supplementary period, washout period and second supplementary period. Subjects were randomly assigned to one of two groups, which drank either fermented milk or placebo in the first supplementary period. These drinks were alternated in the second supplementary period. During each supplementary period, subjects drank 100 g of the designated drink everyday at any time. Subjects were instructed not to take any other milk beverages or fermented foods during the study period. In addition, subjects were instructed not to change their usual diet or level of physical exercise during the study period.

Supplementary drinks The fermented milk drink was prepared as reported previously (Nakamura et al., 1995) with some modifications. Reconstituted 9% (by wt) skim milk (Yotsuba, Hokkaido, Japan) was pasteurized, inoculated with a 3% (by wt) starter culture containing L. helveticus strain CM4, and fermented at 37 1C for 24 h. Aspartame (0.05% by wt; Ajinomoto, Tokyo), 0.25% flavorings and 0.5% stabilizer were added to the fermented milk, which was homogenized at 150 kg cm2, dispensed at 100 g in glass bottles and finally pasteurized at 80 1C for 10 min. One hundred grams of this drink contained 90 g of the fermented milk. Artificially acidified milk was prepared by adding 2.25% days, L-lactic acid to skim milk to adjust the acid content to that of the fermented milk; this placebo drink was prepared in the same way as the fermented milk drink. Both drinks had a similar nutritional composition except for the carbohydrate and energy content, which differed because of the addition of lactic acid to the artificially acidified milk drink (Table 1).

Measurements Actigraphy. It has been shown that actigraphy reflects sleep awakening rhythms with high reliability (Colling et al., 2000; Ancoli-Israel et al., 2003). Throughout the present study, sleep quality was continuously assessed with a micromini type actigraphy instrument (AMI Inc., Ardsley, NY, USA) fitted to the non-dominant wrist of each subject, and initialization of actigraphy and reading of data were performed using Act Millennium. Actigraph analysis software (AW2) was used to analyze the data. The actigraph was collected on the first day of each period, when the data of previous period was read. After charging and initializing the actigraph, it was passed back to the subject within the same European Journal of Clinical Nutrition


102 Table 1 Composition of fermented milk and placebo (artificially acidified milk) drinksa Fermented milk drink

Placebo drink (artificially acidified milk drink)

2.9 3.5 0.7 ND 93.0 25.5

3.0 5.8 0.7 ND 90.6 35.0

Protein (g) Carbohydrate (g) Ash (g) Fat (g) Moisture (g) Energy (kcal)

a Composition per 100 g of supplementary drink. ND, not detectable.

day. Fundamentally, the subjects were equipped with an actigraph all day long throughout the whole study. Each subject filled in sleep diaries during the study, which were collected at the end of each period. Sleep diaries were compared with the results of the actigraph data, and were utilized to improve the accuracy of the actigraph data. The following main outcome variables were compared and analyzed: sleep latency (time from bedtime to sleep-onset time), sleep efficiency (total sleep time as a percentage of sleep-period time), wake episodes (numbers of blocks of contiguous wake epochs) and WASO (wake time after sleeponset).

Sleep health risk index All study participants completed the Sleep-Health Risk Index (SHRI) (Tanaka et al., 2001, 2004) on the final day of each period. From the questionnaire involving life habits and sleep health, five sleep-health risk factors were determined and the total score of each factor score was calculated as the SHRI, where a high score means worse sleep.

The SF-36 health survey The medical outcomes study Short Form 36 (SF-36) is a selfrated measure of functioning, health status and well-being, which has been extensively used for estimating quality of life (QOL) in the general population and in patients with various medical conditions (Ware et al., 1993). In addition, SF-36 seems to be a good instrument to assess QOL in sleep disorders (Hatoum et al., 1998). There are eight component scores (Physical Functioning, Role-Physical, Bodily Pain, General Health, Vitality, Social Functioning, Role-Emotional, and Mental Health). Each component is measured on a 0–100 scale, with a high score reflecting a better QOL. We used the Japanese version of SF-36 version 1.2 which has been shown to be reliable and valid for various patient populations (Fukuhara et al., 1998). All study participants completed this questionnaire on the final day of each 3-week period. European Journal of Clinical Nutrition

Statistics All values are expressed as means7s.e.m.’s. The effect of sequence was evaluated by using Welch’s t-test to compare the sum of first and second supplementary periods between the fermented milk first and the placebo first groups. The effect of period was evaluated by using Welch’s t-test to compare the difference between first and second periods of the fermented milk first group with the difference between second and first periods of the placebo first group. The effect of treatment was evaluated by using Welch’s t-test to compare the difference of first and second periods between the fermented milk first and placebo first groups. A paired t-test was used to compare the baseline and post-intake values of each drink. The P-value was adjusted using the Dunn–Sidak method when required for repetition of these tests. All statistical analysis were two-tailed, and statistical significance was established at Po0.05. Calculations were performed using commercially available statistical software packages, StatMate III (ATMS, Tokyo, Japan).

Results Subject characteristics and side effects The 30 subjects enrolled in the study were randomly assigned to one of two subgroups. One group of subjects took fermented milk first. The other group took placebo first. One person dropped out of the fermented milk first group during the study period because of travel to another country with a time difference of 19 h. As a result, the total number of study subjects became 29 persons. None of the subjects in either group developed any side effects. The baseline characteristics of the two groups are described in Table 2. In the value of each parameter in the baseline period, there was no significant difference between groups. An effect of sequence or period was not detected for the variables of actigraphy, SHRI and SF-36.

Actigraphy Because the data of four subjects were insufficient owing to mechanical trouble with the actigraph in the placebo first group, we analyzed in total the data of 25 persons, corresponding to 14 in the fermented milk first group and 11 in the placebo first group. The value of each actigraphy parameter in the baseline period and after intervention is shown in Table 3. Although there was no significant difference between fermented milk and placebo in all outcome variables of the actigraph, an improvement from the baseline period was observed for the fermented milk in some actigraphy parameters. Sleep efficiency had improved significantly from the baseline period in those taking the fermented milk (P ¼ 0.03), whereas the placebo had no significant effect on sleep efficiency (P ¼ 0.10). The number


103 of awakening episodes decreased significantly from the baseline period in the fermented milk group (P ¼ 0.007), whereas the placebo had no significant effect on this parameter (P ¼ 0.70). As compared with the baseline period, sleep latency and WASO became short slightly in both groups, but these changes were not significant.

Sleep health risk index In the average score of the SHRI, there was no significant difference between the baseline period and either supplementary drink, and between each drink.

Table 2 Baseline characteristics of the two groups Fermented milk first (n ¼ 14)

Placebo first (n ¼ 15)

11 3 72.1475.67 58.8977.79 160.0775.54

12 3 70.675.65 62.47712.56 159.6077.50

Actigraphy variables Sleep efficacy (%) Sleep latency (min) Wake episodes (times) WASO (min)

90.271.8 15.271.8 9.471.1 42.177.8

87.471.8 13.772.1 9.671.1 50.277.2

Sleep–health risk index SHRI

2.8470.36

2.6170.36

SF-36 scores Physical functioning Role-physical Bodily pain General health Vitality Social functioning Role-emotional Mental health

89.6472.11 76.7978.92 76.0074.12 71.8674.38 74.6474.42 89.2973.31 90.4877.09 83.7173.67

92.3372.20 90.0076.58 83.8075.07 69.8573.78 82.4473.36 92.5073.87 91.1176.64 86.4073.59

Male Female Age (years) Body weight (kg) Height (cm)

Abbreviation: WASO, wake time after sleep-onset. Data are presented as the mean7the s.e. of the mean. Groups did not differ significantly with respect to any characteristic.

The SF-36 health survey The mean scores for all SF-36 domains of the baseline period and after intervention are shown in Table 4. We did not observe a significant change between the baseline period and either supplementary drink. Although there was no significant difference between the fermented milk and placebo groups, in seven of the eight domains of the SF-36 the mean scores of fermented milk were higher than those of placebo (Figure 1).

Discussion The purpose of this study was to test the ability to improve sleep and health of CM4 fermented milk and its safety in elderly people. After the fermented milk was consumed for 3 weeks, a significant improvement from baseline in sleep efficiency and number of wake episodes was observed by actigraphy, without any side effects. By contrast, a significant improvement in these parameters was not observed after taking placebo. Taking these results together, we could conclude that fermented milk had a favorable effect on sleep efficiency and number of awakenings. It is known that sleep efficiency will become lower, and brief awakening will become common, with aging (Ohayon and Vecchierini, 2005). The same result has been also observed in an actigraphy study (Yoon et al., 2003). Thus, the possibility of improving these symptoms in the elderly, as observed in this study, is a useful finding. Unlike the actigraphy result, the improvement in SHRI was not significant. However, because the mean SHRI value of the fermented milk group was better than that of the placebo group, this result does not greatly differ from the results of actigraphy. From the results of this study, the fermented milk caused no significant improvement in QOL. But the mean scores of the SF-36 after drinking fermented milk were better than both the baseline value and the placebo value for almost all domains, and a tendency towards improvement from the baseline period in the GH domain was observed in the fermented milk group (P ¼ 0.09), but not in the placebo group (P ¼ 0.83). Thus, we might be able to consider that the

Table 3 Sleep parameters obtained by actigraphy Baseline (n ¼ 25)

Sleep efficacy (%) Sleep latency (min) Wake episodes (times) WASO (min)

Pa

After intervention Fermented milk (n ¼ 25)

Placebo (n ¼ 25)

Baseline vs fermented milk

Baseline vs placebo

91.1871.08* 12.1470.84 8.3270.62* 38.7374.58

91.3770.98 12.2770.85 8.8570.75 39.7274.11

0.03 0.26 0.007 0.14

0.10 0.37 0.70 0.35

88.9771.32 14.5671.36 9.4670.79 45.775.45

Abbreviation: WASO, wake time after sleep-onset. Data are presented as the mean7the s.e. of the mean. a The Dunn–Sidak adjustment for P-value (paired t-test). *Significantly different from baseline, Po0.05.

European Journal of Clinical Nutrition


104 Table 4 Score for all SF-36 domains in the baseline period and after intervention Baseline (n ¼ 29)

Physical functioning Role–physical Bodily pain General health Vitality Social functioning Role–emotional Mental health

Pa

After intervention Fermented milk (n ¼ 29)

Placebo (n ¼ 29)

Baseline vs fermented milk

Baseline vs placebo

91.9071.68 92.2474.74 77.5573.31 75.8372.89 81.3272.66 93.5372.17 91.9574.19 85.2471.99

91.1271.73 87.0775.40 74.7674.05 72.5973.16 77.0173.48 94.4072.41 88.5175.21 83.4572.85

0.75 0.42 0.91 0.09 0.31 0.45 0.99 0.99

0.99 0.94 0.47 0.83 0.81 0.43 0.97 0.74

91.0371.55 83.6275.62 80.0373.37 70.8272.88 78.6872.84 90.9572.58 90.8074.85 85.1072.58

Data are presented as the mean7the s.e. of the mean. a The Dunn–Sidak adjustment for P-value (paired t-test).

Figure 1 Distribution of the different subscales of the SF-36 score for each of the two study drinks. ’ ¼ Fermented milk (n ¼ 29); m ¼ placebo (n ¼ 29).

subjects’ feeling of physical and mental well-being was marginally improved by taking a supplement of fermented milk. No studies have used SF-36 to investigate the improvement in QOL resulting from a fermented milk drink, and thus it could be said that our study is useful in this regard. Overall, these findings indicate that L. helveticus fermented milk more clearly has a clinical benefit as compared with the placebo. Therefore, rather than the effects of substances contained in milk, the effects of fermentation should be considered. The reason for the effects observed in the present study remain unclear. It has been shown that L. helveticus fermented milk contains angiotensin-I-converting enzyme (ACE) inhibitory tripeptides with an antihypertensive effect (Nakamura et al., 1995). There is a report of a psychotropic effect of ACE inhibitor, and the ACE inhibitor Captopril acts European Journal of Clinical Nutrition

as antidepressant (Zubenko and Nixon, 1984), and may be associated with improvement in sleep. Moreover, there is a possibility that somnogenic Muramyl peptide in fermented milk or in the intestine may also have contributed to this result. Muramyl peptide is a cell-wall-derived substance that stimulates host defense systems, causes fever and has a somnogenic function (Krueger and Majde, 1994). As compared with medicines, however, it is thought that the effect of ACE inhibitory tripeptides is mild. Concerning Muramyl peptide, the release mechanism for this peptide is amplified greatly during infection but is thought to operate at a low basal rate under normal conditions. In addition, no subject in our study had fever through all the periods. Thus, it is considered that Muramyl peptide has little influence on this study. Overall, although all of these features have the possibility of contributing to our results, it is thought that they cannot explain the results completely. We can consider that fermentation may form an active substance that controls direct sleep and nerve activity or that fermentation may increase the quantity or absorption of such a substance. It is known that the specific health effects of fermented milk on physical and mental function are based on various bioactive peptides made during the process of the fermentation of milk proteins. The results of this study may suggest that these effects may be due to a new function of known bioactive peptides, or to the function of a previously unidentified bioactive peptide. Like other complementary medicines prescribed for anxiolysis/sedation, such a bioactive peptide may be related to GABAergic or serotonergic neurons or, like melatonin, may regulate the body’s circadian rhythm. The reason why the results were not statistically significant in the comparison between fermented milk and placebo should be interpreted cautiously. Some causes of the observed lack of significance can be considered. The selection of an appropriate placebo for studies investigating fermented milks is problematic because milk has been shown to possess some beneficial effects. Because the baseline value of the elderly subjects was already good, there might be a ceiling effect preventing a significant difference from being


105 reached between the groups. The sleep efficiency of the baseline value in this study was about 90%, which is high as compared with 77.4% in a previous study using actigraphy in healthy elderly people (Yoon et al., 2003). Furthermore, the SF-36 mean scores for our study subjects were more than 10 points higher than the published norms for the Japanese general population aged 65 years and older (Fukuhara et al., 2001). Possibly, we might have been able to observe a significant improvement between drinks if we had specified particular objectives, such as insomnia. Another cause might be that the 3-week intervention period was too short. The study period of fermented milk has varied in past studies from 2 to 4 weeks intervention (Rizkalla et al., 2000; Kullisaar et al., 2003; Marcos et al., 2004); thus, the length of the intervention period in this study should be appropriate. But we have to consider the possibility that the maximum effect of this fermented milk may not have been reached after 3 weeks of intervention. Because we assume that this fermented milk would be taken as a daily intake supplement, longterm effects could appear gradually. Finally, the small sample size in this study may reduce statistical power, which may partially explain our results.

Conclusion This study did not evaluate the potential mechanistic effects of CM4 fermented milk, as the aim was to assess the clinical potential of the probiotic. The results of this study suggest that fermented milk has the potential effect to improve the quality of sleep in the elderly. Thus, systematic investigations are now needed to establish the molecular aspects and mechanisms underlying the positive effect of this probiotic. In addition, further studies of longer duration and larger sample number are necessary to determine the potential beneficial effect of fermented milk.

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