**4. Discussion**

We conducted a double-blind placebo-controlled trial to evaluate the effects of *Euglena* intake on various properties, such as the autonomic nervous system, psychological parameters, and quality of sleep. Our results indicated that the intake of *Euglena* regulates the autonomic nervous system balance and improves various symptoms caused by stress loading. In addition, we observed no adverse events with a maximum intake of 3000 mg of *Euglena* per day, confirming that this dose is safe.

The subjects included in the study were concerned about a lack of motivation and a decline in the quality of sleep. However, the sympathetic nervous system was in a dominant state in these individuals, with a high LF/HF ratio, which is an indicator of the autonomic nervous system balance, exceeding the normal value of 2.0. Previous studies have shown that Uchida–Kraepelin stress affects heart rate variability [31–34]. We selected subjects who felt that taking the Uchida–Kraepelin test was stressful had a substantial increase in their LF/HF ratios. We found that the intake of *Euglena* at 1000 mg suppressed the LF/HF ratio compared with that in the placebo intake group. When the LF/HF ratio was broken down into the components LF and HF, a decrease in LF had a relatively large impact upon 500 mg and 1000 mg Euglena intake. Therefore, *Euglena* may have suppressed the excitation of the sympathetic nervous system. The intake of *Euglena* significantly suppressed the LF/HF ratios in individuals subjected to work stress and appeared to regulate the autonomic nervous system balance. The most effective dose was 1000 mg.

Irritability and tension during work stress were maintained at high levels even 60 min after exposure to work stress in the placebo intake group; however, these parameters were suppressed in the *Euglena* intake groups of 500 mg or more and were significantly suppressed in the 1000 mg intake group, indicating an improvement in the psychological state. In other words, the intake of *Euglena* may have alleviated the tension caused by Uchida–Kraepelin stress loading.

In a POMS2 analysis, which evaluates emotions, the score for friendship (F) was significantly higher in the group taking 500 mg of *Euglena* per day at weeks 4, 8, and 12 after starting intake, both before and after Uchida–Kraepelin stress loading. This suggests that the continuous intake of 500 mg *Euglena* can enhance friendliness. However, this did not occur upon taking *Euglena* at doses other than 500 mg. In SF-36, which evaluates health-related quality of life, a significant difference was observed in daily physical function (role physical), which represents physical fatigue.

Improvement of sleep quality with the intake of *Euglena* was confirmed based on three sleep tests: VAS, OSA, and PSQI. These effects increased in a dose- and time-dependent manners. The parasympathetic nervous system is enhanced at the onset of sleep [35]; accordingly, the improvement in sleep quality by *Euglena* intake was likely due to an improvement in the balance of the autonomic nervous system.

*Euglena* intake also increased the correct answer rate on the Uchida–Kraepelin test. These findings sugges<sup>t</sup> that the continuous intake of *Euglena* may improve concentration and work efficiency.

The autonomic nervous system includes the sympathetic nervous system and the parasympathetic nervous system. It regulates various functions, such as respiration/circulation, digestion/absorption, secretion, and metabolism, and serves an important role in the maintenance of homeostasis in the body. However, when stress signals are transmitted to the hypothalamus in the brain, the autonomic nervous system is excited, depending on the type of stress. An imbalance in the autonomic nervous system due to excessive stress may prevent the proper activation of the sympathetic or parasympathetic nervous system. These conditions may cause excessive irritability, an inability to concentrate, deterioration of sleep quality, weakened immunity, and various ailments, including problems with bowel movements. For example, patients with chronic fatigue syndrome caused by stress experience a decrease in parasympathetic nervous system function and a relative increase in sympathetic nervous system function, depending on the degree of subjective fatigue evaluated by the VAS. This shows that there is a relationship between fatigue and the autonomic nervous system [36]. Therefore, it is important to maintain a well-balanced autonomic nervous system. In this study, the intake of *Euglena* improved the autonomic nervous system balance, thereby improving irritability and tension as well as the quality of daily sleep. Previous studies have evaluated improvements in autonomic nervous system activity after nutritional supplementation in healthy subjects [37–40]. When stress is transmitted to the hypothalamus in the brain, a signal is sent to excite the autonomic nervous system, depending on the type of stress, and a command is simultaneously sent to the pituitary gland to stimulate the thyroid, adrenal medulla, and adrenal cortex via the release of hormones. In addition, signals from the hypothalamus stimulate the adrenal cortex in response to stress; the pituitary gland secretes the adrenocorticotrophic hormone, adrenocorticotropic hormone (ACTH), and the adrenal cortex secretes the hormone cortisol. In this way, the hypothalamus, which controls the autonomic nervous system, also controls the pituitary gland, which secretes various hormones and neurotransmitters. Therefore, a mutual relationship exists whereby an imbalance in the autonomic nervous system a ffects the secretion of hormones and neurotransmitters, thereby a ffecting the balance of the autonomic nervous system. Proteins, B vitamins, and minerals, such as magnesium, are required for hormones and neurotransmitters. A lack of vitamin B6 makes it di fficult for the body to produce serotonin and melatonin, leading to insomnia and autonomic nervous system ataxia. The overconsumption of zinc is related to stress, and a shortage of vitamin B makes it di fficult to produce energy in the mitochondria, which increases fatigue and malaise. *Euglena* consist of a wide variety of nutrients, such as vitamins, minerals, amino acids, and unsaturated fatty acids. The ingestion of *Euglena* assists in replenishing these nutrients in the body, which not only improves autonomic nervous system activity but also normalizes the secretion of hormones and neurotransmitters, contributing to the regulation of the autonomic nervous system balance. In this study, we found no changes in cortisol levels, which suggests that hormone levels are being a ffected rather than cortisol levels and that the stress of Uchida–Kraepelin may not a ffect cortisol production [41]; this warrants further investigation.

Meanwhile, previous studies have used in vitro cell-type-specific calcium imaging to prove that *Euglena* directly induces Ca2+ signaling in dorsal root ganglia (DRG) neurons, suggesting that *Euglena* excites visceral a fferents [42]. Although the mechanism underlying the beneficial e ffects of *Euglena* in our study remains unclear, its ability to excite neurons may contribute to improvements in health-related quality of life [12]. Further studies are needed to better clarify the physiological role of visceral a fferents in response to food constituents.

The storage polysaccharide paramylon in *Euglena* regulates the immune balance. Previous studies of mouse models have confirmed that the ingestion of *Euglena* or paramylon alleviates the symptoms of influenza infection [11], rheumatoid arthritis [43], and atopic dermatitis [44]. The autonomic nervous system is involved in the regulation of immunity, and immunity is generally enhanced in situations where the parasympathetic nervous system is dominant, contributing to defense against infection [16,17]. Cortisol also suppresses the immune system [45]. Previous studies have investigated the ability of paramylon to regulate immunity by binding to Dectin-1 expressed on intestinal immune cells [46,47]; however, it could also mediate immunity by the regulation of the autonomic nervous system and endocrine system.

The results of this study sugges<sup>t</sup> that the intake of *Euglena*-containing food may regulate the autonomic nervous system balance under stress and may have favorable e ffects on work e fficiency, psychological factors, and sleep quality. However, this study had some limitations. First, although this study was conducted with a small homogeneous cohort of individuals from one population, future studies are required to further elucidate these aspects with various ethnicities and people with di fferent backgrounds and stress levels. Furthermore, although some of the e ffects were dependent on intake dose, other e ffects were observed for which an optimal dose may exist independent of intake, thus requiring further investigation. In the future, we would like to further investigate the mechanism underlying these beneficial e ffects as well.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/2072-6643/12/11/ 3243/s1, Table S1: Summary of blood test results. Table S2: Summary of blood pressure and pulse rate test results.

**Author Contributions:** Conceptualization, A.N., K.Y., and N.M.; methodology, A.N. and K.Y.; formal analysis, A.N., K.Y., and A.M.; resources, A.N., K.Y., A.M., and K.S.; original draft preparation and writing, A.N.; review and editing, A.N., K.Y., A.M., K.S., and N.M.; supervision, K.S. and N.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding. **Acknowledgments:** The authors greatly appreciate the useful suggestions and technical support from Oneness support Co. Ltd.

**Conflicts of Interest:** A.N., K.Y., A.M., and K.S. are salaried employees of euglena Co. Ltd., which produced some of the *Euglena* used in this study. All research funding for this study was provided by euglena Co. Ltd. N.M. is the medical advisor of this study, and received honorarium from euglena Co. Ltd. There are no other conflicts of interest.
