Effect of Chlorella-derived multicomponent supplementation on maximal oxygen uptake and serum vitamin B2 concentration in young men

Chlorella is a unicellular green alga that contains high levels of proteins, vitamins and minerals. The present study investigated the effects of a 4-week Chlorella-derived multicomponent supplementation on maximal oxygen uptake and circulating vitamin B2 levels in healthy men. Thirty-four participants were randomly divided into two groups: placebo or Chlorella. Prior to the intervention, we observed that the intake of several minerals and soluble vitamins did not satisfy the nutrient requirements of either group by assessing the frequency of daily food intake. There was a significant negative relationship between the pre-intervention maximal oxygen uptake and serum vitamin B2 concentrations in all subjects (r = −0.372). Maximal oxygen uptake significantly increased after Chlorella supplementation (before vs after, 42.1 ± 1.5 vs 44.9 ± 1.6 ml/kg/min), while serum vitamin B2 concentrations did not (14.6 ± 0.9 vs 14.0 ± 0.9 µg/L). In conclusion, Chlorella-derived multicomponent supplementation increases maximal oxygen uptake in individuals with an insufficient micronutrient status, although there was no association between the increase in aerobic capacity and serum levels of vitamin B2.

Introduction A well-balanced diet is one of the most important factors for being fit and leading a healthy life. In the last few decades, dietary social problems, including skipping breakfast, eating meals irregularly and consuming fast food, have become increasingly common and have led to malnutrition in young individuals. (1)(2)(3) In particular, university students living away from home are at a high risk of poor nutrition. (4) The National Health and Nutrition Examination Survey by the Ministry of Health, Labour and Welfare of Japan reported that many young Japanese men have poor dietary habits, including skipping breakfast or having a low vegetable intake, as compared with women and/or other generations. In particular, the average vegetable intake among Japanese men aged 20-29 years in 2014 was 237 g/day, despite the recommended intake being 350 g/day; only 19% of young Japanese men achieved the recommended level. Fujii et al. (5) reported that Japanese college students who regularly skip breakfast also tend to have a poor vegetable intake. Such dietary problems are not unique to Japan, but also occur throughout the world. (1,2,6) As a solution to such dietary problems in young individuals, dietary supplements may be effective at overcoming nutrient deficiencies by providing a better nutrient balance.
As a result of an evidence-based analysis, the Academy of Nutrition and Dietetics, Dietitians of Canada and American College of Sports Medicine have stated that well-balanced nutrition is necessary for maximal exercise performance. Exercise training increases micronutrient requirements, and a daily multivitamin and multimineral supplement would benefit athletes who have a poor micronutrient status. (7) In our previous cross-over study, a Chlorella-derived multicomponent supplementation increased the aerobic exercise capacity of healthy young individuals. (8) However, our previous study did not explore the nutritional status of the participants or the mechanisms underlying the increase in aerobic exercise capacity.
Chlorella is a unicellular green alga that contains high levels of proteins, chlorophylls, vitamins, minerals and dietary fiber. A lack of vitamin B 1 , B 2 , B 6 and C for 10 weeks decreased the peak oxygen uptake in young individuals. (9) In addition, vitamin B 2 intake is an important factor regarding endurance exercise capacity. (10) Therefore, we hypothesized that Chlorella-derived supplementation would increase maximal oxygen uptake (VO 2 max) by improving the levels of vitamin B 2 and other micronutrients in individuals with an insufficient micronutrient status. To test this hypothesis, we assessed daily food intake frequency and examined the effects of a 4-week Chlorella-derived supplementation on the VO 2 max and circulating vitamin B 2 levels in healthy young men using a double-blinded study design.

Materials and Methods
Subjects and experimental design. A total of 34 young men volunteered to participate in this study. After pre-supplementation tests, the subjects were randomly divided into the placebo (n = 17) and Chlorella (n = 17) groups in a double-blinded manner. During the trial, the participants self-administered 30 tablets of either Chlorella or the placebo per day (15 tablets twice daily, after breakfast and dinner) for 4 weeks. This dosage was in accordance with our previous studies (8,11,12) and the general recommended dosage for Japanese consumers. The participants were asked not to modify their lifestyle during the trial period. Post-supplementation measurements and blood sampling were performed one day after the final tablet intake.
This study was approved by the Ethics Committee of Ryutsu Keizai University. The protocol of this study conformed to the principles of the Declaration of Helsinki. All participants provided written informed consent prior to study initiation.
A power calculation was performed for Pearson's correlation coefficients in all participants and for repeated measures two-way analysis of variance (ANOVA) using G*Power 3. (13) The sample size of this study was enough to detect the correlation and effects of supplementation at 90% power and with an α of 5% when effect A size was assumed as medium. (14) The Chlorella (SunChlorella A; Sun Chlorella Corp., Kyoto, Japan) and placebo tablets used in this study were the same as those used in our previous studies. (8,11,12) Briefly, the Chlorella tablet comprised dried Chlorella pyrenoidosa powder as the main ingredient. The mass of each Chlorella tablet was 200 mg. The nutritional values per 100 g of the tablet were as follows: energy, 399 kcal; water, 5.3 g; protein, 60.8 g; lipid, 9.2 g; saccharide, 6.3 g; dietary fiber, 11.9 g; and ash, 6.5 g (including 5.61 mg of vitamin B 2 ). The main ingredient of the placebo tablet was lactose. The color and shape of the placebo tablet were similar to that of the Chlorella tablet, but the protein (2.0 g), dietary fiber (1.1 g) and ash (2.2 g) contents in the placebo tablet were markedly lower than those in the Chlorella tablet.
Maximal exercise testing. Subjects refrained from alcohol consumption starting on the day before testing, intense physical activity and caffeine consumption starting at 9 p.m. on the day before testing, and drinking and eating excluding water for 1 h before the testing.
Maximal exercise testing involved incremental cycling to exhaustion (4 min at 70 W, with a 30 W increase every min), with monitoring of breath-by-breath oxygen uptake and carbon dioxide production (AE300S; Minato Medical Science, Osaka, Japan), heart rate (HR) calculation using 3-lead electrocardiography (LRR-03; GMS, Tokyo, Japan) and ratings of perceived exertion (RPE, Borg's 6-20 scale). We considered a participant as having achieved maximal exertion when at least two of the following four criteria were met: 1) a plateau in oxygen uptake with increasing exercise intensity (<100 ml/min); 2) achievement of age-predicted maximal HR (±10 bpm); 3) a respiratory exchange ratio of at least 1.15; and 4) an RPE of at least 18 units. (8) In our laboratory, the day-to-day coefficient of variation for VO 2 max was 4.7 ± 4.0%.
Serum vitamin B 2 analysis. Serum vitamin B 2 concentration was measured using a microbiological test. The subjects refrained from alcohol consumption from the day before testing. Blood samples were collected from the antecubital vein by venipuncture after an overnight fast, placed in chilled tubes and centrifuged at 3,000 rpm for 15 min at 4°C. The plasma was stored at −80°C prior to analysis. Serum concentrations of vitamin B 2 were determined using a commercial kit including a microtiter plate coated with Lactobacillus rhamnosus, according to the manufacturer's protocol (ID-Vit ® Vitamin B 2 ; Immundiagnostik AG, Bensheim, Germany).
Dietary intake. Dietary habits were assessed using a validated, brief, self-administered dietary history questionnaire (BDHQ). (15) The BDHQ is a four-page structured questionnaire that enquires about the consumption frequency of 58 food and beverage items, with specified serving sizes described in terms of the natural portion or the standard weight and volume measurement of servings commonly consumed in the general Japanese population. Validation of the BDHQ was performed using 16-day weighed dietary records as the gold standard. (15) With regard to protein, calcium, magnesium, iron, zinc, copper, retinol, vitamin B 1 , vitamin B 2 , niacin, vitamin B 6 , vitamin B 12 and vitamin C, the intake amounts are expressed as the ratio to the recommended dietary amount (RDA) of the Dietary Reference Intake for Japanese 2015. Sodium intake is expressed as the ratio to the estimated average requirement (EAR), as the RDA of sodium was not established in the Dietary Reference Intake. For nutrients without a RDA or an EAR (n-6 fatty acid, n-3 fatty acid, potassium, phosphorus, manganese, vitamin D, vitamin K, αtocopherol and pantothenic acid), the sufficiency rates were estimated based on the adequate intake (AI). The RDA and EAR indicate the amount that would meet the requirement for 97-98 and 50% of the population, respectively. The AI was developed when EAR and RDA could not be set because of insufficient scientific evidence and indicates the amount of nutrients required to maintain a healthy status.
Body mass and fat mass. The subjects refrained from alcohol consumption from the day before testing. Body mass was measured and body fat mass was assessed by bioelectrical impedance analysis after an overnight fast (InBody 430; InBody Japan, Tokyo, Japan).
Statistical analysis. Results are presented as the mean ± SE. Inter-group comparisons for patient variables before supplementation were performed using unpaired t tests. Relationships between two variables were assessed using Pearson's correlation coefficient. To compare the effects of Chlorella tablet intake with placebo supplementation, repeated-measures two-way ANOVA was used. If a significant F value was found, a post-hoc Fisher's exact test was conducted to identify the effects of placebo and Chlorella tablet intake. P values <0.05 were considered statistically significant. Table 1 shows the anthropometric characteristics of the participants. There were no significant differences in the premeasurement data between the Chlorella and placebo groups. Moreover, the height, body mass and body fat mass of both groups did not change significantly after the 4-week intervention. Table 2 shows dietary habits of the two groups before the intervention and the nutrient intakes including Chlorella supplementation. There were no differences in the daily nutrient intake between the groups before the tablet intake period. Several nutrients did not satisfy the nutrient demand (i.e., RDAs, EARs or AIs) in both groups, as follows: potassium, calcium, magnesium, zinc, manganese, vitamin A, vitamin B 1 , vitamin B 2 , vitamin B 6 and vitamin C. On the other hand, sodium was above the nutrient demand in both groups. The energy intake was lower than the demand in both groups, but the intake of protein and fat was higher than the demand. The sufficiency rate of iron, vitamin B 2 , D and K, and niacin increased by ≥20% with Chlorella supplementation.

Results
There was a significant negative relationship between the VO 2 max and serum vitamin B 2 concentration in all participants before the intervention (Fig. 1). The VO 2 max prior to and following the intervention is shown in Fig. 2. There was a significant interaction in the changes between the Chlorella and control groups. The VO 2 max significantly increased following Chlorelladerived supplementation. Changes in vitamin B 2 concentration before and after the intervention are shown in Fig. 3; there was no significant interaction between the Chlorella and control groups. Additionally, the relationship between the change in the VO 2 max and that of the serum vitamin B 2 levels did not reach statistical significance in the Chlorella group (r = −0.311, p = 0.22).

Discussion
We investigated the effects of a 4-week Chlorella-derived multicomponent supplementation on the VO 2 max and circulating vitamin B 2 levels in healthy young men. Prior to the intervention, the daily intake of calcium, magnesium, vitamin B 1 , vitamin B 2 , vitamin B 6 and vitamin C did not satisfy nutrient demands in both groups. However, vitamin B 2 intake was markedly increased in the Chlorella group following supplementation. There was a significant negative relationship between the pre-intervention VO 2 max value and the serum levels of vitamin B 2 in all subjects.   In addition, in the Chlorella group, the VO 2 max was significantly increased following the supplementation, and serum vitamin B 2 levels showed a decreasing trend. Therefore, we concluded that Chlorella-derived supplementation increases the VO 2 max in individuals with an insufficient micronutrient status, although there was no association between the increased VO 2 max and blood levels of vitamin B 2 . In a previous crossover study, we demonstrated that Chlorelladerived supplementation increased the VO 2 max in young men (n = 7) and women (n = 3). (8) In this study, we investigated the effects of a 4-week Chlorella-derived supplementation on aerobic exercise capacity in 34 young men, thus increasing the number of participants and eliminating any confounding effects associated with the female menstrual cycle. In addition, the current study assessed the daily food intake frequency in order to clarify the participants' dietary habits. This study supports the results of the previous study, (8) as a 4-week Chlorella-derived supplementation significantly increased the VO 2 max in young men with inadequate dietary habits.
In the present cross-sectional analysis of pre-supplementation values, we showed for the first time that there was a significant negative correlation between the VO 2 max and serum vitamin B 2 concentrations. These results suggest that individuals with a higher aerobic capacity metabolize more vitamin B 2 than do those with a lower aerobic capacity. Previously, Belko et al. (16,17) reported that various lifestyle modifications, including dietary modification and/or regular exercise, would lead to increased vitamin B 2 demands. Moreover, Manore (18) demonstrated that considerably more vitamin B 2 was required when dieting was combined with exercise. It is possible that the lower circulating levels of vitamin B 2 in individuals with a higher aerobic capacity were due to the higher constitutive usage of vitamin B 2 .
Before the intervention, the sufficiency rate of vitamin B 2 was 67% in the Chlorella group. However, after the Chlorella-derived supplementation, this rate was increased to 86%. No other nutrients demonstrated such a dramatic increase in the sufficiency rate. Therefore, this study subsequently investigated serum vitamin B 2 concentrations. Vitamin B 2 is necessary for the synthesis of two important coenzymes, flavin mononucleotide and flavin adenine dinucleotide. These coenzymes are especially important for the metabolism of glucose, fatty acids, glycerol and amino acids. In a previous study, reduced intake of vitamins B 1 , B 2 , B 6 and C was associated with a decreased aerobic exercise capacity in young men. (9) In addition, Peluchetti et al. (10) demonstrated that vitamin B 2 supplementation dramatically increased aerobic exercise capacity (before: 10.5-14.4 ml/kg/min; after: 30.5-39.3 ml/kg/min) in patients with multiple acyl-coenzyme A dehydrogenase deficiency. These results suggest that vitamin B 2 and other soluble vitamins are important factors associated with the VO 2 max. However, the present study did not show a significant change in circulating vitamin B 2 levels following Chlorella supplementation. Dietary modifications and/or regular exercise increase the vitamin B 2 demand, (16)(17)(18) and the circulating level of vitamin B 2 was lower in participants with a higher aerobic capacity in the present study. Therefore, the additional vitamin B 2 supplied by the Chlorella-derived tablet and the increased vitamin B 2 demand due to an increase in aerobic capacity may have counteracted each other, resulting in no change in the vitamin B 2 concentration.
The National Health and Nutrition Examination Survey by the Ministry of Health, Labour and Welfare of Japan has reported that vegetable intake in Japan has declined in recent years across all generations, and the decline has been particularly pronounced in men. Indeed, some vitamins and minerals (i.e., potassium, calcium, magnesium, zinc, manganese, retinol, vitamin B 1 , vitamin B 2 , vitamin B 6 and vitamin C) were deficient in the participants of the present study. The Academy of Nutrition and Dietetics, Dietitians of Canada and American College of Sports Medicine (7) have suggested that calcium, iron, zinc, magnesium, vitamin B, vitamin C and vitamin E are important nutrients for athletes. The Chlorella-derived supplement used in the present study contained many vitamins and minerals (e.g., vitamin A, B vitamins, vitamin C, vitamin E, calcium, folate, iron, pantothenic acid, potassium, magnesium and zinc). In particular, it should be noted that the sufficiency rate of vitamin B 2 was essentially improved following self-administration of Chlorella-derived tablets.
It is unclear whether vitamin B 2 directly affects aerobic capacity. Future investigations should focus on whether the bioavailability of vitamin B 2 is associated with the observed increase in the VO 2 max following Chlorella-derived tablet intake. Since B vitamins are water-soluble, changes in blood and urine vitamin B 2 concentrations should be examined.
In conclusion, we found a significant negative relationship between the VO 2 max and serum vitamin B 2 concentrations before the intervention. In addition, we observed that a 4-week Chlorelladerived multicomponent supplementation increased the VO 2 max in male university students with insufficient dietary habits, although there was no association between the increase in the VO 2 max and circulating levels of vitamin B 2 .

AI
adequate intake ANOVA analysis of variance BDHQ brief, self-administered dietary history questionnaire EAR estimated average requirement HR heart rate RDA recommended dietary amount RPE ratings of perceived exertion VO 2 max maximal oxygen uptake

Conflict of Interest
Sun Chlorella Corp. provided funding for the study and supplied the test supplements used in the study. TO has received speaker's honorarium and payment for manuscripts not involving this study from Sun Chlorella Corp. AZ-M and SM have no competing interests.