**Olfactory Stimulation with Volatile Aroma Compounds of Basil (***Ocimum basilicum* **L.) Essential Oil and Linalool Ameliorates White Fat Accumulation and Dyslipidemia in Chronically Stressed Rats**

**Da-Som Kim 1,†, Seong-Jun Hong 2,†, Sojeong Yoon 2, Seong-Min Jo 2, Hyangyeon Jeong 2, Moon-Yeon Youn 1, Young-Jun Kim 3, Jae-Kyeom Kim <sup>4</sup> and Eui-Cheol Shin 1,2,5,\***


**Abstract:** We explored the physiological effects of inhaling basil essential oil (BEO) and/or linalool and identified odor-active aroma compounds in BEO using gas chromatography/mass spectrometry (GC–MS) and GC–olfactometry (GC–O). Linalool was identified as the major volatile compound in BEO. Three groups of rats were administered BEO and linalool via inhalation, while rats in the control group were not. Inhalation of BEO for 20 min only reduced the total weight gain (190.67 ± 2.52 g) and increased the forced swimming time (47.33 ± 14.84 s) compared with the control group (219.67 ± 2.08 g, 8.33 ± 5.13 s). Inhalation of BEO for 5 min (392 ± 21 beats/min) only reduced the pulse compared with the control group (420 ± 19 beats/min). Inhalation of linalool only reduced the weight of white adipose tissue (5.75 ± 0.61 g). The levels of stress-related hormones were not significantly different among the groups. The total cholesterol and triglyceride levels decreased after inhalation of BEO for 20 min (by more than −10% and −15%, respectively). Low-density lipoprotein cholesterol levels were lowered (by more than −10%) by the inhalation of BEO and linalool, regardless of the inhalation time. In particular, BEO inhalation for 20 min was associated with the lowest level of low-density lipoprotein cholesterol (53.94 ± 2.72 mg/dL). High-density lipoprotein cholesterol levels increased after inhalation of BEO (by more than +15%). The atherogenic index and cardiac risk factors were suppressed by BEO inhalation. Animals exposed to BEO and linalool had no significant differences in hepatotoxicity. These data suggest that the inhalation of BEO and linalool may ameliorate cardiovascular and lipid dysfunctions. These effects should be explored further for clinical applications.

**Keywords:** *Ocimum basilicum* L.; essential oil; volatile compounds; linalool; stress lipid metabolism

#### **1. Introduction**

Stress is classified as either acute or chronic and can influence the physiological regulation of hormones and inflammatory cytokine secretion through several pathways, involving psychological, social, physical, and chemical factors [1–3]. Chronic stress usually disturbs the autonomic nervous system (ANS), which maintains internal homeostasis responding to changes in the external environment and controls the metabolism of substances in the

**Citation:** Kim, D.-S.; Hong, S.-J.; Yoon, S.; Jo, S.-M.; Jeong, H.; Youn, M.-Y.; Kim, Y.-J.; Kim, J.-K.; Shin, E.-C. Olfactory Stimulation with Volatile Aroma Compounds of Basil (*Ocimum basilicum* L.) Essential Oil and Linalool Ameliorates White Fat Accumulation and Dyslipidemia in Chronically Stressed Rats. *Nutrients* **2022**, *14*, 1822. https://doi.org/ 10.3390/nu14091822

Academic Editors: Daniela Rigano and Paola Bontempo

Received: 8 April 2022 Accepted: 26 April 2022 Published: 27 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

body. Furthermore, the ANS generally regulates the sympathetic and parasympathetic systems, and thus, chronic stress can interfere with the activation of the sympathetic and parasympathetic systems [4]. The deterioration of the ANS usually increases blood pressure, pulse, total cholesterol level, and low-density lipoprotein cholesterol (LDL) levels and decreases high-density lipoprotein cholesterol (HDL) levels. Accordingly, pathologies of the ANS can induce a deterioration in cardiovascular health, leading to hypertension and arteriosclerosis [4,5]. Therefore, researchers have attempted to improve the cholesterol levels and prevent the progression of cardiovascular diseases using natural products with physiological effects [6].

Basil (*Ocimum basilicum Licorice; O. basilicum* L.) is a member of the *Lamiaceae* family. The leaf and stem parts, are used as culinary ingredients and/or as medicinal herbs [7]. Additionally, basil contains a unique fragrance that has been used in the perfume industry. Furthermore, the intake of basil has beneficial effects on the cholesterol level; the intake of basil improves lipid metabolism in high-cholesterol-affected animal models [7]. In addition, orally administered linalool, one of the major compounds in basil, improves cholesterol levels and, when administered by inhalation, induces sedative and relaxing effects [6,8]. Generally, the volatile profiles of essential oil are affected by many factors, such as the geographical area of sampling [9], the variety of/accession to the plants [10], the harvest year [11], the harvest date [12], the extraction system [13], so on. Therefore, the major aroma compound (linalool) of basil is mainly affected by geographical conditions and the harvesting periods, and the concentration of linalool increases according to the flowering periods [14]. In addition, linalool concentration is also affected by the extraction method. In particular, the hydro-distillation extraction method (18.1%) yields a higher concentration of linalool than supercritical fluid extraction (12.6%) [15].

When fragrant products are inhaled, individual fragrance compounds bind to nasal olfactory receptors, and a signal is transmitted to the cerebrum. When a volatile compound is inhaled, it dissolves in the mucus of the nasal mucous membrane and moves to the olfactory epithelium. Subsequently, volatile compounds bind to the olfactory receptors of cilia. Olfactory receptors bind only to certain volatile compounds, and the generated electrical signal reaches the olfactory bulb in the frontal lobe via axons. Therefore, information on individual volatile compounds is delivered to the olfactory cortex and the cerebrum. Individual volatile compounds can be distinguished and recognized according to this signaling pathway [16]. Olfactory stimulation influences the central nervous system (CNS) and ANS activities; thus, olfactory stimulation can control the function of the sympathetic and parasympathetic nervous systems. The involved nerves generally influence energy and lipid metabolism; thus, food intake and cholesterol levels can be controlled by the sympathetic and parasympathetic nervous systems [5,17].

Improvements in lipid metabolism in vivo by the intake of basil and sedative and anti-stress effects of linalool contained in basil have been reported [6–8]. However, the ameliorating effects of inhaling volatile compounds (present in basil) on dyslipidemia caused by chronic stress have not been elucidated. Accordingly, this study observed changes in lipid parameters, stress hormone levels, pulse, body weight, and food intake after inhalation of basil essential oil (BEO) in chronically stressed rats. Furthermore, changes in metabolic parameters following linalool inhalation were observed.

#### **2. Materials and Methods**

#### *2.1. Essential Oil*

The basil used in this study was cultivated in Austria in 2017, and the essential oil was extracted by the distillation method using the leaves (100%). The BEO used in this study was a commercial product, purchased from the Aroma Care Solution (Helga-Stolz GmbH Co., Grafenwoerth, Austria) and stored at 4 ◦C in a dark place until experiments were performed. The grade of this product was for aroma therapy. The experiments in the present study were conducted in 2018–2020.

#### *2.2. Odor-Active Aroma Compounds*

Odor-active aroma compounds (OAACs) in BEO were collected using solid-phase microextraction (SPME) fibers (Supelco Co., Bellafonte, PA, USA), i.e., fibers coated with 100 μm of polydimethylsiloxane (1 cm in size). BEO (1 g) was placed in a glass vial tightly sealed with an aluminum cap. The OAACs were collected in the headspace while heating the sample to 50 ◦C. The SPME fibers were injected into the injector of a gas chromatography–mass spectrometry selective detector (GC–MS; Agilent 7890A & 5975C, Agilent Technologies, Santa Clara, CA, USA) at 220 ◦C, and the analysis was performed after desorption for 10 min. The column was HP-5MS (30 m (length) × 0.25 mm (inner diameter), 0.25 μm (film thickness)), and helium carrier gas was used at 1 mL/min, with a split ratio of 1:10. The initial oven temperature was set at 40 ◦C for 5 min, increased to 200 ◦C at a rate of 5 ◦C/min, and maintained for 10 min. An inlet temperature of 220 ◦C was set in the splitless mode. OAACs, separated by a total ionization chromatogram, were identified using the National Institute of Standards and Technology (NIST) mass spectral library (NIST version 12). Pentadecane (0.005 μg) was used as an internal standard. According to the peak area and concentration of the internal standard, the concentrations of the OAACs in BEO were expressed in μg/mL. To explore the odor-active characteristics of BEO, the volatile profiles were separated by the GC column and assessed using a GC–olfactometry port (GC-O) (ODP 3, Gerstel Co., Linthicum, MD, USA). Odor-active intensity was divided into four levels, with higher levels representing stronger odor-active intensity, as described previously [5].

#### *2.3. Animal Care and Experimental Design*

This study was approved by the Animal Experimental Ethics Committee (Animal protocol #: IACUC-4). Forty-five male Sprague–Dawley rats (4 weeks old) were obtained from Coretec Co., (Busan, Korea). The rats were acclimated to a normal diet for a week and randomly classified into four groups. After classification, chronic stress was applied to all groups for five weeks in total. Chronic mild stress was applied in the first week. Chronic mild stress (CMS) is a complex stress that includes food deprivation, restricted access to food, water deprivation, roommate separation, overnight illumination, and tilting the cage by 45◦. From the second week, the rats were exposed to chronic stress with distilled water (DW) inhalation for 5 min/day in the control group (CON; *n* = 6), chronic stress with linalool inhalation for 5 min/day in the positive control group (POS; *n* = 6), chronic stress with BEO inhalation for 5 min/day in the third group (5 MIN; *n* = 6), and chronic stress with BEO inhalation for 20 min/day in the fourth group (20 MIN; *n* = 6) (Figure 1). Linalool and volatile compounds in BEO flowed at a rate of 8 mL/h, achieved by using a humidifier (Aroma diffuser humidifier; Cactus Co., Shanghai, China).

**Figure 1.** The plan of the animal study showing a week of preliminary breeding, a week of chronic mild stress (CMS), and four weeks of CMS + inhalation + behavior testing.

Food intake and body weight were measured once weekly. The rats were fasted for 16 h before dissection. Blood was collected from the heart using syringes containing 20 mg of ethylenediaminetetraacetic acid. The collected blood samples were kept for 30 min, then centrifuged at 1000 G to separate the serum. Finally, organs and tissues (the liver, kidneys, heart, white adipose tissue, and brown adipose tissue) were extracted and weighed. In addition, the organs and tissues were stored in a −80 ◦C freezer [5].

#### *2.4. Forced Swimming Test*

The forced swimming test was performed weekly with a standard behavioral despair test. Water (25 ◦C) was placed in a chamber (40 × 25 × 26.5 cm) at a height of 16 cm. During each experiment, the animals were placed in the chamber and allowed to swim (mobility). Immobility was assessed after swimming. Immobility was defined as when the animals stood upright and floated without movement, exposing only the head [18].

#### *2.5. Pulse*

The animal's pulse was measured using the tail-cuff method with BP-2000 (Visitech Systems Co., Apex, NC, USA). Eight measurements were taken, excluding the highest and lowest values and deviations. Finally, three measured values were expressed as the average and standard deviation (SD) [5].

#### *2.6. Stress Hormones*

Cortisol (450 nm) in the serum was analyzed using an ELISA kit (YH ELISA Kit, Shanghai Yehua Biological Technology Co., Shanghai, China), and serotonin (450 nm) in brain tissue was analyzed using another ELISA kit (Serotonin ELISA Kit, Bio Vision Co., Milpitas, CA, USA) by absorbance measurement according to the manufacturer's instructions [19,20].

#### *2.7. Analyses of Serum Biomarkers and Hepatotoxicity*

Total cholesterol (500 nm), HDL (500 nm), triglyceride (TG) (550 nm), and hepatotoxicity, including aspartate transaminase (AST) (505 nm) and alanine transaminase (ALT) (505 nm), were analyzed using a commercial kit (Asan Reagents, Asan Pharm Co., Seoul, Korea) by absorbance measurement according to the manufacturer's instructions [5].

#### *2.8. Statistical Analysis*

Experiments were performed in triplicate, and the results are presented as the average and SD. Non-parametric comparison was used to compare paired groups using the Friedman test with chi-square distribution. Differences were considered statistically significant at *p*-values less than 0.05 (SAS Institute Inc., Cary, NC, USA).

#### **3. Results and Discussion**

#### *3.1. Odor-Active Aroma Compounds*

Odor-active aroma profiles were detected using GC–MS and GC–O (Table 1 and Figure 2). A total of 17 aroma compounds were detected in BEO. In particular, four OAACs were identified, including linalool, linalool oxide, menthane, and carvone. Linalool elicits basil essential oil odor activation, and linalool oxide elicits the activation of grass and herb odors. Menthane also elicits herb odor and menthol activation. In addition, carvone elicits lemon odor activation.

Linalool had the highest concentration of OAAC in BEO (Table 1). Linalool is a common and major terpenoid, containing most herbal essential oils and has been identified as a forest-like odor using GC–O [5,21]. In addition, linalool can control the lipid metabolism in vivo [21]. Linalool oxide has shown anxiolytic-like effects in mouse anxiety models via inhalation [22]. Menthene is a hydrocarbon with colorless characteristics and an herb odor [23]. D-Carvone has anti-inflammatory and anti-microbial effects, and this volatile compound was identified as OAAC in essential oils by GC–O [5,24]. In general, the genus *Ocimum* includes approximately 150 species distributed worldwide, and different volatile profiles are characteristic of *Ocimum* species, as reported by a previous study. Importantly, high concentrations of linalool were detected in O. *basilicum* L. (25.6%) and O. *sanctum* L. (21.9%) but not in O. *gratissimum* L. (0.1%) and O. *kilimandscharicum* L. (1.4%) [25].


**Table 1.** Aroma profiles and odor-active aroma compounds in basil essential oil identified using GC–MS and GC–O.

**Figure 2.** Representative aromagram of odor-active aroma compounds (OAACs) in basil (*Ocimum basilicum* L.) essential oil identified by GC–MS and GC–O test.

#### *3.2. Total Food Intake and Total Weight Gain*

Total food intake in the BEO-inhaled and linalool-inhaled group was much lower compared to that in the control group (*p* > 0.05) (Table 2); however, there were no significant differences among all groups. In the case of total weight gain, the 20 min BEO-inhaled group showed significant less weight gain compared to the control group (*p* < 0.05); however, the 5 min BEO-inhaled group and the linalool-inhaled group did not show any significant differences compared to the control group.

**Table 2.** Total food intake and total weight gain during the animal experiment. CON: chronic stressexposed control group; POS: linalool (positive control) inhalation by chronic stress-exposed rats; 5 MIN: BEO inhalation for 5 min by chronic stress-exposed rats; 20 MIN: BEO inhalation for 20 min by chronic stress-exposed rats.


Data are given as mean ± SD values from experiments performed in triplicate. <sup>1</sup> Mean values with different letters within the same row are significantly different according to the non-parametric Friedman test, followed by Dunn's test (*p* < 0.05).

Basil can modulate body weight, and linalool plays an important role as a ligand of peroxisome proliferator-activated receptor α (PPAR*α*) [7,21]. PPAR*α* can modulate fatty acid uptake and fatty acid oxidation and inhibit the occurrence of obesity. Linalool is commonly used for medicinal functions [21]. This study showed that 20 min of BEO inhalation suppressed total weight gain. In contrast, linalool inhalation did not result in decreased body weight. Previous research reported that BEO induced a decrease in body weight [26] and reduced the average body weight [27]. A linalool-containing essential oil has antiobesity effects, including decreasing body weight and/or promoting lipolysis [5,28]. In addition, Baek et al. reported that linalool inhibits body weight gain [6]. However, linalool inhalation only suppressed the average body weight gain in this study. Therefore, the reduction in body weight could be due to the complex effects of the aroma components of BEO, rather than the sole effect of linalool.

#### *3.3. Forced Swimming Test*

Changes in swimming records were measured during the study period (Table 3). During the initial period, no significant differences were observed between the groups. In the final period, the control group had the lowest swimming time among all groups (*p* < 0.05), while the other groups showed increasing swimming time. The BEO-inhaled groups showed increased swimming time in an inhalation time-dependent manner. When comparing the BEO- and the linalool-inhaled groups, the 20 min BEO-inhaled group showed a significant increase in swimming time compared to the control group (*p* < 0.05).

**Table 3.** Forced swimming test during the initial and final periods. CON: chronic stress-exposed control group; POS: linalool (positive control) inhalation with chronic stress-exposed rats; 5 MIN: BEO inhalation for 5 min by chronic stress-exposed rats; 20 MIN: BEO inhalation for 20 min by chronic stress-exposed rats.


Data are given as mean ± SD values from experiments performed in triplicate. <sup>1</sup> Mean values with different letters within the same row are significantly different according to the non-parametric Friedman test, followed by Dunn's test (*p* < 0.05).

Chronic stress can cause oxidative stress, and animals exposed to oxidative stress have an increased immobility period during forced swimming tests [29,30]. In addition, the period of immobility in rats is decreased by reducing oxidative stress [30]. The results of this study also identified differences between stress-exposed rats and stress-relieved rats following the inhalation of BEO and linalool. A previous study indicated that linalool inhalation upregulated plasma biomarkers and gene expression in rat models of stress [31], while another study indicated that BEO ameliorated oxidative stress in rats [32]. In addition, BEO significantly increased the ambulatory activity via the stimulation of the CNS, and this BEO is considered a potent CNS regulator [33].

#### *3.4. Pulse*

During the initial period, no significant differences in pulse were observed between the groups (Table 4). In contrast, during the final measurement, inhalation of BEO for 5 min attenuated the pulse rate compared to that in the control group (*p* < 0.05). Inhalation of BEO for 20 min and of linalool only showed a tendency to decrease the pulse, and changes were not significant (*p* > 0.05).


**Table 4.** Pulse assessment using the tail-cuff method in rats.

Data are given as mean ± SD values from experiments performed in triplicate. <sup>1</sup> Mean values with different letters within the same row are significantly different according to the non-parametric Friedman test, followed by Dunn's test (*p* < 0.05).

The pulse is controlled by the ANS, which includes sympathetic and parasympathetic nerves, and a reduced pulse is associated with decreased sympathetic and increased parasympathetic nerve activity [4]. In this study, inhalation of BEO and linalool resulted in significantly and/or relatively decreased pulse rates. Previous studies indicated that linalool and linalool-containing essential oils attenuated renal sympathetic nerve activity and enhanced parasympathetic nerve activity by olfactory stimulation, and linaloolcontaining essential oil inhalation decreased the pulse rates in rats [28,33]. In addition, inhalation of linalool has a sedative effect in animal models [8], and BEO inhalation also induced a sedative effect by decreasing the arousal response measured on the basis of electroencephalographic activity [34].

#### *3.5. Organ Weights*

The liver, kidney, heart, white adipose tissue (WAT), and brown adipose tissue (BAT) were weighed (Table 5). There were no significant differences in liver weights among the groups. The kidney weights in the 20 min BEO-inhaled group was lower than that of the control group (*p* < 0.05). Meanwhile, inhalation of BEO for 5 min and of linalool induced no significant decrease in liver weight. In terms of heart weight, there were no significant differences between the groups. The control group had the highest WAT weight among all groups. The BEO-inhaled groups showed a decreasing tendency in WAT weights; however, these changes were not significantly different. The linalool-inhaled group showed a decrease in WAT weight compared with the control group (*p* < 0.05). BAT weights were measured in all groups, and there were no significant differences.

WAT is related to oxidative stress, and increased WAT and oxidative stress can increase the metabolic risk [29]. Accumulation of WAT generally increases cardiovascular disorders, being associated with increased levels of TC, LDL, and TG, as well as decreased levels of HDL [17]. This study showed that inhalation of BEO and linalool decreased WAT weight. Linalool inhalation significantly decreased WAT weight compared with the control group (*p* < 0.05). Therefore, the reduction in WAT appeared to occur in a linalool concentrationdependent manner. A previous study reported that linalool reduced WAT weight in mice [6], while another study found that linalool induced lipolysis by upregulating PPAR*α* activity, fatty acid oxidation, and energy metabolism [21]. In addition, linalool treatment significantly reduced lipid accumulation in 3T3-L1 cells [35]. Moreover, research has found decreased fat accumulation following linalool odor stimulation [36].


**Table 5.** Changes in rat organ weights.

Data are given as mean ± SD values from experiments performed in triplicate. <sup>1</sup> Mean values with different letters within the same row are significantly different according to the non-parametric Friedman test, followed by Dunn's test (*p* < 0.05).

#### *3.6. Stress Hormones*

Stress hormones, including cortisol and serotonin, were measured using ELISA kits. Cortisol levels in the control group were the highest among all groups (Table 6). Inhalation of BEO and linalool decreased the cortisol levels; however, these decreases were not statistically significant. The levels of serotonin in the control group were the lowest among all groups. Inhalation of BEO and linalool induced an increase of serotonin; however, these increases showed no significant differences.

**Table 6.** Changes in stress hormones in rats.


Data are given as mean ± SD values from experiments performed in triplicate. <sup>1</sup> Mean values with different letters within the same row are significantly different according to the non-parametric Friedman test, followed by Dunn's test (*p* < 0.05).

#### *3.7. Serum Biomarkers and Hepatotoxicity Indicators*

Serum biomarkers were measured using a commercial kit. TC in the 20 min BEOinhaled group was lower than in the control group (*p* < 0.05) (Table 7). However, there were no significant differences when comparing the control, 5 min BEO-inhaled, and linaloolinhaled groups. In the case of HDL levels, the control and linalool-inhaled groups had the lowest levels compared with the BEO-inhaled groups, regardless of the BEO inhalation time (*p* < 0.05). Thus, BEO inhalation upregulated the HDL levels. The control group had the highest LDL levels among all groups (*p* < 0.05). BEO inhalation ameliorated the levels of LDL, and linalool ameliorated the LDL levels compared to the control group (*p* < 0.05). The TG level in the control group was relatively higher than in the other groups (*p* > 0.05). BEO and linalool inhalation were associated with a decreasing tendency of TG levels. Inhalation of BEO for 20 min showed decreased the TG levels compared to the control group (*p* < 0.05). Meanwhile, linalool was associated with a decreasing trend in TG levels compared to the control group; however, there were no significant changes between the control and the linalool-inhaled group. Regarding the atherogenic index (AI) and cardiac risk factors (CRF) in the control group, inhalation of BEO ameliorated the AI and CRF indices in a time-dependent manner (*p* < 0.05). Inhalation of linalool induced no significant effects on AI or CRF. Inhalation of BEO and linalool significantly decreased (*p* < 0.05) the levels of LHR when compared with the control group. In particular, BEO inhalation for 20 min showed the lowest LHR among all the groups (*p* < 0.05). Hepatotoxicity indicators, including AST and ALT, were measured using a commercial kit. When comparing all groups, the AST and ALT levels did not show any significant differences (Table 8). In addition, the AST/ALT ratio was not significantly different among the groups.

**Table 7.** Effects of basil essential oil and linalool inhalation on lipid metabolism in chronically stressed rats. CON: chronic stress-exposed control group; POS: linalool (positive control) inhalation by chronic stress-exposed rats; 5 MIN: BEO inhalation for 5 min by chronic stress-exposed rats; 20 MIN: BEO inhalation for 20 min by chronic stress-exposed rats.


Data are given as mean ± SD values from experiments performed in triplicate. <sup>1</sup> Mean values with different letters within the same row are significantly different according to the non-parametric Friedman test, followed by Dunn's test (*p* < 0.05).

**Table 8.** Effects of basil essential oil and linalool inhalation on hepatotoxicity in chronically stressed rats. CON: chronic stress-exposed control group; POS: linalool (positive control) inhalation with chronic stress-exposed rats; 5 MIN: BEO inhalation for 5 min b chronic stress-exposed rats; 20 MIN: BEO inhalation for 20 min by chronic stress-exposed rats.


Data are given as mean ± SD values from experiments performed in triplicate. <sup>1</sup> Mean values with different letters within the same row are significantly different according to the non-parametric Friedman test, followed by Dunn's test (*p* < 0.05).

Oxidative stress influences the lipid metabolism [37] and usually increases the prevalence of atherosclerosis by increasing reactive oxygen species, nitric oxygen, and oxidized-LDL production and decreasing the levels of antioxidants [38]. LDL is generally associated with the weight of WAT, and increased levels of LDL can promote cardiovascular diseases such as atherosclerosis and dyslipidemia [5]. In contrast, HDL is associated with antiinflammatory indicators and the presence of antioxidants [5]. Accordingly, HDL plays an important role in cardiovascular health and can reduce the prevalence rate of cardiovascular diseases [39,40]. The results of this study showed that linalool and BEO inhalation decreased the LDL levels in chronically stressed rats. In addition, BEO inhalation ameliorated the levels of HDL in chronically stressed rats, regardless of its concentration. Therefore, BEO and linalool inhalation upregulated the LDL and HDL levels in this study. In particular, the AI and CRF were reduced by BEO inhalation regardless of its concentration (Table 7). A recent study reported that the administration of BEO decreased the levels of TC, LDL, and TG [7]. Similarly, the administration of purple basil essential oil improved hyperlipidemia, lowering triglyceride and total cholesterol levels, similar to our results [31]. Additionally, linalool has cholesterol-lowering, antioxidant, and anti-inflammatory activities [41]. Previous research showed that linalool decreased the LDL levels [6] and activated hepatic PPAR*α* [21]. Therefore, linalool ameliorated dyslipidemia by lowering LDL activity [21]. BEO, a linalool-containing essential oil, has been reported to reduce hyperlipidemia and oxidative stress in rats [31]. Therefore, linalool inhalation played an important role in improving the LDL levels in this study.

The AST and ALT levels are associated with hepatic damage. The AST/ALT ratio is an indicator of liver function impairment [42]. In this study, there were no significant differences in the levels of AST and ALT and in the AST/ALT ratios among all groups. Therefore, inhalation of BEO and linalool had no adverse effects on hepatic and liver function.

#### **4. Conclusions**

In conclusion, these findings suggest that BEO and linalool inhalation suppresses stress responses, including dyslipidemia. Nevertheless, these findings are limited to animal models of chronic stress. Therefore, further research should be performed to investigate the effects of the inhalation of BEO and linalool in clinical trials. Furthermore, these findings can be of interest in the industry field and suggest the use of odor-active aroma compounds in BEO and linalool to suppress stress, without the intake and/or oral administration of health-promoting compounds.

**Author Contributions:** Formal Analysis and Writing-Original Draft Preparation, D.-S.K. and S.-J.H.; Formal Analysis and Writing-Original Draft Preparation, S.Y.; Data Curation, S.-M.J. and H.J.; Writing-Review & Editing, M.-Y.Y., Y.-J.K. and J.-K.K.; Supervision, Project Administration, and Funding Acquisition, E.-C.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded by the Basic Science Research Program, through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2018R1D1A1B07045431).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Anna Oue 1,\*, Yasuhiro Iimura 2, Akiho Shinagawa 2, Yuichi Miyakoshi <sup>1</sup> and Masako Ota <sup>1</sup>**

<sup>1</sup> Faculty of Food and Nutritional Sciences, Toyo University, Gunma 374-0193, Japan

<sup>2</sup> Graduate School of Food and Nutritional Sciences, Toyo University, Gunma 374-0193, Japan

**\*** Correspondence: oue@toyo.jp; Tel.: +81-276-82-9145; Fax: +81-276-82-9033

**Abstract:** The purpose of this study was to test the hypothesis that acute intake of inorganic nitrate (NO3 −) via supplementation would attenuate the venoconstriction and pressor response to exercise. Sixteen healthy young adults were assigned in a randomized crossover design to receive beetroot juice (BRJ) or an NO3 −-depleted control beverage (prune juice: CON). Two hours after consuming the allocated beverage, participants rested in the supine position. Following the baseline period of 4 min, static handgrip exercise of the left hand was performed at 30% of the maximal voluntary contraction for 2 min. Mean arterial pressure (MAP) and heart rate (HR) were measured. Changes in venous volume in the right forearm and right calf were also measured using venous occlusion plethysmography while cuffs on the upper arm and thigh were inflated constantly to 30–40 mmHg. The plasma NO3 − concentration was elevated with BRJ intake (*p* < 0.05). Exercise increased MAP and HR and decreased venous volume in the forearm and calf, but there were no differences between CON and BRJ. Thus, these findings suggest that acute BRJ intake does not alter the sympathetic venoconstriction in the non-exercising limbs and MAP response to exercise in healthy young adults, despite the enhanced activity of nitric oxide.

**Keywords:** beetroot juice; exercise; nitric oxide; sympathoexcitation; venoconstriction

**Citation:** Oue, A.; Iimura, Y.; Shinagawa, A.; Miyakoshi, Y.; Ota, M. Effect of Acute Dietary Nitrate Supplementation on the Venous Vascular Response to Static Exercise in Healthy Young Adults. *Nutrients* **2022**, *14*, 4464. https://doi.org/ 10.3390/nu14214464

Academic Editor: Daniela Rigano

Received: 10 October 2022 Accepted: 21 October 2022 Published: 24 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Dietary nitrate (NO3 −) supplementation with beetroot juice (BRJ) reduces resting blood pressure (BP) in normotensive and hypertensive populations [1–6]. The hypotensive effect of BRJ is likely to be due to both peripheral and central factors. The peripheral factor underlying the hypotensive effect might be related to the vasodilatory impact of the increase in nitric oxide (NO) bioavailability induced by the stepwise reduction of dietary nitrate (NO3 −) to nitrite (NO2 −) and subsequently to NO (i.e., the NO3 <sup>−</sup> → NO2 − → NO pathway) [4,7,8]. In addition to the role of NO as a vasodilator, the increase in NO availability related to the NO3 <sup>−</sup> → NO2 <sup>−</sup> → NO pathway may also alter efferent sympathetic outflow. Indeed, Notay et al. [9] recently reported that acute dietary NO3 − supplementation with BRJ decreased muscle sympathetic nerve activity (MSNA) at rest and blunted the MSNA response to sympathoexcitation via static handgrip exercise in young adults.

Veins have high distensibility and contain approximately 60–70% of the total blood volume at rest [10]. Venous tone is controlled by the sympathetic nervous system and changes with physiological stress (e.g., exercise), thereby leading to alterations in venous volume and/or compliance and contributing to the control of circulatory responses (e.g., BP and cardiac output) [10]. For example, venoconstriction and/or decreased venous compliance occurs sympathetically in the non-exercising limb during exercise [11–16]. In addition to sympathetic control, NO is also an important signaling molecule that contributes to the modulation of venous tone [17,18]. Moreover, in our recent study [19], it is suggested that the increased bioavailability of vasodilator NO associated with dietary NO3 − supplementation with BRJ could contribute to the control of the peripheral vascular tone in

not only arteries but also veins under resting conditions, which may perhaps, in part, be attributable to the hypotensive effect of BRJ. However, the effect of BRJ intake on the venous vascular response to exercise-induced sympathoexcitation has not been investigated. Aging and physiological inactivity might cause stiffness of the veins [20,21], which could be a factor in the pathogenesis of hypertension [22]. Considering these findings, it is also important to understand the hypotensive effect of BRJ from the perspective of the venous vascular system.

Therefore, the purpose of this study was to investigate the effect of acute dietary NO3 − supplementation with BRJ on venous vascular and circulatory responses to static handgrip exercise in humans. Because the scientific evidence indicates that the NO3 <sup>−</sup> → NO2 <sup>−</sup> → NO pathway-related increase in NO bioavailability in response to acute dietary NO3 − supplementation with BRJ causes vasodilation [4,7,8] and attenuates the MSNA response to static handgrip exercise [9], we hypothesized that acute intake of BRJ would attenuate the venoconstriction and BP response to exercise.

#### **2. Materials and Methods**

#### *2.1. Participants*

Sixteen healthy individuals (10 men, 6 women, 22.3 ± 1.4 years, 167.4 ± 9.3 cm, 64.7 ± 13.9 kg) volunteered for this study. None of the women were using oral contraceptives and all were in the self-reported follicular phase (3–10 days after the onset of menstruation) during the experiments. This study was approved by the Human Ethics Committee of Toyo University (TU2019-018-TU2020-H-019) and was conducted in accordance with the Declaration of Helsinki. The purpose, procedure, and risks of the study were explained to the participants, and their written and verbal informed consent was obtained. Throughout the study, the participants were instructed to avoid vigorous exercise, caffeine, and alcohol for 24 h before each visit. For 3 days prior to the main experimental protocol and blood sampling protocol, participants were asked to refrain from high NO3 − foods (e.g., green leafy vegetables and traditional Japanese foods) [23,24]. In addition, because oral bacteria are involved in reducing NO3 − to NO2 − in vivo [25], the participants were asked to abstain from using mouthwash.

#### *2.2. Experimental Design*

Participants visited the laboratory on five occasions. During the first visit, they performed maximal voluntary contraction (MVC) of the left hand using a handgrip dynamometer to determine their 30% MVC. In addition, they were familiarized with the experimental procedure and equipment. Main experiments were carried out at the second and third visits, and blood samplings were performed at the fourth and fifth visits. For the main experiments and blood samplings, participants were assigned in a randomized crossover design to consume BRJ (Beet It®; James White Drinks, Ipswich, UK; 140 mL/day, containing ~8 mmol NO3 −) or placebo control beverage (CON) consisting of prune juice (Sunsweet®; POKKA SAPPORO Food & Beverage Ltd., Nagoya, Japan; 166 mL/day; <0.01 mmol NO3 −). We selected prune juice as the placebo beverage because the NO3 − in prune juice is at a negligible level and the carbohydrate and fiber contents of prune juice are similar to those of BRJ [26,27]. The amount (166 mL/day) of prune juice was calculated to match the energy contained in 140 mL/day of BRJ. A washout period of at least 7 days separated each supplementation period for both the main experiments and the blood sampling.

#### *2.3. Protocol of the Main Experiment*

After arrival at the laboratory, participants were instructed to consume BRJ or CON. Two hours later, all participants rested in the supine position for 20 min before data acquisition in an air-conditioned room (26.4 ◦C ± 0.5 ◦C). The main experiment comprised two protocols: (1) measurement of the changes in venous volume in the non-exercising limbs during the static handgrip exercise, and (2) measurement of the circulatory parameters

during static handgrip exercise. In protocol 1, following the pre-exercise baseline period for 4 min, the static handgrip exercise of the left hand was performed at 30% MVC for 2 min. The cuffs on the right wrist and the right ankle were inflated to 200–220 mmHg at the same time as the start of the baseline period, and the cuffs on the right upper arm and the right thigh were inflated to 30–40 mmHg from the first minute of the baseline period. All cuff inflations were maintained until the end of the exercise. Throughout protocol 1, the changes in venous volume in the non-exercising right forearm and right calf were measured using venous occlusion plethysmography (Hokanson, EC6; D. E. Hokanson, Bellevue, WA, USA). In protocol 2 as well as in protocol 1, static handgrip exercise of the left hand at 30% MVC for 2 min was performed following the 4-min baseline period. Throughout protocol 2, the circulatory parameters were measured in the right middle finger using Finapres NOVA (Finapres Medical Systems BV, Enschede, The Netherlands). Protocol 1 and protocol 2 were performed in random order, and rest periods of at least 20 min were allowed between protocols 1 and 2. In addition, for all protocols, participants controlled their respiratory rate at 10 or 15 breaths per minute, guided by a metronome.

#### *2.4. Blood Sampling Protocol*

To measure the plasma NO3 − concentration, venous blood samples were drawn from an antecubital vein in a seated position before and 2 h after BRJ or CON consumption on a different day from the main experimental protocols.

#### *2.5. Measurements*

Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) were measured noninvasively and continuously from the right middle finger using Finapres NOVA, which was calibrated with the right upper arm cuff and height adjustment. In addition, heart rate (HR) was determined from the BP waveform using the Modelflow software program (Finapres Medical Systems BV, Enschede, The Netherlands).

To assess venoconstriction in the non-exercising forearm and calf during static handgrip exercise of the left hand, the changes in volume in the right forearm and right calf were measured. Inflatable cuffs were wrapped around the right wrist, right upper arm, right ankle, and right thigh, and strain gauges were placed on the sites of maximal thickness in the forearm and calf. Throughout the protocol, the wrist and ankle cuffs were inflated to 200–220 mmHg to arrest the blood circulation of the hand and foot, which have arteriovenous anastomoses, because we wanted to investigate the venoconstriction in the forearm and calf. One minute after the cuff inflation of the wrist and ankle, the cuffs of the upper arm and thigh were inflated to 30–40 mmHg for 3 min, and the volume in the forearm and calf increased until it approached an asymptote, and then static handgrip exercise of the left hand was performed for 2 min. Throughout the protocol (total time, 6 min), the change in volume in the right forearm and the right calf was measured using venous occlusion plethysmography. This method was adopted because when the intravascular pressure of the conduit vein in the limb is maintained at a constant level, the decrease in the limb volume reflects the venoconstriction or the elevated venous vascular tone [15,16]. In addition, the cuff inflation pressure can be considered equivalent to the intravascular pressure of the conduit vein in the limb [28].

Venous blood samples were immediately mixed with EDTA and centrifuged at 3000× *g* rpm for 10 min. Plasma was placed in microcentrifuge tubes and frozen for the subsequent analysis of plasma NO3 − and NO2 − concentrations [29]. The NO3 − in a sample is reduced by a cadmium column to NO2 −, which reacts with a Griess reagent to form a purple azo dye. The NO3 − in a sample needs no reaction and thus reacts with the Griess reagent when the cadmium column is bypassed, as well as when it is used. The dye was developed in a 60 ◦C water bath, the sample was cooled by a 0 ◦C water bath, and its absorbance at 546 nm was detected using a flow-through UV-Vis spectrophotometer (V-750, JASCO Corporation, Tokyo, Japan). Because the plasma NO2 − levels were very low and

below the quantifiable limit (1 μM) in all participants, only NO3 − concentration data are presented in this study.

#### *2.6. Data Analysis and Statistics*

Data are expressed as the mean ± standard deviation. A priori sample size calculation estimated a required sample of 12 participants, assuming a change in venous compliance of 0.031 ± 0.027 mL/dL of tissue/mmHg [30] in a crossover trial with an assigned α of 0.05 and β of 0.2. Pre-exercise baseline values of SBP, DBP, MAP, and HR were defined as the mean value obtained from 1 to 3 min of the baseline period. In addition, pre-exercise baseline values of the forearm and calf venous volume were defined as the average of the last 10 s of the 3-min cuff inflation to 30–40 mmHg. The SBP, DBP, MAP, HR, and limb venous volume values during exercise at 2 min were obtained as the average of the last 1 min of exercise. Relative increases in SBP, DBP, MAP, and HR and relative decreases in venous volume in the forearm and calf with exercise at 2 min from the pre-exercise baseline were calculated. The relative decrease in venous volume was used as the index of venoconstriction.

To compare the changes in SBP, DBP, MAP, HR, and forearm venous volume and calf venous volume with exercise between CON and BRJ, a two-way analysis of variance (ANOVA) with repeated measurements (condition × time) was applied. If the main effect of condition (CON and BRJ), that of time (pre-exercise and during exercise), and/or an interaction effect were detected, post hoc analysis using a paired *t*-test was performed. To compare the plasma NO3 − concentration before and after BRJ or CON supplementation and the plasma NO3 − concentration between BRJ and CON, a paired *t*-test was used. In addition, we calculated the differences in the absolute MAP at 2 min of exercise between CON and BRJ, in the MAP elevation with exercise from pre-exercise (pressor response to exercise) between CON and BRJ, and in the relative decreases in venous volume in the limbs with exercise between BRJ and CON. Using these values, Spearman's rank correlation coefficients were calculated to examine the relationships between venoconstriction and MAP responses to exercise with acute ingestion of BRJ. Statistical significance was set at *p* < 0.05. All statistical analyses were performed using SPSS version 27 (IBM Corp., Armonk, NY, USA).

#### **3. Results**

Figure 1 shows the plasma NO3 − concentration before and after intake of CON and BRJ. The plasma NO3 − concentration increased in all participants after BRJ ingestion (before, 15 ± 6 μM; after, 574 ± 120 μM; *p* < 0.05). However, there was no significant change in the plasma NO3 <sup>−</sup> concentration after CON ingestion (before, 14 ± 3 μM; after, 14 ± 3 μM). Furthermore, there was a significant difference in the plasma NO3 − concentration between CON and BRJ after their intake (*p* < 0.05).

Static handgrip exercise under both CON and BRJ conditions caused similar increases in SBP, DBP, MAP, and HR (Table 1). ANOVA indicated a significant time effect (all *p* < 0.01), and post hoc testing revealed significant differences in SBP, DBP, MAP, and HR between pre-exercise and during exercise (all *p* < 0.05). However, SBP, DBP, MAP, and HR at preexercise or during exercise were similar for CON and BRJ. In addition, the degrees of increases in these parameters during exercise from pre-exercise did not differ between CON and BRJ (Figure 2A–D). In contrast, static handgrip exercise under both CON and BRJ conditions induced similar decreases in venous volume in the non-exercising forearm and calf (Table 1). ANOVA indicated a significant time effect (all *p* < 0.01), and post hoc testing showed significant differences in these parameters between pre-exercise and during static handgrip exercise (all *p* < 0.05). This result meant that sympathetic venoconstriction was obtained during exercise. However, the venous volumes in the forearm and calf preexercise or during exercise were similar for CON and BRJ. In addition, the degrees of the decreases in the venous volume (venoconstriction) in the forearm and calf during exercise from pre-exercise did not differ between CON and BRJ (Figure 2E,F)

**Figure 1.** Plasma nitrate concentration before and 2h after CON and BRJ consumption. Data are shown as the mean ± standard deviation. CON: NO3 −-depleted prune juice; BRJ: beetroot juice. \* *p* < 0.05, significant difference between before and after. † *p* < 0.05, significant difference between CON and BRJ. Data are expressed as values for n = 16 (10 men and 6 women).

**Table 1.** Changes in circulatory parameters and venous volume in non-exercising limbs with static handgrip exercise in CON and BRJ.


Values are mean ± standard deviation. SBP: systolic blood pressure, DBP: diastolic blood pressure, MAP: mean arterial pressure, HR: heart rate. \* *p* < 0.05, significant difference between pre-exercise and during exercise at 2 min. Data are expressed as values for n = 16 (10 men, 6 women).

In the individual data, the difference in the decrease in forearm venous volume (venoconstriction) during exercise between BRJ and CON had no significant relationship with the MAP response to exercise (Figure 3A,B). In contrast, a smaller venoconstriction in the calf during exercise with BRJ rather than CON was significantly associated with a lower MAP elevation with exercise with BRJ but not with absolute MAP at 2 min of exercise (Figure 3C,D).

**Figure 2.** Relative changes in circulatory parameters and venous volume in the non-exercising limbs with static handgrip exercise in CON and BRJ. Percentage increases in the systolic blood pressure (SBP; (**A**)), diastolic blood pressure (DBP; (**B**)), mean arterial pressure (MAP; (**C**)), and heart rate (HR, (**D**)) and percentage decreases in venous volume in the forearm (**E**) and calf (**F**) during exercise from pre-exercise. Data are shown as the mean ± standard deviation. CON: NO3 −-depleted prune juice; BRJ: beetroot juice. Data are expressed as values for n = 16 (10 men and 6 women).

**Figure 3.** Relationship between the mean arterial pressure response and venous vascular response to exercise. Changes in the absolute mean arterial pressure (MAP) at 2 min of exercise (**A**,**C**) and elevated MAP with exercise (**B**,**D**) from BRJ to CON were plotted against changes in decreased venous volume in the non-exercising forearm and calf with exercise from BRJ to CON. Associations were determined with the use of a Spearman's rank correlation coefficient assessment. Data are expressed as values for n = 16 (10 men and 6 women).

#### **4. Discussion**

The new findings in our study are (1) that the increases in SBP, DBP, MAP, and HR and decreases in the venous volume in the non-exercising forearm and calf during static handgrip exercise did not differ between CON and BRJ, despite the elevated plasma NO3 − concentration after BRJ ingestion, and (2) that, in the individual data, a greater attenuation in the decrease in calf venous volume with exercise after BRJ ingestion was significantly associated with a lower MAP elevation with exercise after BRJ ingestion but not with the absolute MAP at 2 min of exercise. These results suggest that the enhanced NO bioavailability induced by BRJ supplementation does not alter sympathetic venoconstriction in non-exercising limbs or the pressor response to exercise in healthy young adults. In addition, although BRJ does not cause a decrease in venoconstriction in all subjects, when the attenuation of venoconstriction in the calf during static handgrip exercise after BRJ ingestion is obtained, the degree of the MAP pressor response to exercise could be reduced to a degree dependent on the attenuation of venoconstriction.

In the present study, the venous volume in the non-exercising forearm and calf decreased with static handgrip exercise under both CON and BRJ conditions, and these decreases in the venous volume did not differ between the two groups (Table 1 and Figure 2E,F), despite the increased plasma NO3 − concentration after BRJ intake. These results suggest that the NO bioavailability increase induced by acute BRJ ingestion does not attenuate the venoconstriction in the non-exercising limbs during exercise, which does not support our hypothesis. This is the first study to investigate the effect of BRJ on the venous vascular response to exercise. Some studies have investigated the effect of NO on the arterial vascular response. For example, sympathetic vasoconstriction in human and animal preparations is inhibited by NO derived from both endothelial nitric oxide

synthase [31] and neuronal nitric oxide synthase [31]. In contrast, several studies have reported that the NO bioavailability associated with dietary NO3 − supplementation (e.g., BRJ) does not alter sympathetic vasoconstrictor responsiveness to exercise [32,33]. Despite differences between arterial and venous vessels and between active and inactive limbs in the previous and present studies, our findings and those of the other studies suggest that exogenous NO augmentation via the NO3 <sup>−</sup> → NO2 <sup>−</sup> → NO pathway after acute BRJ consumption might not affect sympathetic vasoconstriction in either the artery or vein during exercise.

As mentioned above, we found no effect of BRJ on the venous vascular response in the non-exercising limb to exercise. Although we have no conclusive explanation, there are several possibilities for this result. First, in our study, the exogenous production of NO by BRJ was perhaps observed significantly because of increased NO3 <sup>−</sup> by 574 ± 120 μM2h after intake of BRJ, although it may not contribute to the attenuation of venous vascular tone (relaxation of smooth muscle) during exercise for the following reason. Our study participants were healthy young adults who were expected to have normal endothelial function. This meant that the endogenous NO production may have been sufficient to control the venous vascular tone. Indeed, in previous studies investigating the effect of BRJ on arterial endothelial function, flow-mediated dilation (FMD) was improved in participants with impaired endothelial function, including older adults [27,34], overweight or obese men [35], and patients with hypertension [2], but the FMD was very slightly increased [7] or unchanged [4,36] in healthy humans. Second, the inhibition of efferent sympathetic nerve activity induced by the NO increase with BRJ intake might not perhaps be observed in our study because plasma catecholamines during exercise have been reported to not be altered by acute intake of BRJ when compared with placebo [37]. Because the available scientific evidence is limited, we need to further investigate the effects of vasodilator action and inhibition of sympathetic nerve activity induced by the BRJ supplementation-related increase in NO on the venous vascular responses to exercise.

SBP, DBP, and MAP at pre-exercise and during exercise did not differ between CON and BRJ (Table 1). In addition, the degrees of the increases in these parameters with exercise were also similar for CON and BRJ (Figure 2A–D). Consistent with the present findings, previous studies have also reported no effects of acute dietary NO3 − supplementation on resting BP [38,39] and BP during exercise [9,37] in young normotensive individuals. In contrast, some studies reported a reduction in BP following acute dietary NO3 − supplementation with BRJ [1,3,4,40,41]. Similarly, there is no consensus on the hypotensive effect of BRJ. Some possibilities may be considered as to why the effect of dietary NO3 − supplementation on BP differed among studies. First, the individual response to dietary NO3 − supplementation varies widely [41,42]. Second, eating habits might differ among participants. For example, "traditional" foods found in a Japanese diet appear to be high in NO3 − [24]. Because chronic intake of a NO3 −-rich diet might mitigate the ability of dietary NO3 − supplementation to increase the levels of biomarkers of NO synthesis [43], chronic exposure to a NO3 −-rich Japanese diet may have contributed to the lack of a significant effect on BP, even though the participants were requested to refrain from consuming these foods throughout the study period. Finally, aerobic fitness levels might have varied among participants because NO synthase has been reported to be increased by physical activity [44].

Interestingly, in the individual data, a smaller decrease in calf venous volume during exercise with BRJ intake rather than CON was significantly related to a lower MAP elevation with exercise under BRJ conditions (Figure 3D). In other words, if the venoconstriction in the calf during exercise is attenuated by BRJ, the MAP pressor response to exercise may also be lower and in line with the degree of venoconstriction attenuation. In our previous study, in the individual data, a greater increased venous compliance with BRJ tended to be associated with a lower resting BP under BRJ conditions [19]. Based on the present and previous findings, the mechanism for the hypotensive effect of BRJ might be partly attributed to the control of venous vascular tone. Elevated venous stiffness appears to at least somewhat be a factor in the pathogenesis of hypertension [22]. Thus, it is very important to understand the influence of nutritional component(s) with hypotensive effects on venous vascular control, and our present results may boost the development of interventions to improve and maintain vascular health.

This study has several limitations. First, the venous vessel is modulated by an active factor that indicates activation of the sympathetic nerve [12,14–16] and by a passive factor that shows the change in volume-flow dependence [45]. The decrease in limb volume has been reported to reflect the venoconstriction or the elevation of venous tone when the intravascular pressure of the large vein in the limb is maintained at a constant level [15,16]. In addition, the cuff inflation pressure can be considered equivalent to the intravascular pressure of the large vein in the limb [28]. Considering these findings, we believe that the decreases in venous volume in the forearm and calf under cuff pressure of a constant subdiastolic BP of 30–40 mmHg in the present study were caused by sympathetic activation so that this decreased venous volume in limbs reflects the venoconstriction. Second, we did not quantify NO2 − concentrations. This is important because the conversion of NO3 − to NO2 − is necessary for biological effects to occur [4]. However, because there is evidence that a significant increase in the plasma NO3 − level is accompanied by an increase in NO2 − concentration in healthy young adults [40,46,47], we believe that the plasma NO2 − concentration was also elevated after consumption of BRJ in our study. Finally, because the small number of limited participants might have introduced a degree of sampling bias, several variables can interfere with the results and conclusions inferred. Thus, our results cannot be generalized, and the conclusion of our study is applicable to a limited extent to the healthy young adults who participated in this study.

#### **5. Conclusions**

In this study, we investigated the effect of acute dietary NO3 − supplementation with BRJ on the venous vascular response and circulatory responses to exercise. Our findings suggest that acute BRJ supplementation leads to an increase in the plasma NO3 − concentration but does not change venoconstriction in the non-exercising limbs or the MAP response to static exercise in young healthy adults. In addition, although BRJ does not cause a decrease in venoconstriction in all subjects, when venoconstriction during exercise is attenuated after BRJ ingestion, the MAP pressor response to exercise may be reduced to a degree dependent on the attenuation of venoconstriction.

**Author Contributions:** Conceptualization, A.O.; Methodology, A.O., Y.I., A.S., Y.M. and M.O.; Data analysis, A.O., Y.I. and A.S.; Writing—Original Draft Preparation, A.O.; Writing—Review and Editing, A.O., Y.I., A.S., Y.M. and M.O.; Funding Acquisition, A.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by a grant from the Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C) (21K11561).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Human Ethics Committee of Toyo University (TU2019-018-TU2020-H-019).

**Informed Consent Statement:** Informed consent was obtained from all participants involved in the study.

**Acknowledgments:** We would like to express our gratitude to everyone who volunteered to participate in this study.

**Conflicts of Interest:** The authors have no financial conflict of interest to declare.

#### **References**


**Karolina Jakubczyk 1,\*, Patrycja Kupnicka 2, Klaudia Melkis 1, Oliwia Mielczarek 1, Joanna Walczy ´nska 1, Dariusz Chlubek <sup>2</sup> and Katarzyna Janda-Milczarek <sup>1</sup>**


**Abstract:** The fermented tea beverage Kombucha is obtained through a series of biochemical and enzymatic reactions carried out by symbiotic cultures of bacteria and yeasts (SCOBY). It contains organic acids, vitamins, amino acids, and biologically active compounds, notably polyphenols, derived mainly from tea. Kombucha exhibits a range of health-promoting properties, including antioxidant or detoxifying effects. This fermented beverage is traditionally brewed with black tea, but other types of tea are used increasingly, which may have significant implications in terms of chemical composition and health-promoting effects. In this preliminary study, we investigated the content of micronutrients (manganese (Mn), copper (Cu), iron (Fe), chromium (Cr) and zinc (Zn)) by the ICP-OES method in Kombucha prepared with black, red, green and white tea at different time points of fermentation (1, 7, 14 days). It should be noted that the composition of separate ingredients such as tea, leaven or sugar has not been studied. Kombucha had the highest content of zinc—0.36 mg/L to 2.08 mg/L, which accounts for between 3% and 26% of the RDA (Recommended Dietary Allowance) for adults, and the smallest amounts of chromium (0.03 mg/L to 0.09 mg/L), which however represents as much as between 75% and 232% of the RDA. It has been demonstrated that the type of tea as well as the day of fermentation have a significant effect on the concentrations of selected minerals. Kombucha can therefore supplement micronutrients in the human diet.

**Keywords:** kombucha; fermentation; microelements

#### **1. Introduction**

Kombucha is a low-alcohol beverage made by fermenting a sugared tea infusion with symbiotic cultures of bacteria and yeasts (SCOBY), commonly called "tea fungus". This complex community comprises acetic acid bacteria (AAB) (*Gluconobacter: G. entanii, G. oxydans, Acetobacter: A. xylionoides, A. aceti, A. pasteurianus*)*, Komagataeibacter (K. intermedius, K. rhaeticus*), lactic acid bacteria (LAB) (*Lactobacillus* and *Leuconostoc*)*,* and yeasts (*Schizosaccharomyces pombe, Zygosaccharomyces bailii, Saccharomyces*). Its dynamics are still not fully understood [1–7].

Kombucha fermentation is a combination of three fermentation processes: alcoholic, lactic and acetic acid. The bacteria present in the tea fungus are responsible for the production of acetic acid, while yeasts, representatives of the osmophilic type, induce the breakdown of sucrose. The resulting product, glucose, is a substrate for both lactic fermentation and alcoholic fermentation [4,8]. Under the influence of lactic acid bacteria, glucose is converted into lactic acid. During alcoholic fermentation, on the other hand, glucose is converted into ethyl alcohol, releasing carbon dioxide. Ethanol produced during the breakdown of glucose is oxidised by acetic acid bacteria to acetic acid and acetaldehyde (Figure 1).

**Citation:** Jakubczyk, K.; Kupnicka, P.; Melkis, K.; Mielczarek, O.; Walczy ´nska, J.; Chlubek, D.; Janda-Milczarek, K. Effects of Fermentation Time and Type of Tea on the Content of Micronutrients in Kombucha Fermented Tea. *Nutrients* **2022**, *14*, 4828. https://doi.org/ 10.3390/nu14224828

Academic Editors: Daniela Rigano and Paola Bontempo

Received: 23 September 2022 Accepted: 11 November 2022 Published: 15 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** Types of fermentation in Kombucha. Created with BioRender.com.

*Acetobacter* are also responsible for the oxidation of glucose to glucuronic and gluconic acid, the key detoxifying agents in Kombucha. The process is also accompanied by the production of cellulose, which is part of the tea fungus [7]. Thus, sweetened tea is transformed into Kombucha by a process involving three types of fermentation, whose activity and dominance changes over time. Initially, the beverage is rich in glucose, followed by alcohol, while the final stage is dominated by organic acids, including acetic acid.

The elementary ingredients of the traditional recipe are black tea and white sugar [9]. Sucrose is the main source of carbon in Kombucha fermentation due to its uncomplicated structure and ability to provide simple carbohydrates for microbial metabolic pathways, as well as its low cost and easy availability [1,10]. However, Kombucha is more and more often made with green, red and white teas or herbal infusions instead of black tea [7], and with coconut sugar, cane sugar, maple syrup or honey instead of white sugar [1,10,11]. The entire Kombucha production process takes place at room temperature over 7–14 days, during which it acquires its distinct chemical and organoleptic characteristics. The flavor of the finished tea beverage is described as mildly sour, fruity, fizzy, resembling that of cider [12–14].

Research to date indicates that the fermented tea beverage contains numerous bioactive substances, originating from the material used, mainly tea, but also resulting from the enzymatic transformations of organic compounds carried out by microorganisms. The bioactive compounds include vitamins (E, K, B, C), amino acids (especially theanine, a derivative of glutamine), polyphenolic compounds, i.e., catechins and flavonoids, and a variety of minerals [4,7,15–17].

It is worth noting that Kombucha has been hailed as a functional fermented beverage with antioxidant, antimicrobial, antioxidant, anti-diabetic properties, reducing cholesterol levels, supporting immune and digestive function, and also stimulating liver detoxification [7,17–19]. However, the presence and the amounts of nutrients in Kombucha, and accordingly its beneficial effects, are determined by a number of factors, such as the parameters of the fermentation process, e.g., time, or the ingredients used [12].

In addition, Kombucha is gaining popularity as one of the novelties offered by fermented food manufacturers. The valuable composition of such foods translates into manifold biological effects in consumers' bodies [20]. As a result of the biochemical transformations of organic compounds by microorganisms, it is possible to obtain foods with not only extended shelf life and microbiological stability, but also a higher nutritional value. The fermentation process largely breaks down the antinutrients in food, e.g., phytates, enhancing the potential for utilizing nutrients of key importance in the human diet. Also, fermented foods may be better tolerated by people with certain food sensitivities and intolerances [21]. Recent scientific reports confirm the interactions between microorganisms and plant products [22,23]. The microbiota present in the product is responsible for "digesting" plant material into absorbable active small molecules, which then induce physiological changes in the body [24–27]. Research findings show that fermentation of tea residues can significantly increase the antioxidant activity (up to 3.25 times), as well as the polyphenol concentrations (5.68 times) of Kombucha. Interestingly, green tea residues showed a stronger effect than those of black tea [26].

Even though Kombucha is gaining increasing recognition and the range of flavors available on the food market continues to expand, the detailed composition and the effects of individual fermentation parameters or type of tea on the properties of this product are not fully understood [12]. A few scientific articles report that Kombucha is a source of micronutrients, but the data are incomplete. Hence, the aim of this study was to evaluate for the first time the content of selected micronutrients in Kombucha beverages made with black, green, white and red tea at different time points of fermentation.

#### **2. Materials and Methods**

#### *2.1. Plant Material*

The material consisted of four types of leaf tea (*Camellia sinensis*): black, green, white and red (Pu-ERH) originating in China.

#### *2.2. Preparation of Kombucha*

The Kombucha cultures in the present article were purchased from a commercial shop. Kombucha bacterium component belongs to the strains of *Acetobacter*, while the yeasts are *Saccharomyces cerevisiae* and *Zygosaccharomyces*. One hundred grams of sugar (100.0 g/L, 10.0%), eight grams of tea (8.0 g/L, 0.8%) and 1 L of hot distilled water (90 ◦C) were added to the flask. The solution was infused for 10 min in a sterile conical flask. After cooling to 30 ◦C, the tea decoction was filtered into clean glass bottles and Kombucha pellicle (100.0 g/L, 10.0%) and one hundred milliliters of leaven from a previous culture (100.0 mL/L, 1.0%) were added (Figure 2).

**Figure 2.** Material and methods—Preparation of Kombucha and laboratory analyses. Created with BioRender.com.

#### *2.3. Fermentation of Kombucha*

Kombucha culture was kept under aseptic conditions. Fermentation was carried out by incubating the Kombucha culture at 28 ± 1 ◦C for 1, 7 and 14 days. Replicates were prepared so that each replicate was completely collected after its stipulated period of fermentation. The Kombucha obtained was filtered and analyzed.

#### *2.4. Determining Elements Content in Infusions*

#### Sample preparation:

The samples were mineralized using the MARS 5 CEM microwave digestion system. The volume of the sample given to research was 0.8 mL. The samples were transferred to clean polypropylene tubes, 2 mL of 65% HNO3 (Suprapur, Merck, Darmstadt, Germany) was added to each vial and each sample was allowed 30 min pre reaction time in the clean hood. After completion of the pre-reaction time, 0.5 mL of non-stabilized 30% H2O2 solution (Suprapur, Merck, Darmstadt, Germany) was added to each vial. Once the addition of all reagents was complete, the samples were placed in special Teflon vessels and heated in the microwave digestion system for 35 min at 180 ◦C (15 min ramp to 180 ◦C and maintained at 180 ◦C for 20 min). At the end of digestion all samples were removed from the microwave and allowed to cool to room temperature. In the clean hood, samples were transferred to acid-washed 15 mL polypropylene sample tubes. A further 5-fold dilution was performed prior to ICP-OES measurement. A volume of 2 mL was taken from each digest. The samples were spiked with an internal standard to provide a final concentration of 0.5 mg/L Ytrium, 1 mL of 1% Triton (Triton X-100, Sigma, Kawasaki, Japan) and diluted to the final volume of 10 mL with 0.075% nitric acid (Suprapur, Merck, Darmstadt, Germany). Blank samples were prepared by adding concentrated nitric acid (500 μL) to tubes without a sample and subsequently diluted in the same manner described above. Multielement calibration standards (ICP multi-element standard solution IV, Merck, Darmstadt, Germany) were prepared with different concentrations of inorganic elements in the same manner as in blanks and samples. Deionized water (Direct Q UV, Millipore, Burlington, MA, USA, approximately 18.0 MΩ) was used for preparation of all solutions. Sample determination:

Samples were analyzed using inductively coupled plasma optical emission spectrometry (ICP-OES, ICAP 7400 Duo, Thermo Scientific), which is often utilized to measure the concentrations of mineral nutrients as well as heavy metals and allows simultaneous measurements of many different elements, also in plant samples [28,29]. ICP-OES with a concentric nebulizer and cyclonic spray chamber was used to determine the content of micro and macroelements. The analysis was performed in both radial and axial modes. The wavelengths used in the analysis were: Zn 206.200, Cr 205.560, Mn 257.610, Cu 224.700, Fe 259.940.

Validation was performed by evaluating the following: NIST SRM 8414 reference material (National Institute of Standards and Technology, USA), limit of detection (LOD), and the recovery of internal standard (yttrium). To eliminate possible interference, the emission lines were selected empirically in pilot measurements. This model of validation is often used in ICP-OES studies, also those regarding plant samples [29]. The recovery of Y was within 90–106%. The R2 values for all standard curves were in the range between 0.998 and 1.000.

#### *2.5. Statistical Analysis*

All determinations were carried out in at least three replicates. Statistical analysis was performed using Stat Soft Statistica 13.0 and Microsoft Excel 2017. Distributions of values for individual parameters were analyzed using the Shapiro-Wilk test. Since the distribution of continuous variables deviated from normal, the Kruskal-Wallis test was used to evaluate the differences between the studied parameters. Spearman's correlation test was used to determine the correlations between the parameters studied. Results were expressed as mean values and standard deviation; however, median values and quartile ranges were used for statistical analyses. Differences were considered significant at *p* ≤ 0.05.

#### **3. Results**

Analysis of Kombucha beverages prepared with black, green, white and red tea at different time points of fermentation revealed the presence of five trace elements (Tables 1–3). The content of identified micronutrients manganese (Mn), copper (Cu), iron (Fe), chromium (Cu) and zinc (Zn) was quantified. It should be noted that the composition of separate ingredients such as tea, leaven or sugar was not studied. The general pattern of mineral concentrations in the Kombucha samples was as follows: Zn > Mn > Fe > Cu > Cr.


**Table 1.** Analysis of reference material Bovine Muscle NIST-SRM 8414.

LOD—limits of detection, %RSD range—relative sample deviation.


**Table 2.** The microminerals content (Mn, Cu) and Recommended Dietary Allowances (RDA) in Kombucha.

Different numbers (a–l) in the columns represent statistically significant differences \* *p* < 0.05 between particular types of Kombucha (1, 7, 14 days of fermentation) and tea: a—BK 0, b—BK 7, c—BK 14, d—GK 0, e—GK 7, f —GK 14, g—WK 0, h—WK 7, <sup>i</sup> —WK 14, <sup>j</sup> —RK 0, k—RK 7, <sup>l</sup> —RK, BK—Kombucha prepared from black tea, GK—Kombucha prepared from green tea, RK—Kombucha prepared from red tea, WK—Kombucha prepared from white tea.

The content of manganese in Kombucha ranged from 0.43 mg/L to 1.40 mg/L and was dependent on both fermentation time and type of tea used (Table 2). Statistically significant differences are presented in Table 1. The lowest values were observed in Kombucha prepared with black and white tea, while the highest were found in green tea Kombucha. Irrespective of the type of tea used, the highest results were observed on day 14 of fermentation. One liter of the beverage can cover from 19% to 61% of manganese requirement for men and 24% to 78% for women.


**Table 3.** The microminerals content (Fe, Cr) and Recommended Dietary Allowances (RDA) in Kombucha.

Different numbers (a–l) in the columns represent statistically significant differences \* *p* < 0.05 between particular types of Kombucha (1, 7, 14 days of fermentation) and tea: a—BK 0, b—BK 7, c—BK 14, d—GK 0, e—GK 7, f —GK 14, g—WK 0, h—WK 7, <sup>i</sup> —WK 14, <sup>j</sup> —RK 0, k—RK 7, <sup>l</sup> —RK, BK—Kombucha prepared from black tea, GK—Kombucha prepared from green tea, RK—Kombucha prepared from red tea, WK—Kombucha prepared from white tea.

The content of copper in Kombucha depended on both fermentation time and the tea used (Table 2) and ranged from 0.01 mg/L to 0.25 mg/L. Kombucha brewed with red tea had the lowest levels of copper, while that prepared with black tea had the highest. Irrespective of the type of tea used, the highest results were observed on day 14 of fermentation, except for white tea (Table 2). Between 7% and 28% of the requirement for this micronutrient was met for both men and women. With respect to this element, there were no statistically significant differences between Kombuchas made with different types of tea on days 1 and 7 of fermentation. The only differences observed were on day 14, leading to the conclusion that the type of tea does not affect copper content in the early days of fermentation.

The content of iron in Kombucha ranged from 0.18 mg/L to 0.46 mg/L and was dependent on both fermentation time and type of tea used (Table 3). The lowest iron levels were observed in Kombucha brewed with green tea, while the highest were found in white tea Kombucha. One liter of the beverage covers as little as 1.8% to 4.6% of the iron requirement for men, and 1% to 2.5% for women.

The chromium content in Kombucha ranged from 0.03 mg/L to 0.09 mg/L and was dependent on both fermentation time and type of tea used (Table 3). The lowest levels were observed in Kombucha brewed with green tea and red (7 days of fermentation). Irrespective of the type of tea used, the highest results were observed on day 14 of fermentation. The requirement for this element was covered at 75% to 232% for both men and women.

The content of zinc in Kombucha ranged from 0.36 mg/L to 2.08 mg/L and was likewise dependent on both fermentation time and type of tea used (Table 4). The lowest levels were noted in Kombucha brewed with white tea, while the highest were found in black tea Kombucha. Irrespective of the type of tea used, the highest results were observed on day 14 of fermentation. The requirement for zinc was covered at 3% to 19% for men and 5% to 26% for women. The mineral content was almost invariably highest on day 14 of fermentation regardless of the type of tea. There were no statistically significant differences between Kombuchas made with different teas on days 1 and 7 of fermentation, so initially the type of tea does not have a significant effect the content of this element.


**Table 4.** The microminerals content (Zn) and Recommended Dietary Allowances (RDA) in Kombucha.

Different numbers (a–l) in the columns represent statistically significant differences \* *p* < 0.05 between particular type of Kombucha (1, 7, 14 days of fermentation) and tea: a—BK 0, b—BK 7, c—BK 14, d—GK 0, e—GK 7, <sup>f</sup> —GK 14, g—WK 0, h—WK 7, <sup>i</sup> —WK 14, <sup>j</sup> —RK 0, k—RK 7, <sup>l</sup> —RK, BK—Kombucha prepared from black tea, GK—Kombucha prepared from green tea, RK—Kombucha prepared from red tea, WK—Kombucha prepared from white tea.

In addition, the content of micronutrients was analyzed independently of the day of fermentation, taking into account the type of tea used to prepare Kombucha. The highest concentrations of Zn, Cu and Cr were found in the beverage made with black tea. In the case of manganese, the highest concentration of this element was observed in Kombucha brewed with green tea, while that of iron in the Kombucha prepared with red tea (Table 5). The only statistically significant differences were observed for manganese. For the other elements, the type of tea did not affect their content.


**Table 5.** The microminerals content in different type of Kombucha.

Different letters (a, b, c, d) in the columns represent statistically significant differences \* *p* < 0.05 between particular type of Kombucha (1, 7, 14 days of fermentation) and tea: a—BK, b—GK, c—WK, d—RK, BK—Kombucha prepared from black tea, GK—Kombucha prepared from green tea, RK—Kombucha prepared from red tea, WK—Kombucha prepared from white tea.

Additionally, significant positive correlations were found between some micronutrients (Table 6); however, this relationship was variable for different types of Kombucha. Negative significant correlations were also found for Kombucha prepared on the basis of red tea: Zn vs. Cu and Cu vs. Fe (Table 6).


**Table 6.** Spearman's rank correlation between micronutrients for different types of Kombucha.

\* *p* < 0.05, BK—Kombucha prepared from black tea, GK—Kombucha prepared from green tea, RK—Kombucha prepared from red tea, WK—Kombucha prepared from white tea.

Regardless of the type of Kombucha, a weak but statistically significant correlation was found between time and the content of Mn (0.279), Zn (0.348) and a moderate, significant relationship between time and the content of Fe (0.423) and Cr (0.447).

Regardless of the day of fermentation, quite strong relationships between the time and concentration of selected mineral compounds were shown in Table 7. Therefore, it can be concluded that both the fermentation process and the time significantly affect the chemical composition of this drink.


**Table 7.** Spearman's rank correlations between fermentation time and microminerals content for different types of Kombucha.

\* *p* < 0.05, BK—Kombucha prepared from black tea, GK—Kombucha prepared from green tea, RK—Kombucha prepared from red tea, WK—Kombucha prepared from white tea.

#### **4. Discussion**

Kombucha fermented tea is becoming increasingly popular, not only for its sensory properties, but also for its health-promoting benefits. In addition, the drink is classified as a functional food or nutraceutical [7,30,31].

Our study confirms that Kombucha can be a source of micronutrients: chromium (Cr), manganese (Mn), copper (Cu), zinc (Zn), and iron (Fe). The general pattern of mineral concentrations in the Kombucha samples was as follows: Zn > Mn > Fe > Cu > Cr. Our analysis was carried out at different time points of fermentation (days 1, 7 and 14). In addition, for the first time, different types of leaf tea were used as the base of the beverage: black, green, red and white tea. We have shown that the content of selected micronutrients is dependent not only on the day of fermentation but also on the type of tea used.

Micronutrients are minerals which, although present in trace amounts, are essential for the normal development and functioning of the human body. Given that cells do not have the ability to synthesize trace elements, they must be supplied with food. A properly balanced diet should contain essential minerals in such quantities that the total supply is adequate to meet demand, as both deficiency and excess can cause a range of dysfunctions and disorders [32,33]. The search for and analysis of different types of new foods can add valuable information about a source of micronutrients.

Manganese plays an important role in development, digestion, reproduction, antioxidant defense, energy production, immune response and regulation of neuronal activity [34]. The adult requirement for this mineral ranges from 1.8 mg (women) to 2.3 mg (men) [35]. Our analysis showed that manganese (Mn) concentrations ranged from 0.43 mg/L to 1.40 mg/L, accounting for 19% to 61% of the requirement for this element for men and 24% to 78% for women. The highest manganese content was found in the green tea Kombucha from day 14 of fermentation.

Copper is a component of superoxide dismutase and thus influences free radical decomposition reactions. Moreover, it is responsible for enzymatic reactions and the synthesis of collagen and neurotransmitters [36]. The RDA for adults is 0.9 mg [35]. The amount of copper (Cu) in our study ranged from 0.06 mg/L to 0.25 mg/L on day 14 of fermentation, representing between 7% and 28% of the RDA for both sexes. The highest content of this element was detected in the beverage made with black tea on day 14 of fermentation.

Iron, an essential mineral for health and life, is responsible for the synthesis of hemoglobin—a protein found in erythrocytes, which carry oxygen molecules from the lungs to peripheral tissues and support immune and nerve functions [37]. To prevent iron deficiencies, a daily intake of 10 to 18 mg of this mineral is recommended, depending on life stage and sex [35]. Our Kombuchas contained between 0.18 mg/L and 0.46 mg/L of iron (Fe), representing between 1.8% and 4.6% of the RDA for men and between 1% and 2.5% for women. The highest iron content was found in the beverage prepared with white tea on day 14 of fermentation. The results of the analysis for each beverage revealed differences in the content of minerals, correlated with fermentation time as well as the tea used, and the differences were statistically significant (*p* < 0.05).

Chromium is an essential nutrient for normal metabolism of glucose, protein and fat. It enhances insulin sensitivity in tissues and also participates in intracellular redox reactions. The recommended daily intake of this mineral for men and women aged 19–50 years is 0.04 mg [35,38]. Our results showed that the chromium content in Kombuchas made with infusions from different types of tea ranged from 0.03 mg/L to 0.09 mg/L, meaning that Kombucha can cover as much as 75% to 232% of the RDA for both men and women. The highest content of this mineral was detected in the product made with green and red tea on day 14 of fermentation. Consuming even small amounts of Kombucha will correct deficiencies of this element.

Zinc (Zn) is a key element in many processes, from cell growth and differentiation, to regulating immune system function and modulating mechanisms related to learning and memory [39]. The recommended daily intake is 11 mg for men and 8 mg for women [35]. Zinc was detected in amounts ranging from 0.36 mg/L to 2.08 mg/L on day 14 of black tea fermentation, which accounts for 3% to 19% of the requirement for men and 5% to 26% for women. In addition, it has been demonstrated that *Acetobacter aceti* bacteria biotransform chromium and zinc and increase their amounts. These properties are used in the treatment of diabetes due to their hypoglycemic effect [40].

Minerals such as Mn, Zn and Cu can find be found in plant protection products, fertilizers, pesticides and fungicides. Their presence in the product may be related to agricultural practices in use and the content of these elements in phytosanitary products [41].

Kombucha, despite its long tradition, is not adequately researched. There are few studies analyzing the mineral content of this beverage, particularly in terms of micronutrients. It should be emphasized, however, none of them take into account the different time points of fermentation and type of tea.

Ivanišová et al. assessed the chemical composition and antioxidant, antimicrobial and sensory properties of Kombucha made with black tea on day 7 of fermentation, and their findings appear to be consistent with the present study. Their Kombucha, however, was prepared in a different way. The infusion was made by boiling 1 liter of water, 5 g of black tea leaves (Darjeeling, India) and 30 g of white sugar, left to steep for 15 min and

fermented at 22 degrees Celsius for 7 days [30]. In our study, we used 1 liter of water, 100 g of white sugar and 8 g of tea. In the study by Ivanišová et al., the content of manganese was 1.57 mg/L, copper 0.14 mg/L, iron 0.31 mg/L, zinc 0.53 mg/L and chromium was not detected during the analyses. Our results were: 0.67 mg/L for manganese, 0.13 mg/L for copper, 0.24 mg/L for iron, 0.74 mg/L for zinc and 0.04 mg/L for chromium. The most pronounced differences were noted in the content of manganese and the presence of chromium, but this may be related to the use of material of a different origin [30]. The researchers conclude that the content of minerals valuable for the human body increases with fermentation time. In a comparison of the chemical composition and properties of tea and Kombucha, fermented tea proved to be more valuable, and its antioxidant activity was several times higher. During fermentation, the content of the essential elements Fe, Mn, Zn and Ni increased significantly, too. In addition, the authors of the study emphasize that due to the absence of harmful elements, Kombucha is safe to consume [30].

Jayabalan et al. analyzed the chemical composition of the tea fungus (SCOBY) used to brew black tea Kombucha. Their study also made reference to time points, with tests carried out on days 7, 14 and 21 of fermentation. Biochemical properties, including mineral content, increased throughout fermentation time, reaching maximum values on day 21 [2]. Among micronutrients, the highest concentrations in dried tea fungus were found for zinc and manganese. However, the finished Kombucha drink was not analyzed. The results are consistent with our report.

It is worth noting that similar findings were obtained in our earlier study focusing on fluoride ions. In that case, too, longer fermentation resulted in a higher fluoride concentration in the beverage [14].

Similarly, a comparative analysis of sweet black tea vs. Kombucha showed that fermentation significantly increases the content of selected minerals [42]. Concentrations of Zn, Cu, Fe, Mn, Ni and Co were determined. Tests for certain toxic elements showed that Pb and Cr were present in very small amounts, while Cd was not found. Among micronutrients, the highest concentrations were noted for manganese (0.462 ug/mL), iron (0.353 ug/mL), copper (0.237 ug/mL), and the lowest for zinc (0.154 ug/mL) and chromium (0.001 ug/mL). These results are very similar to our observations, with similar concentrations of Mn, Cu and Fe. The Cr content in our study was slightly higher, but the biggest differences were found for Zn. According to Bauer-Petrovska and Petrushevska-Tozi, the levels of copper, iron, manganese, nickel and zinc increase due to the metabolic activity of Kombucha [42].

Tea as such is an important source of elements in the human diet [43]. What is more, the type or species of plant can significantly affect the mineral content in the beverage. This is related, among other things, to the individual differences between species, the diversity of production processes of the plants concerned and the type of soil in which they grow, the capacity of the plants to store nutrients, the types of pollution, climate or geographical location [43–45].

Brzezicha-Cirocka et al. analyzed 118 black teas, determining the concentrations of 14 elements. In terms of micronutrients, the highest concentration was found for Mn, along with the highest percentage of the RDA (15%) per daily intake of this beverage [46]. Similar results were also obtained by Koch et al. where, for all black teas tested, mineral contents were ranked in the following order: K > Ca > Mg > Mn > Fe > Na > Zn > Cu. It has been shown that mineral composition can be significantly affected by the origin of black tea, defined not only as a country, but also a region or province [45]. Differences in mineral composition have been noted between black and green teas, and the authors pointed out that apart from production-related factors, mineral content can be also influenced by soil conditions, location, rainfall, altitude, genetic characteristics of the plant, and age of the tea leaves [47].

Even though all teas used in this study are derived from the same plant species, *Camellia sinensis*, and come from a single producer in China, they differ considerably in their production processes, as also demonstrated in this paper. Depending on the process

followed to obtain the final product, different types of tea can be distinguished. The main types are black tea, white tea, green tea and red tea. Processing treatments affect the color, aroma, taste, intensity, and also the chemical composition of the tea infusion [48]. Black tea is obtained by complete oxidation of tea leaves. After harvesting, the leaves are left to wither completely. During this time, they are also crushed and rolled to speed up the oxidation process. Once they have acquired a sufficiently dark color, they are dried at a high temperature. Red tea is made from green buds and young leaves. Immediately after harvesting, they are heated to inactivate the enzymes. While still moist, the leaves are rolled and then dried in the sun. This type of tea also undergoes fermentation during prolonged storage in high humidity conditions [49]. White tea is obtained from the buds of the tea plant harvested in the spring. Oxidation takes place only where the leaves are damaged, and it is minimal. However, the leaves for white tea are not immediately subjected to a drying process, but are allowed to rest freely, which allows for the activation of enzymatic processes in the leaves, i.e., enzymatic oxidation. Green tea is not oxidized. To make it, withering tea leaves are steamed or pan-fried (Figure 3) [50]. The general principles for obtaining dried tea are very similar, but small differences in production result in distinct flavors and aromas, as well as the chemical composition of the infusion [50].

**Figure 3.** Types of teas and their production process. Created with BioRender.com.

In our study, irrespective of the day of fermentation, the content of individual micronutrients in Kombucha varied, with the type of tea being the determining factor. The highest concentrations of Zn, Cu and Cr were observed in the beverage prepared according to the traditional recipe, i.e., using the fully oxidized black tea. In the case of manganese, the highest concentration of this element was observed in Kombucha brewed with green tea, while that of iron in the Kombucha prepared with red tea. White tea, which undergoes the least amount of processing, only drying, gentle rolling and light oxidation, has the lowest concentrations of micronutrients. Hence, one of the factors determining the final mineral composition is the processing of the leaves themselves. Tea oxidation appears to be particularly important, as Kombucha made with black tea contained the highest levels of minerals.

It is also worth noting that the micronutrient content increased with the day of fermentation of the beverage, reaching the maximum level on day 14. Today, a significant number of manufacturers embrace the use of fermentation in food production, due to its positive effects on enhancing biosafety, extending shelf life and functional properties [46]. It appears that in most cases, the content of both macro- and micro-nutrients in foods significantly increases during fermentation [51].

The fermentation process increases the bioavailability of micronutrients and trace elements through the degradation of insoluble metal cation complexes and anti-nutritive substances such as oxalates, tannins and phytates [52]. These compounds are hydrolyzed by enzymes (e.g., phytases) produced by microorganisms such as lactic acid bacteria and yeast that make up SCOBY. In addition, the synthesis of lactic acid during fermentation, causing pH changes, provides the conditions necessary for the activation of microbial enzymes, thus contributing to the intensification of their action [53,54]. This mechanism is confirmed by the results of studies by Castro-Alba et al. which showed a correlation between increased availability of iron, zinc and calcium in fermented quinoa flour (3.6, 4.0 and 3.5 times, respectively) and a reduction in phytate levels [55].

Sometimes, microorganisms use individual elements for their own metabolism as a substrate to initiate the fermentation process or the synthesis of secondary metabolites, including vitamins and polyols [56]. We also observed these changes in our study, but they were strongly related to the type of tea and the day of fermentation, which could be related to the activity of microorganisms [51]. Ivanišová et al. reported a decrease in Ca and Pb concentration in the Kombucha drink. Cultures of Kombucha microorganisms show the ability to detoxify the drink, as these bacteria are considered biosorbents. These SCOBY microorganisms have properties that allow them to accumulate and bind heavy metal contaminants on their cellular structure [30]. The results of Mamisahebei et al. showed that the Kombucha cultures used in the beer brewing process are very effective in removing heavy metals such as arsenic, chromium and copper. The content of Co did not increase in Kombucha, probably due to its inclusion in vitamin B 12, as the B vitamins (mainly B 1, B 6 and B 12) are mainly produced during the fermentation process [57].

Scientific studies have confirmed the potential of LAB strains to improve the bioavailability of minerals. The results confirm that the LAB strains LAB *L. fermentum* B4655, *L. plantarum* B4495, *L. casei* B1922, *L. bulgaricus* CFR2028 i *L. acidophilus* B4496 reduced the content of phytic acid when used to ferment soy milk at 37◦ C for 24 h. In addition, the results show an increase in Mg and Ca levels in fermented soy milk compared to control [58]. Bahaciu et al. [59] showed that germination and fermentation (Lactobacillus) of soybeans for four days at 25 ◦C led to an increase of 40.87%, 43.41%, 59.56% and 53.4% for Zn, Mg, Fe and Ca, respectively, which are higher than the values obtained for germination alone. Additionally, fermentation (4 or 10 h at 30 ◦C) of ground quinoa seeds *with L. plantarum* 299v significantly reduced the phytic acid content and improved the bioavailability of minerals such as Ca, Fe and Zn [60]. This direction should be extended in future scientific research.

The limitations of this preliminary study include the lack of composition analysis of the discrete ingredients such as tea, leaven and sugar. These results will provide a complete understanding of the biochemical processes in Kombucha. Nevertheless, it should be noted that Kombucha, especially when subjected to a longer fermentation process, contains more organic acids, and thus its pH is strongly acidic. Moreover, the produced CO2 can start to accumulate between the drinks (Kombucha) and the biofilm (SCOBY). This can prevent the transfer of nutrients and thus block the continuity of chemical changes in the reaction environment. It should therefore be consumed in limited quantities, diluted with water or fermented for a shorter period of time [7].

#### **5. Conclusions**

Our findings clearly show that the type of tea used to make Kombucha has a significant impact on the micronutrient content of the final product. In addition, fermentation time also determined the levels of selected minerals. Irrespective of the type of tea, the highest results were observed mainly on day 14 of fermentation. Kombucha had the highest content of zinc (0.36 mg/L to 2.08 mg/L), which accounts for between 3% and 26% of the RDA for adults, and the smallest content of chromium (0.03 mg/L to 0.09 mg/L), which, however, represents as much as between 75% and 232% of the RDA. Black tea proved to be the best source of Zn, Cu and Cr; green tea was rich in Mn; while red tea had the highest iron content. In conclusion, Kombucha, particularly based on black tea, can supplement micronutrients in the human diet.

**Author Contributions:** Conceptualization, J.K.; methodology, J.K.; formal analysis, O.M., J.-M.K. and J.K.; investigation, J.K., P.K. and D.C.; writing—original draft preparation, K.M., J.W. and J.K.; writing—review and editing, J.K. and J.-M.K.; supervision, J.K.; project administration, J.K.; funding acquisition J.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Pomeranian Medical University in Szczecin.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

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