**1. Introduction**

Dyslipidemia is a disorder of the lipid and lipoprotein metabolism characterized by too-high or too-low blood lipid levels [1]. Abnormal serum lipid levels increase the risk of cardiovascular diseases and can be caused by elevated total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), or triglyceride (TG) levels, or low high-density lipoprotein cholesterol (HDL-C) [2]. Globally, cardiovascular diseases currently represent the primary cause of mortalities, including within Thailand [3]. In fact, the World Health Organization (WHO) [4] estimates that cardiovascular diseases will claim the lives of approximately 23.6 million people by the end of 2030. Regarding Thailand specifically, the Medical Record Section, Service Division, of the Bhumibol Adulyadej Hospital reported that the number of patients with dyslipidemia continues to increase steadily each year. Currently, the recommended treatment options for dyslipidemia include lifestyle modifications and drug therapy [5,6]; however, lipid-lowering drugs can induce adverse side effects. Hence, dietary changes, including reduced fat diets, and increased vegetable and fruit intake, which influence the plasma lipid profile [7], may represent effective alternatives to drug therapy.

Vegetables and fruits contain dietary phytochemicals, including flavonoids, carotenoids, vitamins, minerals, and dietary fiber, which decrease dyslipidemia and cardiovascular risk [8–11]. Indeed, their consumption is inversely correlated with plasma TC and LDL-C levels [11]. Suwimol et al. [7] investigated the effect of vegetable and fruit intake on plasma lipid profiles and oxidative status and reported that the consumption of eight servings of vegetables and fruits per day significantly reduces LDL-C and lipid peroxidation via malondialdehyde (MDA) levels. However, the sensory aspects of fruits and vegetables (i.e., color, smell, flavor, texture) can dissuade patients from consuming adequate amounts. Therefore, altering these characteristics by blending or mixing may improve uptake. Moreover, the combination of vegetables and fruits with various forms of probiotics, such as probiotic powder or probiotic fermented milk, can provide beneficial probiotic and dietary fiber [12].

Probiotics comprise a group of bacteria that are generally recognized as safe (GRAS) [13] for use in food and human health products, including for the treatment of various diseases [14] while also decreasing the risk of dyslipidemia. *L. paracasei* are widely utilized as probiotics or synbiotics to improve clinical outcomes. Chiu et al. [15] reported that, in a hamster model, ingestion of *L. paracasei* NTU 101 fermented milk, along with a highcholesterol diet, significantly reduced serum cholesterol levels, compared to the control group. Furthermore, Dehkohneh et al. [16] reported significantly reduced serum cholesterol levels in Wistar rats following consumption of *L. paracasei* TD3 with a high-fat diet.

The current 30-day randomized controlled trial therefore aims to investigate the effects of consuming vegetable and fruit juice (VFJ), with probiotic *L. paracasei*, on physical parameters (body weight, BW; body mass index, BMI; and waist circumference, WC) and biological markers (lipid profile, lipid peroxidation, oxidative stress enzymes, and bile acid (BA) level) in dyslipidemia patients at Bhumibol Adulyadej Hospital. The findings will show that the intake of VFJ with *L. paracasei* could provide health benefits by improving lipid profiles, lipid peroxidation, and oxidative stress enzyme activity levels.

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

#### *2.1. Materials*

Organic vegetables and fruits from the same plantations and the same farm, i.e., green lettuce (*Lactuca sativa*), Chinese celery (*Apium graveolens* var. *secalinum*), cherry tomato (*Solanum lycopersicum* var. *cerasiforme*), onion (*Alium cepa*. Linn), and lime (*Citrus aurantifolia* (Christm. and Panz.) Swing) were obtained from King Fresh Farm CO., LTD. (contact farming) located in Samut Sakhon, Thailand. Only apple (*Malus pumila*), was not organic, which was obtained from a market in Bangkok, Thailand. The selected raw material requirements were as follows: (i) an edible vegetable or fruit, (ii) of a low price, (iii) available throughout the year, (iv) had rich source of bioactive compounds, and (v) had a favorable taste in amixed juice.

#### *2.2. Probiotics Lactobacillus paracasei*

The powder form of a probiotic *L. paracasei* that is on the list of notifications of the Ministry of public health, Thailand (2011) was provided by Innovation Center for Holistic Health, Nutraceuticals and Cosmeceuticals, Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand. A total of 10 g of probiotic powder was contained within an aluminum foil sachet. The sachet of probiotic powder was kept in the fridge (4–6 ◦C) with the cell survival rate ≥ 90% for 3–6 months of storage.

## *2.3. Preparation of Vegetable and Fruit Juice (VFJ) with and without Probiotic Lactobacillus paracasei*

Organic fresh vegetables and fruits were washed under running tap water and drained in a sieve to remove excess water before creating the juice. Only apples were peeled and immersed in a mixture of 0.5% (*v*/*v*) vinegar and 0.5% (*w*/*v*) saline for 15 min. The fruits and vegetables were then sliced, weighed, and prepared according to the recipe shown in Table 1. The composition was blended using a 1500-watt blender (Buono, Model BUO-127799T, Taipei, Taiwan) for 1 min. The resulting juice was then poured into 250 mL plastic bottles and stored at 4–6 ◦C before consumption on the same day of preparation. For the VFJ with *L. paracasei*, 10 g of the *L. paracasei* probiotic powder was added to the bottle. The final concentration of the probiotic was 10<sup>9</sup> CFU per bottle.


**Table 1.** Ingredients of vegetable and fruit juice (VFJ).

#### *2.4. Determination of Bioactive Compounds and Nutritions in Vegetable and Fruit Juice (VFJ)*

The antioxidant activity of VFJ was measured using a modified ABTS (2, 20-Azinobis (3-ethylbenzothiazoline-6-sulfate)) assay, following the method of Saenjum et al. [17]. In brief, the ABTS◦+ aqueous solution was prepared by the mixture of 7.0 mM ABTS stock solution (Merck, Darmstadt, Germany) and 2.45 mM potassium persulfate (RCl Labscan, Bangkok, Thailand). The mixture solution was incubated in the dark for 16 h at room temperature. Then, the working solution was created by mixing 1.0 mL of ABTS◦+ aqueous with 50 mL deionized water to attain an absorbance of 0.70 ± 0.05 at 734 nm using a spectrophotometer. The reaction mixture contained 2000 µL of the ABTS◦+ working solution and 100 µL of the sample or positive control using Trolox (Merck, Darmstadt, Germany). Then, the incubation was continued for 3 min at room temperature. The results were shown as µg Trolox equivalent per mL.

Other nutritional information of VFJ was analyzed by the Institute of Nutrition, Mahidol University, Nakhon Pathom, Thailand.

#### *2.5. Ethical Considerations*

The Ethics Committee of Bhumibol Adulyadej Hospital approved the study protocol (approval number IRB 68/61). In addition, the study was reviewed and approved by the Thai Clinical Trials Registry (TCTR) Committee (TCTR identification number is TCTR20220109001; https://www.thaiclinicaltrials.org/show/TCTR20220109001 (accessed on 9 January 2022). Patients voluntarily decided whether to participate the clinical trial or not. The research team clearly explained the purpose and methodology of this study. All the participants approved of the study procedure and provided their consent before enrollment.

#### *2.6. Participants and Study Design*

The participant population was recruited from patients of Bhumibol Adulyadej Hospital, Bangkok, Thailand. The inclusion criteria included: ≥18 years of age; an LDL-C level between 130 and 160 mg/dL; and had never undergone medical treatment for dyslipidemia. The exclusion criteria included: family history of dyslipidemia; had undergone gastrointestinal surgery; diagnosed with metabolic disorder, cardiovascular disease, thyroid disorder, kidney disease, or liver disease; regular consumption of prebiotic, probiotic, and/or nutritional supplements; currently taking drugs that may affect lipid metabolism; smoking; alcohol consumption during the trial.

The 20 eligible participants were randomized to the probiotic or placebo groups using random allocation software. The participants were blinded to group assignments. During

the 30-day intervention period, the Nutrition Section of Bhumibol Adulyadej Hospital provided both groups with a regular diet in lunch boxes for three meals per day, along with two bottles of VFJ; the probiotic group received VFJ with probiotic *L. paracasei* and the placebo group received VFJ without probiotic *L. paracasei*. Each participant drank the juice daily 30 min before lunch and dinner. A flow diagram is shown as Figure 1. pital provided both groups with a regular diet in lunch boxes for three meals per day, along with two bottles of VFJ; the probiotic group received VFJ with probiotic *L. paracasei* and the placebo group received VFJ without probiotic *L. paracasei*. Each participant drank the juice daily 30 min before lunch and dinner. A flow diagram is shown as Figure 1.

dyslipidemia. The exclusion criteria included: family history of dyslipidemia; had undergone gastrointestinal surgery; diagnosed with metabolic disorder, cardiovascular disease, thyroid disorder, kidney disease, or liver disease; regular consumption of prebiotic, probiotic, and/or nutritional supplements; currently taking drugs that may affect lipid me-

The 20 eligible participants were randomized to the probiotic or placebo groups using random allocation software. The participants were blinded to group assignments. During the 30-day intervention period, the Nutrition Section of Bhumibol Adulyadej Hos-

*Appl. Sci.* **2022**, *12*, x FOR PEER REVIEW 4 of 13

tabolism; smoking; alcohol consumption during the trial.

**Figure 1.** The study flow diagram and enrollment. **Figure 1.** The study flow diagram and enrollment.

until analysis within a month.

#### *2.7. Outcome Assessment*

*2.7. Outcome Assessment*  At baseline and the study endpoint, BW, BMI, and WC physical parameters were measured. Additionally, the lipid profile was assessed based on blood samples collected at the Chemistry and Immunology Laboratory, Department of Pathology, Bhumibol Adulyadej Hospital. Lipid peroxidation and oxidative stress enzymes were measured from blood samples, and bile acid (BA) levels were measured from fecal samples at the Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University (Figure 2). Ten milliliters of blood were collected from each participant after an 8 h overnight fast and stored in an icebox before being transported to the laboratory. A 10 g fecal At baseline and the study endpoint, BW, BMI, and WC physical parameters were measured. Additionally, the lipid profile was assessed based on blood samples collected at the Chemistry and Immunology Laboratory, Department of Pathology, Bhumibol Adulyadej Hospital. Lipid peroxidation and oxidative stress enzymes were measured from blood samples, and bile acid (BA) levels were measured from fecal samples at the Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University (Figure 2). Ten milliliters of blood were collected from each participant after an 8 h overnight fast and stored in an icebox before being transported to the laboratory. A 10 g fecal sample was also collected from each participant and stored in a plastic container at 4–8 ◦C before being sent to the laboratory the next day. The fecal samples were stored at −20 ◦C until analysis within a month.

sample was also collected from each participant and stored in a plastic container at 4–8 °C before being sent to the laboratory the next day. The fecal samples were stored at −20 °C

**Figure 2.** The study schedule of the clinical trial. **Figure 2.** The study schedule of the clinical trial.

#### 2.7.1. Physical Examination 2.7.1. Physical Examination

BW and BMI were measured using a digital weighing scale (Tanita, Model 1584, Tokyo, Japan). WC was measured and recorded using a tape measure (Hoechstmass, Sulzbach, Germany). BW and BMI were measured using a digital weighing scale (Tanita, Model 1584, Tokyo, Japan). WC was measured and recorded using a tape measure (Hoechstmass, Sulzbach, Germany).

#### 2.7.2. Lipid Profile 2.7.2. Lipid Profile

Lipid profile, TC, LDL-C, HDL-C, and TG, levels were measured using an automatic analyzer (Model Cobas®8000, Roche Diagnostics, Mannheim, Germany). Lipid profile, TC, LDL-C, HDL-C, and TG, levels were measured using an automatic analyzer (Model Cobas®8000, Roche Diagnostics, Mannheim, Germany).

#### 2.7.3. Lipid Peroxidation

2.7.3. Lipid Peroxidation MDA, the main marker for lipid peroxidation, was determined using a thiobarbituric acid reactive substances (TBARS) assay according to the modified method of Zeb and Ullah [18] and Atasayar et al. [19]. Serum samples (50 μL) were reacted with thiobarbituric acid (TBA) (Sigma-Aldrich, St. Louis, MO, USA) and trichloroacetic acid reagent (TCA; Merck, Darmstadt, Germany) at 100 °C for 30 min and then placed in cool water. The reaction was measured at 532 nm using a multi-mode microplate reader (SpectraMax M3, MDA, the main marker for lipid peroxidation, was determined using a thiobarbituric acid reactive substances (TBARS) assay according to the modified method of Zeb and Ullah [18] and Atasayar et al. [19]. Serum samples (50 µL) were reacted with thiobarbituric acid (TBA) (Sigma-Aldrich, St. Louis, MO, USA) and trichloroacetic acid reagent (TCA; Merck, Darmstadt, Germany) at 100 ◦C for 30 min and then placed in cool water. The reaction was measured at 532 nm using a multi-mode microplate reader (SpectraMax M3, San Jose, CA, USA).

#### 2.7.4. Oxidative Stress Enzymes

San Jose, CA, USA).

2.7.4. Oxidative Stress Enzymes Oxidative stress enzyme activities, including superoxide dismutase (SOD) activity, catalase (CAT) activity, and glutathione peroxidase (GPx) activity, were evaluated in the Oxidative stress enzyme activities, including superoxide dismutase (SOD) activity, catalase (CAT) activity, and glutathione peroxidase (GPx) activity, were evaluated in the serum samples.

serum samples. The SOD activity assay was modified from that by Kaya et al. [20] using the inhibition of nitroblue tetrazolium (NBT) reduction by the xanthine/xanthine oxidase system as a superoxide generator. The sample was supplemented with a 0.1 mL mixture of ethanol (RCI Labscan, Bangkok, Thailand) and chloroform (Merck, Darmstadt, Germany) (ratio 5:3, *v*/*v*) and centrifuged at 4000 × *g* for 10 min. The ethanol phase of the supernatant was evaluated. One unit of SOD was defined as the amount of enzyme causing 50% inhibition The SOD activity assay was modified from that by Kaya et al. [20] using the inhibition of nitroblue tetrazolium (NBT) reduction by the xanthine/xanthine oxidase system as a superoxide generator. The sample was supplemented with a 0.1 mL mixture of ethanol (RCI Labscan, Bangkok, Thailand) and chloroform (Merck, Darmstadt, Germany) (ratio 5:3, *v*/*v*) and centrifuged at 4000× *g* for 10 min. The ethanol phase of the supernatant was evaluated. One unit of SOD was defined as the amount of enzyme causing 50% inhibition of the NBT reduction rate. SOD activity was inhibited in units per milliliter of the sample.

of the NBT reduction rate. SOD activity was inhibited in units per milliliter of the sample. The CAT activity was determined using a method modified from that by Kaya et al. [20]. A reaction of 750 μL of 0.059 M hydrogen peroxide (Merck, Darmstadt, Germany) and 25 μL of plasma sample or catalase standard (0–700 units/mL) was mixed for 3 min. Kinetic reactions were detected using a spectrophotometer at 240 nm. CAT activity was The CAT activity was determined using a method modified from that by Kaya et al. [20]. A reaction of 750 µL of 0.059 M hydrogen peroxide (Merck, Darmstadt, Germany) and 25 µL of plasma sample or catalase standard (0–700 units/mL) was mixed for 3 min. Kinetic reactions were detected using a spectrophotometer at 240 nm. CAT activity was calculated by comparison with the catalase enzyme standard curve and expressed as units per milliliter.

The modified method of GPx activity was performed according to Rush and Sandiford [21]. The reaction was carried out by mixing β-nicotinamide adenine dinucleotide phosphate (β-NADPH) (Sigma-Aldrich, St. Louis, MO, USA), 1.0 mM sodium azide solution (Sigma-Aldrich), 200 mM glutathione (Sigma-Aldrich, St. Louis, MO, USA), 10 units/mL glutathione reductase (Sigma-Aldrich), and 50 mM phosphate buffer (pH 7.0) with 0.4 mM ethylene diamine tetraacetic acid (Loba Chemie, Mumbai, India). Next, 12.5 µL of plasma was added to the reaction. A mixture without plasma was used as a blank sample. GPx activity was determined using 0.042% hydrogen peroxide as the substrate. The reaction was followed using a spectrophotometer at 340 nm intervals for 15 s for 5 min. The activity of plasma GPx was determined by measuring the rate of oxidation of NADPH, and the result was reported as units per milliliter of plasma.

## 2.7.5. Bile Acid

Bile acid levels were determined in feces using a commercially available IDK® Bile acid kit (Immundiagnostik AG, Bensheim, Germany) according to the manufacturer's protocol and as previously studied [22]. Fifteen milligrams of fecal sample was added to a screw-capped tube containing 1.5 mL of buffer (dilution factor of 1:100). The sample was homogenously shaken and left to stand for 10 min. The mixture sample (10 µL) was added to the reagent in the strip and incubated for 5 min at 25 ◦C. The absorbance of the sample was measured at 405 nm using a multimode microplate reader.

#### *2.8. Statistical Analysis*

Data analysis was performed using STATA version 15.1 (StataCorp, College Station, TX, USA) for Windows licensed to the Faculty of Pharmacy, Chiang Mai University. Demographic characteristic data were evaluated using an independent *t*-test. A paired *t*-test was used to assess differences within the treatment groups. Gaussian regression analysis was performed to assess the effects of treatment between groups.

#### **3. Results**

#### *3.1. Nutrition Information of the Vegetable and Fruit Juice (VFJ)*

The antioxidant activity in VFJ was reported as 244.68 µg Trolox equivalent/mL, which was expressed as 60.97% of inhibition. Table 2 displays the nutritional values of the VFJ without probiotic *L. paracasei*, such as energy, moisture, protein, total carbohydrate, total dietary fiber, soluble dietary fiber, insoluble dietary fiber, vitamin and mineral, total polyphenol, flavonoid, and carotenoid according to the Association of Official Agricultural Chemists (AOAC) test method. The amount was reported by the Institute of Nutrition, Mahidol University, Nakhon Pathom, Thailand.


**Table 2.** Nutritional value of the vegetable and fruit juice (VFJ).


**Table 2.** *Cont.*

<sup>1</sup> mg eq GA represents milligram equivalent gallic acid.

#### *3.2. Participant Characteristics*

The participant characteristics are shown in Table 3. Of the 20 participants, 17 were female and 3 were male with ages ranging from 27 to 58 years. All eligible participants were randomized into two groups (*n* = 10 per group). The placebo group comprised one (10%) man and nine (90%) women, with ages ranging from 27 to 54 years, while two (20%) men and eight (80%) women were assigned to the probiotic group (33–58 years old). No patients withdrew from the study before the endpoint, and no serious adverse events were reported during the intervention period. No statistically significant differences were observed in the demographic characteristics of participants between the two groups at baseline.

**Table 3.** General demographic characteristics of the participants.


Data are presented as mean ± standard error. The *p*-value was at 95% confidence interval. The population was analyzed using an exact probability test, and the continuous data were analyzed using a *t*-test. BMI = body mass index, WC = waist circumference, LDL-C = low-density lipoprotein cholesterol.

#### *3.3. Effect of the Vegetable and Fruit Juice (VFJ) with and without Probiotic L. paracasei on Physical Parameters*

BW, BMI, and WC showed no considerable differences at baseline and endpoint within the placebo group. Moreover, within the probiotic group, no considerable changes were observed in BW or BMI; however, the WC exhibited a decreasing trend (*p* < 0.10; Table 4). Moreover, no significant differences were observed between the two groups in terms of BW, BMI, or WC, at the endpoint (Table 5).

**Table 4.** Physical examination parameters and biological markers in plasma and feces of the participant at the baseline (Day 0) and endpoint (Day 30).



**Table 4.** *Cont.*

Data are presented as mean ± standard error. \*, \*\*, and \*\*\* were significantly different *p* < 0.10, *p* < 0.05, and *p* < 0.01, respectively, within each group at different times of this study. BW = body weight, BMI = body mass index, WC = waist circumference, TC = total cholesterol, LDL-C = low-density lipoprotein cholesterol, HDL-C = high-density lipoprotein cholesterol, TC = triglyceride, MDA = malondialdehyde, CAT = catalase, GPx = glutathione peroxidase, SOD = superoxide dismutase, BA= bile acid.

**Table 5.** Gaussian regression analysis of physical examination parameters and biological markers for the probiotic group compared with the placebo group at the endpoint (Day 30) of the study.


Data were compared to the placebo group at endpoint (Day 30). The *p*-value was at 95% confidence interval. \* and \*\* were significantly different *p* < 0.10 and *p* < 0.05, respectively. BW = body weight, BMI = body mass index, WC = waist circumference, TC = total cholesterol, LDL-C = low-density lipoprotein cholesterol, HDL-C = high-density lipoprotein cholesterol, TC = triglyceride, MDA = malondialdehyde, CAT = catalase, GPx = glutathione peroxidase, SOD = superoxide dismutase, BA= bile acid.

#### *3.4. Effect of the Vegetable and Fruit Juice (VFJ) with and without Probiotic L. paracasei on Lipid Profile*

No significant differences were observed in the TC, LDL-C, HCL-C, TG, or TG/HDL-C ratio (*p* > 0.05) at the endpoint in the placebo group compared to the baseline value (Table 4). Meanwhile, significant declines in the probiotic group were observed in TC, LDL-C, TG, and TG/HDL-C ratio, with a significant increase in HDL-C (Table 4). The parameters of TC, LDL-C, TG, and TG/HDL-C ratio significantly declined: (*p* < 0.05), (*p* < 0.01), (*p* < 0.05), and (*p* < 0.05), respectively. In contrast, HDL-C showed a significant increase (*p* < 0.01). Moreover, although the differences in TC, LDL-C, TG, and TG/HDL-C ratio between the probiotic and placebo groups (Table 5) were not statistically significant, HDL-C was significantly higher in the probiotic group at the endpoint (*p* < 0.01).

#### *3.5. Effect of the Vegetable and Fruit Juice (VFJ) with and without Probiotic L. paracasei on Lipid Peroxidation*

MDA levels in the plasma of the placebo group at the baseline (Day 0) and endpoint (Day 30) were 0.18 ± 0.07 and 0.07 ± 0.01 µM. While the MDA levels of the probiotic group at the baseline (Day 0) and endpoint (Day 30) were 0.19 ± 0.07 and 0.06 ± 0.01 µM, the statistical data showed a significant difference (*p* < 0.10) (Table 4). However, the MDA levels did not differ significantly between the probiotic group and the placebo group at the endpoint (Table 5).

## *3.6. Effect of the Vegetable and Fruit Juice (VFJ) with and without Probiotic L. paracasei on Oxidative Stress Enzyme*

Plasma levels of CAT, GPx, and SOD in the placebo group did not differ significantly (*p* > 0.05) between baseline and endpoint (Table 4). Meanwhile, in the probiotic group, CAT levels were significantly increased (*p* < 0.01), whereas GPx exhibited an increased trend (*p* < 0.10). However, the SOD levels were not significantly different after the endpoint compared to the baseline of the study period. Moreover, no significant differences were detected in CAT, GPx, or SOD levels between the groups (Table 5).

## *3.7. Effect of the Vegetable and Fruit Juice (VFJ) with and without Probiotic L. paracasei on Bile Acids Level*

Fecal levels of BA did not differ significantly within the placebo group between timepoints and were 30.06 ± 3.65 µmol/L at the baseline and 29.29 ± 2.16 µmol/L at the endpoint. Meanwhile, a trend toward significantly increased BA levels (*p* < 0.10) was detected in the probiotic group at the endpoint compared to baseline (Table 4). Moreover, BA levels were significantly higher (*p* < 0.05) in the probiotic group compared to the placebo group at the endpoint (Table 5).

#### **4. Discussion**

In the present study, the selected recipe of VFJ was analyzed in terms of its properties, especially the antioxidant activity, which was related to dyslipidemia and oxidative stress in patients with dyslipidemia [23]. The nutritional value of VFJ, including total dietary fiber, soluble dietary fiber, insoluble dietary fiber, vitamins and minerals, total polyphenols, flavonoids, and carotenoids makes it a potential alternative to conventional medicine for lowering dyslipidemia. Indeed, many reports have verified its ability to reduce dyslipidemia risk by altering the TC, LDL-C, HDL-C, and TG levels. Specifically, soluble dietary fiber has been shown to reduce TC and LDL-C levels [24,25], potentially by increasing BA excretion, thus delaying reabsorption in the intestine [26]. Additionally, the production of short-chain fatty acids by microbial fermentation of dietary fiber in the colon lowers LDL-C levels [27].

Several studies have reported that probiotic bacteria can improve lipid profiles. Fuentes et al. [28] studied the efficacy of *L. plantarum* capsule (containing 1.2 <sup>×</sup> <sup>10</sup><sup>9</sup> CFU of a mixture strain CECT 7527, CECT 7528, and CECT 7529) in hypercholesterolemic patients for 12 weeks. The level of TC in the *L. plantarum* group significantly decreased to 13.6% compared to a placebo group. Ahn et al. [29] suggested that the consumption of a mixed probiotic powder (*L. curvatus* HY7601 and *L. plantarum* KY1032) for 12 weeks decreased 18.3% of TG level compared to a group that consumed the powder without probiotic strains.

Therefore, the VFJ and VFJ containing probiotic bacteria may present useful complementary medicine for patients with dyslipidemia presenting with mild symptoms. In particular, the VFJ offers the benefit of not requiring heat during the preparation process, thereby retaining the full nutrition content of the ingredients. The consumption of VFJ with and without probiotic *L. paracasei* on physical parameters was discussed in term of BW, BMI, and WC. WC was found to significantly decrease in the probiotic group on day 30 compared to baseline. Our results are consistent with those of Zhang et al. [30], who showed that obese participants exhibited a significant reduction in WC after probiotic consumption. Meanwhile, no effect was observed on BM or BMI. Similarly, in a study by Michael et al. [31], obese participants showed reduced WC compared to the placebo group participants after consuming a mixture of probiotic *Lactobacillus* and *Bifidobacterium* (50 billion per day) for six months.

The result of this study showed that the lipid profile of the placebo group (consumption of VFJ without probiotic *L. paracasei*) was not significantly different between baseline and endpoint. However, the lipid profile of the probiotic group (consumption of VFJ with probiotic *L. paracasei*) showed that the level of TC, LDL-C, TG, and TG/HDL-C ratio was significantly declined to 5.59, 6.00, 24.14, and 30.83%, respectively, while the level of

HDL-C was significantly increased to 8.33% at the endpoint, when compared with the baseline. Overall, the VFJ containing probiotic *L. paracasei* could improve lipid profile in the participant with dyslipidemia. This might be associated with bile salt hydrolase (BSH) produced by probiotics, which is the enzymatic deconjugation of bile acids by hydrolysis of conjugated bile acids into deconjugated bile acids [32]. Deconjugated bile acids are less soluble and are reabsorbed in the intestinal lumen less than conjugated bile acids, leading to the excretion of larger amounts of free bile acids in feces [30]. Thus, the deconjugation of bile acids results in a reduction in serum cholesterol by increasing the demand of cholesterol for de novo synthesis of bile acids to replace those lost in feces [33]. The outcomes of lipid profile are related to the previous literature of Mohamadshahi et al. [34]. They studied the consumption of conventional yogurt and probiotic yogurt (containing *L. acidophilus* La-5 and *B. lactis* Bb-12) in diabetic patients. The subjects had a daily intake of 330 g of yogurt for 8 weeks. The result was confirmed that the probiotic yogurt caused a decrease in LDL-C/HDL-C ratio and significantly increased HDL-C levels. Oxidative stress is an imbalance in the cells between the system of reactive oxygen species (or free radicals); production and accumulation lead to the damage of cellular structures such as lipids, proteins, and nucleic acids [35]. Therefore, the harmful effects can respond with dyslipidemia. The lipid peroxidation is one of the possible outcomes of aberrant free radicals, of which MDA is the primary marker. In the current study, the MDA level was significantly decreased to 68.42% in the probiotic group, from 0.19 ± 0.07 mg/dL at the baseline to 0.06 ± 0.01 mg/dL at the endpoint. These findings agree with the results of a previous study that reported decreased MDA levels in the serum and liver of hyperlipidemic rats following *L. casei* supplementation [36]. Moreover, the ability of an antioxidant defense system is based on main enzymatic components, including CAT, GPx, and SOD. For the probiotic group, the levels of CAT and GPx were significantly increased to 8.07 and 31.75% at Day 30 compared to Day 0. However, the level of SOD was an insignificant difference after the endpoint compared to the baseline of the study period. Similarly, within a clinical trial, Chamari et al. [37] reported that probiotic yogurt decreased oxidative stress in healthy women within a clinical trial. That is, intake of the probiotic yogurt for six weeks significantly increased CAT activity compared to the normal yogurt. Kleniewska et al. [26] found that the supplement containing probiotic *L. casei* (4 <sup>×</sup> <sup>10</sup><sup>8</sup> CFU) and prebiotic Inulin (400 mg) might be an advantageous influence on the antioxidant properties of healthy human plasma after 7 weeks. Briefly, the symbiotic could be a significant increase in CAT activity; however, the activity of SOD and GPx was an insignificant increase compared to a control group. The result was slightly dissimilar because of the difference in probiotic bacterial strain. These results could show that VFJ with probiotic bacteria affected oxidative stress. Several mechanisms explained the probiotics affecting antioxidant activity and reduced damages caused by oxidation. The probiotics have their antioxidant enzymatic system, especially SOD. Furthermore, the probiotic strain was able to provoke the system of antioxidation in the host and increased the antioxidant enzymatic activity. As a result, they can prevent or decrease the seriousness of intestinal pathology caused by reactive oxygen species [38]. Moreover, the metabolites of antioxidant activity were produced by probiotics. For example, butyrate, as SCFA, was generated by the fermentation of microbiota in the small intestine and/or a final section of the small intestine led to induce antioxidative enzymes [39]. Wang et al. [38] reported that the probiotics *Lactobacillus* and *Bifidobacterium* were able to produce lactic acid, acetic acid, and propionic acid, resulting in a lower intestinal pH, maintaining a balance of the gut microbiota. The probiotics can also regulate the growth of harmful bacteria, which may contribute to reduced oxidative stress.

BAs or bile salts are synthesized from cholesterol in the liver and, subsequently, become conjugated to glycine or taurine, which increases their solubility [40,41]. Approximately 200–600 mg of BA is produced daily in the human liver and excreted in feces [40]. The alteration in the BA level of the probiotic group was significant increased by 19.40%. Thus, based on the marked increase in BAs within the probiotic group of the current study, it could be inferred that the BSH activity of *L. paracasei* reduced the TC, LDL-C, TG, and

TG/HDL-C ratio, resulting in increased fecal BAs [33,42]. The BAs that are lost in the feces are then regenerated from cholesterol, via de novo synthesis, in the liver to maintain a constant BA pool [40,41], thus, reducing serum cholesterol levels [33].

The level of lipid profile, lipid peroxidation, oxidative stress enzyme, and bile acid, especially the TG/ HDL-C ratio, can indicate a risk rate of dyslipidemia [43]. These available results could be explained in more details. The treatment period should be extended to more than 30 days and/or the amount of VFJ and probiotic *L. paracasei* should be adjusted to a higher level: more than 500 mL/day and 10<sup>9</sup> CFU/day, respectively. Further research is warranted to investigate the impact of the consumption of VFJ with probiotics on patients who have higher LDL-C levels or who have been administered lipid-lowering drugs to prove the effectiveness of the juice with probiotics combined with a medical treatment.

## **5. Conclusions**

According to the present study, the intake of VFJ with *L. paracasei* at the dosage of 2 <sup>×</sup> <sup>10</sup><sup>9</sup> CFU/day for 30 days could serve as an effective alternative strategy for the primary prevention of dyslipidemia in Bhumibol Adulyadej Hospital patients who had not undergone medical treatment. Specifically, intake of VFJ with *L. paracasei* could provide health benefits by improving lipid profiles, lipid peroxidation, and oxidative stress enzyme activity levels. The level of TC, LDL-C, TG, and TG/ HDL-C ratio significantly decreased in the probiotic group (consumption of VFJ with *L. paracasei*) compared to the placebo group (consumption of VFJ without *L. paracasei*). While the HDL-C marker was a high level in the probiotic group. Moreover, the level of MDA in the probiotic group was significantly decreased compared to the placebo group. However, the activity of CAT and GPx significantly increased in the participant group of VFJ with *L. paracasei* consumption. The BA level in feces significantly increased in the probiotic group. Therefore, intake of VFJ with *L. paracasei* could be improved to benefit health in terms of the lipid profile, lipid peroxidation and oxidative stress enzymes.

**Author Contributions:** Conceptualization, P.S., S.S. and C.C.; methodology, P.S., S.S., N.L. and C.C.; formal analysis, P.S.; data curation, P.S.; investigation, P.S., S.S., N.L., C.C., N.M. and E.K.; writing—original draft preparation, P.S.; writing—review and editing, P.S., S.S., N.L., C.C. and N.M.; supervision, S.S., N.L. and C.C.; funding acquisition, P.S., S.S. and C.C.; project administration, S.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Research and Invention Center of Bhumibol Adulyadej Hospital, Bangkok, Thailand, and the Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand. Part of this research was supported by the Innovation Center for Holistic Health, Nutraceuticals, and Cosmeceuticals, Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand.

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

**Informed Consent Statement:** Not applicable.

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

**Acknowledgments:** The authors would like to express their gratitude to Bhumibol Adulyadej Hospital, Royal Thai Air Force, Bangkok, Thailand and Chiang Mai University, Chiang Mai, Thailand for research support. This research was partially supported by Chiang Mai University.

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

#### **References**


*Communication*
