Barley, which is rich in β-glucans, is known to exhibit hypocholesterolemic effect.
The study aimed to investigate the hypocholesterolemic effect of yogurt containing barley β-glucan (BBG) and probiotic bacteria in rats fed on a cholesterol-enriched diet.
The methodology was based on adding of 0.75% BBG to skim milk (SM) powder. Four treatments of yogurt were formulated, wherein the first treatment was produced from SM without the addition of BBG and fermented by yogurts starter (YS). The second treatment was produced from SM with the addition of 0.75% BBG, and fermented by YS. The third treatment was produced from SM without the addition of 0.75% BBG, and fermented by Bifidobacterium lactis plus Lactobacillus acidophilus. The fourth treatment was produced from SM with addition of 0.75% BBG, and fermented by Bifidobacterium lactis plus Lactobacillus acidophilus. All formulations were evaluated for their effect on plasma lipids, liver lipids, lipid peroxidation, and the fecal excretion of bile acids in rats.
The results indicated that yogurt containing probiotic bacteria and BBG was more effective in lowering of plasma and liver cholesterol levels than other treatments. The fecal excretions of bile acids and lipid peroxidation were markedly promoted in yogurt formulated with BBG and probiotic bacteria compared with the positive control group. The results showed an inverse relationship between the fecal excretions of bile acids and the levels of total cholesterol in the plasma from rats fed on a high-cholesterol diet.
The inclusion of BBG and probiotic bacteria in the diet of rats fed on high-cholesterol diet had health-promoting impacts on the levels of plasma and liver lipids. Yogurt with Bifidobacterium plus L. acidophilus and supplemented with BBG were effective in lowering the levels of cholesterol in plasma and liver lipids, while the excretion of bile acids in the feces was enhanced. These hypocholesterolemic effects of yogurt preparations containing BBG and probiotic bacteria could create an effective and economic contribution in treating hypercholesterolaemia.
There is an increasing interest in the popularity of functional foods due to the increase in consumer awareness and demand . The applications of probiotics in food products have been well established throughout generations. The interest in the microorganisms in the recent years emanated from the discovery of their salubrious impact in lowering plasma cholesterol. The amount and type of fat consumed are important to the etiology of several chronic diseases, such as obesity, cardiovascular diseases, and cancer [2–4]. The serum cholesterol level is widely considered as a contributory risk factor for the development of cardiovascular diseases (CVD) such as atherosclerosis, CHD and stroke. The World Health Organization (WHO) has predicted that by 2030, CVD will remain the leading causes of death and affect approximately 23.6 million people globally .
The reported health promoting ability of lactic acid bacteria (LAB) in humans and livestock include cholesterol-lowering effect , inhibition of pathogenic microorganisms , immune-modulation , and neutralization of food mutagens produced in the colon and halting of intestinal dysfunction . Some bacterial species excrete bile salt hydrolase, leading to increased bile excretion in feces . However, other reports are contradictory and fail to show hypocholesterolemic effects of probiotics [11–13]. Consequently, this area remains controversial.
β-Glucan is a functional bioactive ingredient comprise a group of β-D-glucose polysaccharides found in the cell walls of cereals, yeast, bacteria, and fungi, with different properties dependent on the source . Food and Drug Administration (FDA) has approved β-glucan (3 g/day) to qualify for the coronary heart disease (CHD) claim FDA . Fortification of foods with β-glucan is of great interest. Attempts to fortify foods with β-glucan including pasta, tea cakes , muffins , bread , and beverages have been reported [17–20]. Worrasinchai et al.  stated that β-glucans is poorly absorbed in the human digestion tract and, therefore, β-glucans could be used as a non-caloric food and can be used in foods as a thickener, water retention, or oil bending agent and an emulsion stabilizer [2, 22–26].
β-glucans is known to exhibit hypocholesterolemic impact. Increased intestinal viscosity is thought to be crucial for cholesterol lowering. Concentration, structure, and molecular weight, play important roles in β-glucan functionality . β-glucans decreased serum total cholesterol (18.9%), and LDL-cholesterol (24.3%) in Sprague-Dawley rats . Addition of β-glucan also resulted in greater bile acid excretions. It was suggested that the hypocholesterolemic effects of β-glucan might be due to the enhancement of CYP7A1 expression resulting from increased fecal excretion of bile acids. On the other hand, β-glucan extracted from oat and barley did not affect cholesterol metabolism in young healthy adults .
Yogurt is traditionally made from fermenting milk. The belief in the beneficial influence of yogurt on human health and nutrition has existed in many civilizations over a long period of time . Milk fat plays an important role in the texture, flavor and color development of dairy products. Although the manufacture of low-or non-fat dairy products was carried out for many years, the use of fat replacers in the manufacture of dairy products is still novel. β-glucans use in food is interesting, especially in yogurt wherein non-fat yogurt enriched with β-glucan could be used as health promoter for many people suffering from diseases. There are limited reports on the fortification of yogurt with barely β-glucan (BβG). Sahan et al.  studied the effects of adding β-glucans on yogurt properties but they used very low levels of β-glucans (0.05%) from a hydrocolloidal composite of β-glucans. Vasiljevic et al.  studied the growth and metabolic activity of probiotic organisms in β-glucans enriched yogurt and reported that addition of oat β-glucans resulted in improved probiotic viability and stability. Brennan and Tudorica  found that β-glucans (0.5%) addition improved serum retention and viscoelastic nature of yogurt.
There is little information about the hypocholesterolemic effects of yogurt fortified with probiotic bacteria and BBG as a fat replacer. This study was undertaken to provide such information on the hypercholesterolemic effect of yogurt containing probiotic bacteria and BBG in rats fed on a high-cholesterol diet.
2Materials and methods
Spray-dried skim milk (SM) powder (type low heat, grade A) was obtained from local market (Giza, Egypt). Hull-less barley was obtained from Institute of Field Crop Research (Agricultural Research Centre, Giza, Egypt).
2.2Extraction of barley β-glucan (BBG)
BBG was extracted from hull-less barely flour according to Benito-Román et al. . Hull less barley flour was weighted and added to an Erlenmeyer flask, then water was added wherein the liquid: solid ratio was 10 : 1 and pH of water was 6. Erlenmeyer flask transferred to a water bath, incubated for 3 h at 55°C and the flour suspended at the high stirring rate. The clear supernatant was collected by centrifugation at 5500 rpm and 4°C for 10 min. BBG was extracted from the clear supernatant after adjusting the concentration with absolute ethyl alcohol to 30%. The precipitated BBG was freeze-dried (Christ BETA 2–16, Osterode am Harz, Germany). The final product was packed in a plastic container and keep at –20°C until used.
2.3Bacterial strains and culture preparation
Yogurt cultures of Streptococcus salivarius sub sp. thermophilus, and Lactobacillius dulbrueekii sub sp. bulgaricus were obtained from Egyptian Microbial Culture Collection (EMCC) at Cairo Microbiological Resources Centre (MIRCEN, Faculty of Agriculture, Ain Shams University, Egypt). Bifidobacterium lactis Bb-12, and Lactobacillus acidophilus LA-5 (freeze-dried) were obtained from Chr. Hansen laboratories (Copenhagen, Denmark). Strains were cultured on deMan, Rogosa and Sharpe (MRS, Difico Laboratories) agar plates. Anaerobic strains were kept in an anaerobic jar (Anaerogen, Oxoid). Fully grown colonies were stored on plates at 4°C until needed with sub culturing on a monthly basis. For long-term conservation of strains, spore or cell suspensions were kept in cry vials at –80°C with 90% glycerol as cryoprotectant. Lactobacilli were cultivated in MRS broth and bifidobacteria were grown in MRS broth with some supplementation with cysteine and incubated for 24 h at the suitable growth temperature. An appropriate volume of overnight culture was used to inoculate 150 mL cultures and incubated for 24 h at 37°C in an anaerobic jar (Anaerogen, Oxoid). The culture was prepared by adding a few mg of the subculture to 100 mL of previously reconstituted and sterile (121°C/2 min) skimmed milk (10% total solids). This mixture was incubated at 42°C until the onset of gelatin. Two mL of culture from this passage were transferred into 100 mL of sterile SM at 42°C and the culture was incubated until a gel had just formed. This second culture was used for the propagation of a bulk culture (1 L) for inoculation of the different treatments. Bulk cultures were prepared 24 h before the production of yogurt.
2.4Preparation of yogurt
Preliminary studies were carried out to select the suitable concentrations of BBG that can be used in the production of low-fat yogurt. Different amounts (0.25%, 0.5%, 0.75%, and 1%) of BBG were incorporated in the yogurt mixtures to substitute fat in milk. The yogurt samples were sensory evaluated and the results indicated that the 0.75% BBG had the highest score. Based on the preliminary studies, 0.75% of BBG was added to SM powder to produce low-fat yogurt in the further investigation.
Fresh whole cow’s milk was obtained from the Dina company (Giza, Egypt). Yogurt was produced as control samples according to Singh et al. . The yogurt was prepared by blending with 13% (w/v) non-fat milk solids and water at 50°C. The mixture was divided into four portions. The first portion was heated at 80°C for 15 min and cooled to 45°C, then inoculated with 3% (v/v) of Streptococcus salivarius sub sp. thermophilus plus Lactobacillus dulbrueekii sub sp. bulgaricus and abbreviated as YSM (treatment I). The second portion was mixed with 0.75% BBG, heated at 80°C for 15 min and cooled to 45°C, then inoculated with 3% (v/v) of Streptococcus salivarius sub sp. thermophilus plus Lactobacillus dulbrueekii sub sp. bulgaricus and abbreviated as YSMBBG (treatment II). The third portion was heated at 80°C for 15 min and cooled to 45°C, then inoculated with 3% (v/v) of probiotic bacteria (Bifidobacterium lactis Bb-12 plus Lactobacillus acidophilus LA-5) and abbreviated as PYSM (treatment III). The forth portion was mixed with 0.75% BBG, heated at 80°C for 15 min and cooled to 45 °C, inoculated with 3% (v/v) from probiotic bacteria (Bifidobacterium lactis Bb-12 plus Lactobacillus acidophilus LA-5) and abbreviated as BYSMBBG (treatment IV). All treatments were incubated at 42°C for 4 h until the pH reached 4.5. The yogurt samples were stored in the refrigerator at 5°C till further analysis.
2.5Counts of yogurt cultures, Lactobacillus acidophilus and Bifidobacterium lactis
Streptococcus thermophilus was enumerated using of M17 agar according to Ravula and Shah . L. delbrueckii subsp. bulgaricus was enumerated using of MRS agar. Enumeration of Lactobacillus acidophilus and Bifidobacterium lactic Bb 12 was performed using selective MRS agar (Himedia) under anaerobic incubation at 37°C for 3 days. Plates were incubated an aerobically at 37°C for 48 h. Table 1 indicates the viable count yogurt cultures, Lactobacillus and bifidobacteria in the experimental products.
|Treatment||Microbial count (107 cfumL-1)|
|Yoghurt culture||Probiotic culture|
|S. thermophilus||L. bulgaricus||B. Bifidium Bb 12||L. acidophilus LA5|
aThe data presented are the means of three replicate trials. bND: not determined.
Forty-eight weaning male albino rats (average weight 65–80 g) were obtained from Institute of Ophthalmology Research (Giza, Egypt). The animals were housed individually in well aerated cages with screen bottoms and fed on a basal diet as described by Reeves et al. . All the experiments using animals were performed under the permission of Ethical Committee of ARC (protocol 2014-15, 25 March 2014). Salt and vitamin mixtures were prepared as described in AOAC . Temperature and humidity were maintained at 25°C and 60%, respectively wherein food and water were provided. The animals were randomly sorted into six groups each group contain 8 rats. Group one was fed on control diet according to Reeves et al. , and considered as negative control group. Group two was fed on control diet with addition of 0.5 g cholesterol for each 100 g diet plus 50 mL water and considered as positive control group. Group three fed on control diet with addition of 0.5 g cholesterol for each 100 g diet plus 50 mL yogurt starter (YS). Group four fed on control diet with addition of 0.5 g cholesterol for each 100 g diet plus 50 mL yogurt enriched with 0.75% BBG (YSSMBBG). Group five was fed on the control diet with addition of 0.5 g cholesterol for each 100 g diet plus 50 mL from yogurt fermented by Bifidobacterium lactis plus Lactobacilus acidophilus (PYSM). Group six feed on control diet with addition of 0.5 g cholesterol for each 100 g diet plus 50 mL from yogurt fermented by Bifidobacterium lactis plus Lactobacillus acidophilus and included 0.75% BBG (PYSMBBG). The composition of diets and animal groups is presented in Table 2. During the experimental period (7 weeks) rats were kept separately in well-aerated cages. The diet consumed and body weight, organs weight were recorded at the end of the experimental period.
|Basel diet (cholesterol-free diet)*||Negative control (I)||100 g basel diet+50 mL water|
|Basel diet and cholesterol-enriched diet||Positive control (II)||99.5 g basal diet+0.5 g cholesterol+50 mL water|
|Cholesterol-enriched diet+YS (YSSM)**||III||99.5 g basal diet+0.5 g cholesterol+50 g (YSSM) yogurt|
|Cholesterol-enriched diet+(YSSM+BBG)||IV||99.5 g basal diet+0.5 g cholesterol+50 g (YSSM+BBG) yogurt|
|Cholesterol-enriched diet+probiotic yogurt (PYSMM)||V||99.5 g basal diet+0.5 g cholesterol+50 g (PYSMM) yogurt|
|Cholesterol-enriched diet+probiotic yogurt with BBG (PYSMBBG)||VI||99.5 g basal diet+0.5 g cholesterol+50 g (PYSMBBG) yogurt|
2.6.2Blood sampling and serum analysis
All feed was removed and blood samples were collected from rats within different treated groups from the orbital venous plexuses by a capillary tube at the end of experimental period. Blood serum was separated by centrifugation at 3000 rpm for 15 min to obtain plasma, which was kept frozen at –20°C until analysis. Total cholesterol (TC) in serum, high density lipoprotein cholesterol (HDL), and triglycerides (TG) were determined according to Trinder . Low density lipoprotein cholesterol (LDL) and very low density lipoprotein cholesterol (VLDL) were calculated as follows: VLDL+LDL cholesterol = TC – HDL. TC and TG were extracted from the liver according to Fernandez and McGregor , and measured by the method of Trinder (1969).
2.6.3Determination of lipid peroxidation
Malondialdehyde (MDA) level in plasma was determined as an index of lipid peroxidation because MDA is an end-product of unsaturated fatty acid peroxidation and can react with thiobarbituric acid to form a colored complex called the thiobarbituric acid reactive substance (TBARS). TBA reactivity was assayed by the method of Erdincler et al. .
2.7Feces collection and determination of bile acids
Feces were collected on the last 2 days of the feeding period and kept frozen (–25°C) until analyzed for bile acids. The rats were sacrificed and the liver, heart, kidney and spleen were excised and weighed. The liver was washed with an ice-cold saline solution and stored at 25°C. Total bile acids (TBA) value was determined according to Onning et al. . TC of feces was determined as described by Rudel and Morris . To extract bile salts, 1.0 g of feces were suspended in chloroform:methanol (1 : 1, v/v), then extracted at 60°C for 60 h. The extract was evaporated and dissolved in methanol to measure TBA.
Statistical analysis was performed by running student t-test using Stat view 512 software (1986). Chi-square was performed to compare between the controls and experimental yogurt. Significant effects were declared p < 0.05.
3Results and discussion
3.1Body weight and food intake
Data in Table 3 presents the total food intake by the animal groups at the end of the experiment. Data indicated that there was a lower food intake in rats that fed on each yogurt supplement compared to the control groups. The groups fed on the diet supplemented with yogurt (group III), yogurt plus BBG (group IV), yogurt with probiotic bacteria (group V) and probiotic bacteria plus BBG (group VI) showed a significant increase (p < 0.05) in body weight. All groups showed also differences in food efficiency (Table 3). The rats fed on the yogurt-supplemented diets demonstrated a lower food intakes compared to the control groups. However, the body weights of the rats treated with the yogurt were significantly higher than those of the control, due to the ingestion of yogurt, resulting in a decrease in food intake and an increase in body weight. The results were in agreement with the results obtained by Abd El-Gawad et al. ; Al-Sheraji et al. ; Mahrous et al. , and Ogunremi et al. .
|group||weight (g)||weight||intake (g)||efficiency|
|Negative control (I)||73.6±3.8||223.7±13.7||150.1±18.1||743.0±23.5||20.2±1.8|
|Positive control (II)||72.7±2.9||238.9±12.8||166.2±21.7||789.8±23.9||21.1±3.2|
*Food efficiency (%) = (body weight gain/food intake)×100.
3.2Body weight gain and organs weight
Data in Table 4 presents the effects of feeding on diet contained yogurt with BBG and probiotic bacteria on rats fed on a cholesterol-high diet. The group fed on cholesterol-high diet had the highest liver weight (6.985 g), while basal diet group had the least (3.925 g). There were significant differences in the liver weight between the group fed basal diet and the respective groups fed on cholesterol-high diet. There was a decrease in the liver weight for groups fed on cholesterol-high diet and supplemented with yogurt containing YS, probiotic bacteria and BBG. However, there is no significant difference was recorded. There were no significant differences in the weight of the heart for the six animal groups. There was a significant difference in kidney weight between rats fed the positive control diet and other groups. Similar results were reported by Abd-Al-Gawad et al. ; Al-Sheraji et al. ; Mahrous et al. ; Sharifuzzaman et al.  who reported that the incorporation of oat β-glucan and other fibers improve the body weight gain of animals due to the improve in the growth and performance. High cholesterol in diets induced an increase in liver weight suggested to be due to the accumulation of lipid masses in liver cells.
|Treatment/animal group||Weight after 7||Body weight||Organ weight (g)|
|weeks (g)||gain (g)||Liver||Kidney||Heart||Spleen|
|Basel diet (I)||223.7±13.7||150.1±18.1||3.92±0.2||0.90±0.1||0.72±0.0||0.42±0.1|
3.3Effect of feeding on the levels of plasma and liver lipids
Table 5 presents the effect of feeding different yogurt preparations on the levels of plasma and liver lipids in rats. The feeding of cholesterol-high diet for 7 weeks significantly increased the levels of serum TC, TG and LDL. Cholesterol-high diet in groups fed on probiotic bacteria and BBG had lower TC, TG and LDL. The serum TC levels in the groups fed yogurt supplemented with BBG (groups II, III, IV, V and VI) were significantly different from the positive control group with mean values of 145.7, 126.6, 131.8 and 120.7 mg/100, respectively. The cholesterol-high diet group had a serum TC content of 264.8 mg/100 mL. The serum TG and LDL-C levels in the respective groups also showed similar trends as serum TC. The control group that fed on the basal diet had serum TG and LDL values of 112.9 and 82.9 mg/100 mL, respectively. The data indicated that the diet contained yogurt supplemented with BBG and probiotic bacteria had effects on reduction of TC, TG and LDL. On the other hand, there were no significant differences in the plasma HDL levels between the negative control group and other experimental groups at the end of the experimental period. The data in Table 5 shows that the content of liver TC in the positive control group (7.36 mg/g) was significantly higher than that in the negative control group (2.93 mg/g). There were significant differences between groups fed on the positive control diet and those fed on basal diet and yogurt with supplemented with BBG. The basal diet supplemented with yogurt contained probiotic bacteria and BBG (groups III, IV, V and VI) were more effective in lowering the levels of liver TC than other diets. The obtained results agree with the results obtained by Abd Al-Gawad et al. , who found that yogurt or soy yogurt containing probiotic bacteria was more effective in the lowering of plasma and liver TC levels. Probiotic lactobacillus strains have also been reported to have hypocholesterolemic effect in rats  Singh et al. . The content of liver TG was significantly higher in rats fed on the positive control diet than those fed the negative control diet (Table 5). In contrast, the levels of liver TG in rats fed the experimental diet groups were significantly lower than in rats fed on the positive control diet. These results are in agreement with those reported by Kheadr et al.  and Xie et al.  who suggested that the yogurt fermented by Bifidobacterium lactis Bb-12 plus Lactobacillus acidophilus LA-5 and supplemented with BBG reduced TC in rat liver tissues.
|animal group||TC (mg/100 mL)||TG||HDL-cholesterol||VLDL+LDL-||Cholesterol||Triglycerides|
|(mg/100 mL)||(mg/100 mL)||cholesterol||(mg/g)||(mg/g)|
|Basel diet (I)||121.4±8.90||44.60±4.6||38.5±1.9||82.90±1.8||2.93±0.1||16.87±0.4|
*Values are given as means±SD from eight rats in each group.
3.4Effect of feeding on fecal bile acid excretion
Data in Table 6 show the effect of feeding on different yogurt preparations on the fecal bile acid excretion in rats. There was no significant difference in the levels of total bile acids between rats fed the negative control and positive control diets. The rat groups fed on yogurt supplemented with BBG and probiotic bacteria excreted significantly higher levels of bile acids than either the positive or negative control diets. The excretion of bile acids in rats fed on the yogurt supplemented with BBG and probiotic bacteria were significantly higher than those fed on yogurt with YS diets. These results demonstrated an inverse relationship between the level of bile acids excreted in the feces and the total levels of TC, LDL and VLDL in rats treated with diet contained probiotic bacteria and BBG. The results obtained in this study suggest that hepatic cholesterol metabolism could be altered to supply more cholesterol for bile acid synthesis. Several studies reported that Bifidobacterium spp. and Lactobacillus spp. enhanced both the secretion of bile acids and the activity of cholesterol 7-α-hydroxylase, a rate-limiting enzyme in the synthesis of bile acids  (Singh et al., 2015). Moreover, an in vitro study conducted by Tahri et al. demonstrated that bifidobacteria cells can eliminate cholesterol from the growth culture medium through both assimilation and coprecipitation with deconjugated bile salts. In this way, bifidobacteria increases the fecal excretion of bile salts. Similar results were obtained by Mahrous et al. , and Ivey et al.  who found that the incorporation of oat β-glucan with bifidobacteria and Lactobacillus in yogurt and synbiotic products increased the excretion of fecal bile acids in rats. This finding and our results suggest that hepatic cholesterol metabolism could change in order to provide more cholesterol for synthesis of bile acids.
|Treatment/animal group||Cholic acid||Chenodeoxycholic acid||Deoxycholic acid||Lithocholic acid||Total bile acids|
|(mg/100 g)||(mg/100 g)||(mg/100 g)||(mg/100 g)||(mg/100 g)|
|Basel diet (I)||204,6±2.9||35.6±1.8||20.7±0.9||0.9±16.8||277.7|
*Values are given as means±SD from eight rats in each group.
Table 7 presents the effect of feeding different yogurt formulations on lipid peroxidation in rats. Plasma MDA level was significantly (p < 0.05) increased in hypercholesterolaemic animals of the positive control group as an expression of the oxidative stress caused by a cholesterol-rich diet. Nagao et al.  has suggested that the accumulation of fat in the body might result in a high susceptibility to lipid peroxidation. The plasma MDA decreased significantly in all other groups that contained yogurt culture, BBG and probiotic bacteria (treatments III, IV, V and VI). Similar results were obtained by Ou et al. ; Uskova and Kravchenko, ; Harisa et al. and Singroh et al.  who found that the fermented dairy drink containing L. casei 114001 reduced MDA content in the blood plasma of Wistar rats. L. acidophilus has been shown to significantly decrease the elevation of MDA in diabetic rats wherein the inhibitory percentages of S. salivarius ssp. thermophilous ATCC 19258 and L. delbrueckii ssp. bulgaricus ATCC 11842 on plasma and liver lipid peroxidation were 57% and 41%, respectively. Lin and Chang  found that the inhibitory percentages of B. longum ATCC 15708 and L. acidophilus ATCC 4356 on plasma lipid peroxidation were 34% and 29%, respectively. The obtained results were agree with the results that obtained by Lin and Chang  and Abubakr et al.  who reported that LAB and probiotic bacterial strains possess highly antioxidant properties. Therefore, it could be said that the yogurt with probiotic and LAB supplemented with BBG has antioxidant properties.
|Treatment/animal||Blood MDA||Liver MDA|
|Basel diet (I)||9.12±0.4||9.43±1.0|
*Values are given as means±SD from eight rats in each group.
Our results demonstrated that the inclusion of BBG and probiotic bacteria in the diet of rats fed on high-cholesterol diet had noticeable health-promoting impacts on the levels of plasma and liver lipids. Yogurt samples with Bifidobacterium plus L. acidophilus and supplemented with BBG were very effective in lowering the levels of TC in plasma and liver lipids, while the excretion of bile acids in the feces was enhanced. The treatments containing yogurt fermented with Bifidobacterium plus L. acidophilus and supplemented with BBG have also antioxidant properties. These hypocholesterolemic effects of yogurt preparations containing BBG and probiotic bacteria could create an effective and economic contribution in treating hypercholesterolaemia, if these effects could be confirmed in human volunteers.
Conflict of interest statement
The authors declare no conflict of interests.
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