Potential of barley enriched yogurt to improve probiotic growth for the management of hypercholesterolemia

ABSTRACT This study aimed to evaluate the effect of barley bran and flour-enriched yogurt against hypercholesterolemia in rats. Purposely, barley-enriched yogurts were prepared, and their quality characteristics including physicochemical, viscosity, textural and sensory analysis of functional yogurts were performed. The enrichment of 6% barley flour and barley bran in yogurt improved the viscosity, textural, physicochemical and sensory attributes. The barley bran and flour-enriched yogurt improved the growth of probiotics in T3 and T6. A hypercholesterolemic rats study was conducted and observed that lipid profile improved and glucose significantly decreased in response to functional yogurts. The improvement in functions of ALT (71.83 ± 1.61, 67.03 ± 1.67 and 62.33 ± 2.08 U/L), AST (245.17 ± 1.04, 231.33 ± 3.06 and 223.60 ± 2.79 U/L) and ALP (271.33 ± 1.53, 208.83 ± 1.26 and 189.37 ± 0.78 U/L) was observed G1, G4, and G5 groups, accordingly. It was concluded that barley bran exhibited greater potential than barley flour in the improvement of yogurt quality and hypercholesterolemia management. GRAPHICAL ABSTRACT


Introduction
Cardiovascular disorders (CVDs) are the leading cause of higher death rates worldwide.People's hectic lifestyle has shifted their concern toward fast foods, including fried, roasted, and many junk foods having higher amounts of fats.After meeting the body's requirements, these higher amounts of fats accumulate in the body tissue reservoirs and result in fats-linked severe health issues such as hypercholesterolemia, high blood pressure, multiple cardiovascular disorders, higher glycemic index, and higher levels of triglycerides, LDLs levels, and oxidative stress.Hypercholesterolemia among the top fat-related ailments can be defined as increased serum lipids levels.It plays a significant role in raising the rate of CVDs. [1]In this concern, various scientists and investigators are making continuous efforts in the development of several modified foods capable of meeting the current requirements of consumers for healthy lifestyles.These modifications to foods include a reduction in calorie intake and a reduction in higher amounts of cholesterol and saturated fats in various food products. [2]Lowdensity lipoprotein cholesterols (LDL-ch) show atherogenic effects beyond 100 mg/dL.Hence, as the LDL cholesterols levels are increased, the disease risks are also increased. [3]n recent years, functional foods have gained tremendous attention due to their health benefits.Functional foods enriched with prebiotics, especially dietary fibers, are well known for their utilization in biotechnological applications involving fermentation processes globally.These foods significantly contribute to consumers' health by preventing and reducing the risks of health hazards. [4]Considering the role of dietary fibers, barley β-glucans have a central focus on their bioactive and functional properties.β-glucans are glucose polymers consisting of 1 → 3 and 1 → 4 linkages which contribute to their unique functional attributes, physical characteristics, and some rheological properties, i.e., Water holding and stability capacity solubility, and viscosity. [5]These unique properties of barley are the main reason for diverting the attention of scientists to its utilization in the processing and development of various products.Yogurt is considered a good source of probiotics.Adding dietary barley fibers in yogurt development improves viscosity and textural characteristics and reduces syneresis.
Therefore, yogurt containing probiotics prepared from barley or its components considerably impacts bacterial attraction and depicts the optimistic effect of probiotic-electrocyte relationships. [6]his study was focused on developing probiotics-based barley flour and barley bran-enriched yogurt to lessen hypercholesterolemia levels.This research aimed to select the most effective formulation with the addition of barley bran and barley flour and the manufacturing procedure to develop the functional yogurt.Moreover, the effects of these functional yogurts were evaluated on hypercholesterolemia-induced Sprague Dawley rats.

Procurement of raw material
The present research work was carried out at the Faculty of Food Science and Nutrition, Bahauddin Zakariya University, Multan.For this purpose, barley bran and barley grains were procured from the local market.MRS Agar and M17 agar were purchased from Merck Sigma-Aldrich® Solutions, Darmstadt, Germany.

Pretreatment and storage of raw material
The fine flour was prepared by grinding barley grains using a small grinder (Model No:51822, National Home Appliances, Pakistan).Then, the flours were packaged in zip lock bags and stored at room temperature for further study.

Proximate and physicochemical analysis of barley bran, barley flour and yogurt
The yogurt samples were prepared with the incorporation of barley bran and barley flour in different concentrations.They were analyzed for their composition, such as protein, ash, moisture, fiber, fat, and Nitrogen free extracts, according to their respective procedures provided by AOAC. [7]

Addition of barley flour and bran in yogurt
The yogurt was prepared by adding barley flour and bran, and disposable cups with proper lids were used to pack the yogurt.Then, it was stored at 4ºC in the refrigerator for further analysis.The concentrations of barley bran and flour used for enrichment are given below in Table 1.

Sensory evaluation of yogurt
The prepared yogurt was examined by a panel of 15 judges of faculty members, staff, and students for quality traits such as mouth feel, taste, appearance, texture color, and overall acceptability.The 9-point hedonic scale designed by the American Dairy Science Association was utilized to evaluate the sensory attributes of enriched yogurt samples. [8]The questionnaire containing all information regarding the yogurt sample was provided to the judges to collect their opinion.

Viscosity and texture analysis
The viscosity of all the yogurts was measured using a viscometer (Brookfield LVDVE-230) with modifications provided by Amerinasab et al.. [9] All readings were observed in Millipascal Second (mPa.S) Unit.A food texture analyzer (FRTS 100N, Imada, Japan) was used to evaluate yogurt's texture profile (stiffness).The cylinder probe with a 20 mm diameter was used to evaluate the stiffness of the yogurt.The penetration strength at the geometrical center of the yogurt sample was 30 mm, and the penetration speed was 5 mm/s. [10]termination of probiotics i.e., Streptococcus thermophilus and Lactobacillus bulgaricus The dilution of the 10 g yogurt sample was done decimally in peptone base sterile water (0.01%) in Petri plates, and streptococcus thermophilus was grown on media M17 agar supplemented with lactose (10% w/v) and sterile water (5%) at 45°C/2-days with triplicate plates.However, MRS agar media was used to count Lactobacillus bulgaricus in triplicate samples with the same environmental condition by adjusting pH to 5.2. [11]

Experimental animal model
The role of barley bran and barley flour yogurts was assessed on hypercholesterolemia by planning an experimental model.For this reason, 30 rats were acquired (5 in each group) and kept in an animal room of the Faculty of Food Science and Nutrition, Bahauddin Zakariya University, Multan.Rats were acclimatized by providing water and a regular diet for a week.

Composition of experimental diets and induction of hypercholesterolemia
According to the procedure reported by Zulet et al. [12] model, a high cholesterol diet-induced hypercholesterolemia with some modifications.The standard diet contained 0% cholesterol.The complete composition of the diet is given in Table 2.

Experimental protocol
There were six groups, G 0, G 1, G 2 , G 3, G 4, and G5, in which rats were distributed, and each of them consisted of four rats (Table 3).In the experimental trial of 4 weeks, the regular diet was given to G 0 alone as a normal group and high-cholesterol diet was given to other groups to induce hypercholesterolemia.After 2-weeks, baseline measurements were recorded, and dietary requirements were adjusted according to the hypercholesterolemia induction level.After inducing hypercholesterolemia, the G 1 group was fed a regular diet without any treatment.G 2 and G 3 were also given 4% barley bran and 4% barley flour yogurt along with a standard diet.Moreover, G4 and G5 groups were given 4% barley bran and 4% barley flour yogurt along with a cholesterol-containing diet to evaluate their effects on hypercholesterolemia.

Biological assessment
Body weight was evaluated weekly.After the completion of the trial period, the body weight was measured.Blood samples were obtained, and parameters such as serum lipid profile and liver enzymes (ALT, AST, and ALP) were considered for the biological assessment of Hypercholesterolemia. [13]

Determination of serum triglycerides
Liquid triglyceride (GPO-PAP) method of Fadhilah et al. [14] was followed to determine triglycerides in serum.

Determination of serum lipid profile
The CHOD-PAP method of Kaur et al. [15] was followed to estimate the serum total cholesterol level of the rats.Estimating the level of HDL and LDL was done by following the method reported by Aslam et al.. [16]

Determination of glucose
Plasma glucose from the serum samples of rats was measured on the BTS-350 spectrophotometer through glucose assay (glucose oxidase/peroxidase endpoint analysis technique). [17]

Statistical analysis
The statistical analysis was carried out on Statistics 8.1 software.The results of all parameters were evaluated through a randomized design and 2-way factorial design by the Tukey test at the significance level (p < .05).

Results
The current study was planned to analyze the proximate composition of barley bran and barley flour yogurts.In addition, the hypocholesterolemic impacts of barley bran and barley flour-enriched yogurt were evaluated.This study was divided into two parts.The first part was based on the proximate and physicochemical analysis of formulated yogurts.In contrast, the second part considered the comparative efficacy of barley flour and barley bran yogurt in Sprague Dawley rats to evaluate the potential effects of barley flour and barley bran yogurt against hypercholesterolemia.

Proximate analysis of barley bran and barley flour
The proximate analysis of barley bran and barley flour was conducted to evaluate their nutritional value.The moisture contents in barley flour and barley bran observed were 10.72 ± 0.15 and 8.82 ± 0.15%, accordingly.The ash contents were lower in barley flour (2.69 ± 0.12%) than in barley bran (4.09 ± 0.18%), as shown in Table 4.

Physicochemical analysis of barley bran and barley flour enriched yogurt
The yogurt was prepared by adding barley bran and barley flour in different concentrations to evaluate its physicochemical properties.For this purpose, the Fat, proteins, pH, TSS, and acidity of these enriched yogurt samples were measured.The highest fat percentage was observed higher in T 0 with a value of 3.2 ± 0.14%.The fat percentages observed in T 1 , T 2 , and T 3 were at par with T 4 , T 5 , and T 6 accordingly, as presented in Table 5.The highest protein contents were observed in T 5 and T 6 with values of 3.0 ± 0.01 and 3.0 ± 0.01%, accordingly.Moreover, T 0 and T 2 showed similar results to T 4 and T 3 accordingly.However, T 1 (2% barley-enriched yogurt) showed the least protein content.The highest pH was observed in T 0 and T 4 with values of 4.5 ± 0.01 and 4.5 ± 0.12%, respectively, followed by T 3 .The mean pH values in T 2 and T 6 were similar such as 4.3 ± 0.02 and 4.3 ± 0.01, respectively.Moreover, the value observed for pH in T 1 (4.2 ± 0.1) was at par with T 6 (4.2 ± 0.23).The TSS observed in T6 was highest, followed by T 3 , T 5 , T 2 , T 1 , T 4, and T 0. The acidity percentage was the same in all treatments with negligible variations except T 4 (0.6 ± 0.02%).

Impact of barley on texture and viscosity and texture of yogurt
There was a non-significant difference among the lower angles of the groups while upper angles showed significant differences, i.e., the lowest value of T1 was 74.50 ± 4.84°.There was a non-significant difference among the lower viscosity of the studied groups, and upper viscosity had significant differences.For instance, T2, T3, T4, and T5 showed almost the same values of lower angle among all treatment groups.However, T6 (6% barley bran) showed the least value of lower angle when compared to T1 (control).T1 showed a slight decrease in its value at the upper angle compared to all other treatment groups and the control group (Table 6).T3 (6% barley flour) showed the least value, whereas T6 (6% barley bran) showed the highest value of upper viscosity.The textural analysis showed that the texture of yogurt was improved by adding bran and barley flour.The highest force was observed on T3 (1.33 ± 0.12 N), followed by T6 (1.20 ± 0.10 N), and the least force was observed on T0 (0.80 ± 0.00 N).

Impact of barley on probiotics growth in yogurt
The cultures of probiotics were used in yogurt to evaluate the relationship between probiotics and the addition of barley bran and barley flour in yogurt.The selected probiotics, i.e., Lactobacillus bulgaricus  and Streptococcus thermophilus, were determined every week during the 21 days study period.The results indicated that the highest growth of Lactobacillus bulgaricus was observed in T6 (6% barley bran yogurt), followed by the T3 (6% barley flour), T5 (4% barley bran), T2 (4% barley flour), T4 (2% barley bran), T1 (2% barley flour) and T0 (control).The bacterial count (CFU/g) was increased in all groups from the 0-7 th day.Similarly, a significant increase in all groups and a minor decrease in T6 were observed on the 14 th day.The CFU/g in all groups was decreased while increasing in T5 and T6 on the 21 st day of storage, as shown in Figure 1.The results exhibited that the various concentrations of barley bran and barley flour improved the growth rate of Lactobacillus bulgaricus in all yogurts samples.The microbial count of Streptococcus thermophilus in all yogurts was also carried out at intervals.The negligible increase of S. thermophilus count (CFU/g) in the control group was observed, while a significant increase in all other treated groups was noticed.However, the S. thermophilus exhibited the highest growth in T6 and least in T2 among the treated groups during the storage study, as shown in Figure 2.

Sensory evaluation of yogurt
The sensory analysis was carried out through the questionnaire, and the 9 points hedonic scale was used to collect the judges' responses about the sensorial characteristics such as appearance, color texture, taste, mouth feel, and acceptability of enriched yogurt samples.The mean values of judges' scores suggested that T 0 (8.8 ± 0.11) was ranked 1 st regarding its appearance, followed by the T5 (8.36 ± 0.35), T 2 (8.3 ± 0.37), T3 (7.02 ± 0.75), T1 (6.5 ± 0.27), T4 (5.89 ± 0.1) and T 6 (4.73 ± 0.06).The highest scores were observed in T 0 for color.The points given to T2 were at par with the T5.Moreover, the descending  trend was observed for color in T 1 , T 3 , T 4 , and T 6 .After T 0 (control), T 5 Yogurt was suggested best for texture, followed by T 2 , T 1 , T 3 , T 6 , and T 5 .The scores for taste were highest in T5, followed by T0, T2, T4, T6, and T3.The highest value for mouth feel was observed in T 2 , followed by T5 and T 0 .However, the points suggested for T 3 were at par with T 4 , and the least points were observed for T 1 .The overall acceptability was calculated for each sample using all properties.The points for overall acceptability suggested that T2 and T5 were at the highest rate of acceptability with approximately the same values, followed by the T0.However, T1, T4, and T6 were at the lower level, as shown in Table 7.

Effect of enriched yogurt treatments on organs and body weight
The analysis of organ weight is imperative to evaluate the possible beneficial effect of any component during various studies.The organ weight comparison is an important factor along with body weight differences of experimental animals (rats).It is a crucial indicator for the analysis of the effect of any component on an experimental subject, i.e., considerable variations in the weight of organs can be observed between untreated and treated rats without any morphological changes.The highest heart weight was observed in G1, followed by G2, G0, G3, G4, and the least in G5.The highest and lowest values for liver weight were observed in G1 and G0.However, mean liver weights for G2, G4, and G5 were at par.Likewise to heart and liver weight, the highest body weight was observed in G1, followed by G5, G0, and G2, and the lowest was observed in the G3 group, as shown in Table 8.

Effect of enriched yogurt on liver enzymes
The effect of functional yogurt was observed on liver enzymes.The group (G1) fed on a cholesterolcontaining diet showed the highest value (71.83 ± 1.61 U/L) of ALT, and G3 showed the lowest value  (37.25 ± 1.09 U/L) of ALT, which was almost similar to the value of G2 (37.42 ± 0.95 U/L).However, the groups (G4 and G5) fed on a cholesterol diet + barley flour yogurt and cholesterol diet + barley bran yogurt-containing diet showed higher results due to increased cholesterol levels.The same trend was observed in the AST and ALP enzyme values, as presented in Table 9.The same trends were due to their functions which were interlinked with each other.

Effect of enriched yogurt on lipid profile
The effect of barley flour and barley bran-enriched yogurts on the lipid profile of the experimental subjects was evaluated by measuring the TGs (mg/DL), TCs (mg/DL), HDL (mg/DL), LDL (mg/DL), VLDL (mg/DL), Glucose (mg/DL) from their blood samples given in Table 10.The Group (G1) was observed with the highest amounts of TGs (69.43 ± 6.54), but G3 and G5 consuming groups showed a significant reduction in TGs compared to their respective controls.The TCs reduction was considerable in G2 compared to G0 and G5 compared to G1.The same trends were observed in HDLs, LDLs, VLDLs, and glucose concentrations.

Discussion
Barley bran and flour are good sources of dietary fibers i.e., β-glucans play a key role in the management of hypercholesterolemia. Barley bran and flour are the potential sources of prebiotics which enhanced the activities of the probiotics in dairy products, especially yogurt.In the current study, the proximate analysis of the yogurt prepared by the addition of barley bran and barley flour in various concentrations was carried out.Protein, fat, and NFE contents were higher in barley flour than in barley bran.However, the percentage levels of fiber were higher in barley bran (26.00 ± 0.58%) than in barley flour (15.96 ± 0.37%).Barley's composition of this study was in accordance with the study of El-Taib et al.. [18] The Physico-chemical analysis i.e., fat, proteins, pH, TSS, and acidity of these enriched yogurt samples were analyzed.Yogurts depicted the functional properties, beneficial for the management of hypercholesterolemia.The results of Mohammed [19] were correlated to this study.Mohammed explored the 16.62% TSS of yogurt.Elsanhoty et al. [20] reported 11.98%to 12.13% of total solids.Similarly, the viscosity of textural analysis depicted that the addition of barley bran and flour improved the texture of the yogurt.Various studies barley indicated that β-glucan in it promotes the viscosity of fermented dairy products such as yogurt (Zhao et al. [21] ; Xiao et al. [22] ; Shah et al. [23] ).
Hamid and Doosh [24] reported in a study that the addition of β-glucan improved the rheological properties of yogurt, i.e., viscosity and water-holding capacity, and sensory evaluation attributes of fatfree yogurt, especially the treatment with an additional rate of 0.4% of β-glucan.The increase in force on treated yogurts was due to the barley bran and barley flour addition which improved the texture of yogurt and enhanced its stability.It was reported by Kaur and Riar [25] that the addition of β-glucan significantly (P ≤ .05)improved whey separation (syneresis), viscosity, texture profile, and sensory characteristics during storage.The colony-forming units of probiotics i.e., Lactobacillus bulgaricus and Streptococcus thermophilus, were noticed every week during the 21 days study period.The results indicated that the increasing microbial growth rate was observed from the 0-14 th day and reduced on the 21 th day in most of the groups.Dancīte et al. [26] conducted a study on milk fermentation through Lactobacillus delbrueckii ssp.Bulgaricus and Streptococcus thermophilus with barley flour and grain were additionally added.This study reported that flour from non-germinated and 24 and 36 hours germinated barley grain, enriched yogurt with dietary fiber (from 0.89 to 1.77 g•100 kcal-1) promoted the growth of lactic acid bacteria in the product, increased the viscosity, and shortened fermentation time.Hasani et al. [27] conducted a study and reported the incorporation of barley bran in low-fat yogurt containing Lactobacillus ssp.significantly affected viable probiotic bacteria in comparison with the control group.Lee and Lee [28] investigated the effects of barley flour added on the quality characteristics of yogurt during the fermentation.The addition of barley flour has been shown to affect the fermentation efficacy of lactic acid bacteria in yogurt.Lee et al. [29] reported that β-glucan from barley contributes to microorganism development in yogurt.
The sensory analysis has suggested that T2 and T5 yogurt contained an optimal concentration of barley flour and barley bran, which could potentially improve yogurt's sensory characteristics.Kaur and Riar [25] provided similar results of barley bran yogurt which were similar to our study.In this study, the effect of β-glucans present in barley on the alteration of sensory characteristics, i.e., flavor, taste, color, and acidity of yogurt, was evaluated.Consequently, Hamid and Doosh [24] prepared functional yogurt by adding barley bran in different concentrations and conducted a sensory evaluation for the same attributes considered in our study.The study revealed that 1.2%barley bran was rated the lowest point, and the points increased with the increase in barley bran concentrations.A study by Hasani et al. [27] reported that with an increase in barley bran concentration, taste, appearance, and texture, scores decreased significantly.
The efficacy study of barley bran and yogurt-enriched, yogurt resulted from their hypocholesterolemic impact on rats.The higher increase in the heart, liver, and body weight of the G1 group was due to the accumulation of cholesterol in the body's heart, liver, and adipose tissues.However, the results suggested that the G3 diet containing a regular diet and barley bran yogurt was more effective in inhibiting cholesterol accumulation in the body's adipose tissue.Moreover, a lesser increase in body weight of G5 was observed compared to G1 and G4 after the entire study period.It is concluded that barley bran yogurt significantly prevents the accumulation of cholesterol in adipose tissues in both conditions, whether consumed with high-cholesterol diets or with a regular diet.The inhibition of the increase in organ or body weight was supported by the study by Nie and Luo, [ [30] Diet containing a regular diet + barley bran yogurt and a cholesterol diet + barley bran yogurt is more effective than those containing barley flour yogurts on the functions of liver enzymes.Li et al. [31] studied the effects of barley on liver enzymes and resulted that it influenced the liver enzymes in rats.Mohammed et al. [32] suggested that enrichment of (G4) 4% long-chain inulin and 90 mg kg −1 Fe 2 (SO 4 ) 3 in equal amounts (1:1) in yogurt had considerable restorative effects in restoring hematocrit and hemoglobin levels along with controlling the liver by maintaining RBCs count.Moreover, the enzymatic activities, iron contents of the liver, and antioxidant properties were also improved by G4 Diet.The histological results indicated that G4 improved the structures of the liver, which were at par with the control group.It was noticed that barley bran-enriched yogurts significantly reduced cholesterol levels in all groups.However, the effect of barley flour was slightly less than barley bran.The improvement in lipid profiles with the addition of barley and its various parts was studied by different scientists. [15]Moreover, Rizvi et al. [33] studied similar trends of HDL cholesterol improvements in an animal model.Shoukat and Sorrentino, [34] explored the effects of barley β-glucans and probiotics bacteria enriched yogurt by testing the plasma of rats given higher cholesterols and depicted a negative association between excretions of bile acids and cholesterol levels.

Conclusion and recommendations
It was concluded that barley bran addition was more effective than barley flour in improving the growth and function of probiotics and preventing hypercholesterolemia via reducing higher cholesterol levels and maintaining their normal ranges.The barley bran was proved to be more effective in improving the activity of liver enzymes which significantly aids in the reduction of anemia.Moreover, the barley bran improved quality attributes i.e., physicochemical characteristics, viscosity and texture of functional yogurts.The barley bran and barley flour-based functional foods significantly contribute to the nutraceutical industry to cure various ailments.Consequently, further trials on barley bran are needed to ensure its safety and benefits to developed products meant for human consumption.

T 0 =Figure 1 .
Figure 1.Effects of Barley Flour and Barley Bran on Lactobacillus bulgaricus Growth.

Table 4 .
Proximate Analysis of Barley Flour and Barley Bran.

Table 5 .
Physicochemical Analysis of Yogurt.

Table 6 .
Impact of barley constituents on viscosity and texture of yogurts.

Table 7 .
Effects of Barley Flour and Barley Bran on Streptococcus thermophilus Growth.Mean Values of Sensory Analysis of Yogurt.

Table 8 .
Comparisons Test of Organs and Body Weight.

Table 9 .
Effect of Enriched Yogurt on liver enzymes.

Table 10 .
Effect of Barley Enriched Yogurt on Liver Enzymes.