Hypocholesterolemic effects of diets containing different levels of kishk as a dried fermented milk–whole wheat mixture in experimental rats

==Abstract==

====Background====

''Kishk''  is a popular traditional functional food in Egypt. This study was performed to investigate the effects of different levels of ''kishk''  as a dried fermented milk/whole wheat mixture on growth performance, relative weight of organs, lipid profile, and some biochemical parameters in rats fed a cholesterol-rich diet.          

====Methods====

Forty male rats were assigned to five groups, each consisting of eight rats. The first one presents the negative control group that received the basal diet, while the second group that serves as the positive (+) control group received a high-cholesterol diet (HCD). The last three groups received HCD supplemented with 10%, 20%, and 30% of ''kishk'' .          

====Results====

Rats fed diets containing various levels of ''kishk''  for 8 weeks had significantly (''p''  < 0.05) lower body weights compared with the rats of both negative and positive groups. The liver/body weight ratio significantly increased in rats fed HCD compared with the control rats. Incorporation of ''kishk''  into the HCD at levels of 10%, 20%, and 30% significantly (''p''  ≤ 0.05) decreased the change of liver/body weight ratio by 14.46%, 17.51%, and 18.78%, respectively, when compared with the HCD group. Results also indicate that rats fed HCD had a state of dyslipidemia, compared with the negative control group. Administration of HCD supplemented with various levels of ''kishk''  for 8 weeks significantly (''p''  < 0.05) attenuated the increases in serum cholesterol, low-density lipoprotein cholesterol, triglyceride concentration, and atherogenic indices, and increased high-density lipoprotein cholesterol in a dose-dependent manner compared with the HCD group. Activities of liver enzymes (alanine transferase and aspartate transferase) as well as kidney function parameters (urea, uric acid, and creatinine) were elevated in the HCD group compared with the negative control group.          

====Conclusion====

Consumption of HCD supplemented with various levels of ''kishk''  for 8 weeks induced a significant protective effect reflected in the reductions of the serum levels of aspartate transferase and alanine transferase, as well as kidney functions (uric acid, urea, and creatinine).  

==Keywords==

biochemical ; function food ; hypercholesterolemia ; kishk ; whole wheat

==1. Introduction==

Hypercholesterolemia is the presence of high levels of cholesterol in the blood [[#bib1|[1]]] . It is a form of “hyperlipidemia” (elevated levels of lipids in the blood) and “hyperlipoproteinemia” (elevated levels of lipoproteins in the blood) [[#bib1|[1]]] . Hypercholesterolemia has emerged as a strong risk factor for cardiovascular disease (CVD). CVD is a severe problem in developed and developing countries. The World Health Organization (WHO) estimated that 17.3 million people died from CVD in 2008 and warned that 23.6 million people will die annually from CVD by 2030. CVD is a disease mainly caused by atherosclerosis [[#bib2|[2]]] . One of the risk factors of atherosclerosis is hypercholesterolemia [[#bib3|[3]]]  and [[#bib4|[4]]] , and low-density lipoprotein cholesterol (LDL-C) is the major cause of onset of the atherogenic process [[#bib2|[2]]] . Total cholesterol (TC) can be broken down into a diagnostic lipoprotein profile, including high-density lipoprotein (HDL), LDL, intermediate-density lipoproteins, very-low-density lipoprotein, chylomicron remnants, and triglycerides (TG). Patients at an increased risk of coronary artery disease frequently exhibit an atherogenic lipoprotein phenotype characterized by elevated plasma levels of both TG-rich lipoproteins and small, dense LDL and low concentrations of HDL-C. Recently, in a large observational study, the calculated non-HDL plasma cholesterol concentration (the sum of the cholesterol contents of LDL, intermediate-density lipoprotein, and very-low-density lipoprotein) was a stronger predictor of cardiovascular events than plasma cholesterol alone [[#bib5|[5]]]  and [[#bib6|[6]]] . Improvement in the predictability of coronary artery disease on inclusion of very-low- and intermediate-density lipoprotein cholesterol emphasizes the proatherogenic nature of TG-rich lipoproteins and their remnant particles. Control of cholesterol levels through therapeutic drugs have significantly reduced the risk of developing atherosclerosis and associated CVDs [[#bib7|[7]]] , [[#bib8|[8]]] , [[#bib9|[9]]]  and [[#bib10|[10]]] . Notably, statins, a class of cholesterol-lowering drugs inhibiting cholesterol synthesis, have most widely been prescribed for treating hypercholesterolemia and reducing CVDs [[#bib8|[8]]]  and [[#bib10|[10]]] . However, adverse effects associated with therapeutic drugs, such as myopathy, liver damages, and potential drug–drug interaction, have been reported [[#bib11|[11]]] , [[#bib12|[12]]] , [[#bib13|[13]]] , [[#bib14|[14]]] , [[#bib15|[15]]]  and [[#bib16|[16]]] . Therefore, development of additional therapies for controlling cholesterol levels is warranted, especially for those with better safety profiles. Functional foods have played an important role in the food industry during the past decade. These foods provide basic nutrition as well as health benefits such as disease prevention, or may delay the evolution of chronic disorders. Components in these foods can produce physiological benefits or remove compounds that may pose a health risk [[#bib17|[17]]] . Recent researches have focused on the search for functional foods for combating chronic diseases such as cancer, CVDs, and Type 2 diabetes [[#bib18|[18]]] . Fermented foods are an important part of diet in many parts of the world and are known from ancient times. Traditional dried fermented milk/cereal foods are widely used in the diet of people in the Middle East, Asia, Africa, and some parts of Europe [[#bib19|[19]]] . ''Kishk''  is one of the traditional food products in Egypt. It is a fermented milk–wheat mixture stored in the form of dried balls in Egypt  [[#bib20|[20]]] . ''Kishk''  is a balanced food with excellent preservation quality, richer in B vitamins than either wheat or milk, and well adapted to hot climates by its content of lactic acid, and it has a therapeutic value  [[#bib20|[20]]] , [[#bib21|[21]]]  and [[#bib22|[22]]] . ''Kishk''  product is a highly nutritious food, having a protein content of about 25.3%. It is highly digestible and of high biological value  [[#bib23|[23]]] . The main objective of the present study was to evaluate the hypocholesterolemic effect of diets containing different levels of ''kishk''  as a dried fermented milk/cereal mixture in rats.      

==2. Materials and methods==

===2.1. Ethics statement===

This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85–23, revised 1996). Experimental design and animal handling procedures were approved by the ethical committee of the Food Technology Research Institute, Agricultural Research Center, Giza, Egypt. Every effort was made to minimize the number of animals and their suffering.

''Kishk''  was purchased from the local market in Giza, Egypt. ''Kishk''  was ground in an electric grinder (Braun Model 1021), passed through a 150 μm mesh sieve, and stored in glass containers at 4°C for further use. The chemical composition and energy of ''kishk''  as determined by the Standard Association of Official Analytical Chemists (AOAC) methods (2000) are shown in  [[#tbl1|Table 1]] . Cholesterol and cholic acid were purchased from Sigma Chemical Co., Ltd (St Louis, MO, USA). The kits of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), Triglyceride (TG), transaminase enzymes [aspartate transferase (AST) and alanine transferase (ALT)], and kidney function parameters (creatinine, urea, and uric acid) were obtained from Biodiagnostic Co. (Dokki, Giza, Egypt). All chemicals used were of analytical reagent grade.

<span id='tbl1'></span>

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|+

Table 1.

Proximate composition and calorie content of ''kishk'' .                  

|-

! rowspan="2" | Component
! colspan="2" | ''Kishk''
|-

! %
! Energy (kcal/100 g)
|-

| Moisture
| 8.97
| —
|-

| Protein
| 18.64
| 74.56
|-

| Ash
| 7.30
| —
|-

| Crude fiber
| 7.57
| —
|-

| Fat
| 5.19
| 46.71
|-

| Total carbohydrate*
| 61.30
| 245.2
|-

| Total energy
| colspan="2" | 366.47
|}

* By difference.

===2.2. Animals and experiment design===

Forty male Albino rats with an average weight of 190–200 g were obtained from experimental animals of the Food Technology Research Institute, Agricultural Research Center, Giza, Egypt. The animals were pair housed in plastic cages at 25 ± 2°C and a humidity of 55 ±  6% with a 12-hour light–dark cycle. Food and water were provided ''ad libitum''  throughout the study. All the rats were acclimated to the basal diet for 2 weeks to stabilize the metabolic conditions before carrying out feeding experiments. The basal diet was formulated according to the AOAC methods  [[#bib24|[24]]] , and consisted of casein (15%), corn oil (10%), cellulose (5%), salt mixture (4%), vitamin mixture (1%), and starch (65%). Casein is characterized by its low content of sulfur-containing amino acids; hence, L methionine was added to the basal diet at the level of 4.6 g/kg diet. The composition of the vitamin and salt mixtures used was similar to that reported by AOAC [[#bib24|[24]]]  and Reeves et al [[#bib25|[25]]] , respectively. The rats were randomly divided into five groups (''n''  = 8). The first one presents the negative control group, which was fed only the basal diet, while the second group, which serves as the positive (+) control group, was fed a high-cholesterol diet (HCD) containing 98.75% basal diet, 1% cholesterol, and 0.25% cholic acid. The third, fourth, and fifth groups were fed HCD supplemented with 10%, 20%, and 30% of ''kishk''  respectively. The animal experiments conducted according to the recommendations of the Guide for the Care and Use of Laboratory Animals, published by the US National Institute of Health (NIH Publication No. 85–23, revised 1996). Experimental design and animal handling procedures were approved by the ethical committee of the Food Technology Research Institute; Agricultural Research Center, Giza, Egypt. Every effort was made to minimize the number of animals and their suffering.      

===2.3. Biological evaluation===

Daily food intake and weekly body weights were recorded during the experimental period (8 weeks). At the end of the experiment, total food intake, body weight gain, and food efficiency ratio were calculated.

===2.4. Relative organ weight (g) of experimental rats===

At the time of sacrifice, the hearts, livers, and kidneys of the experimental rats were identified, removed, rinsed with physiological saline solution, and dried by tissue papers. Weights of organs with respect to their body weights were immediately recorded.

===2.5. Blood sampling===

At the end of experimental period (8 weeks), rats were sacrificed after an overnight fast. The blood of each rat group was collected, centrifuged (3,500 rpm, 10 minutes, 4°C) to obtain the sera, and kept in a deep freezer (–25°C) until analysis.

===2.6. Biochemical analyses===

TC, HDL-C, LDL-C, and TG were determined according to the methods of Roeschlau et al [[#bib26|[26]]] , Assmann [[#bib27|[27]]] , Levy [[#bib28|[28]]] , and Fossati and Prencipe [[#bib29|[29]]] , respectively.      

===2.7. Atherogenic indices===

The atherogenic index (AI) and the cardiac risk factor were calculated using the following formulas: AI = TC – HDL-C/HDL-C [[#bib30|[30]]] , and cardiac risk factor = TC/HDL-C [[#bib31|[31]]] . Activities of alanine aminotransferase (ALT) (E.C. 2.6.1.2) and aspartate aminotransferase (AST) (E.C. 2.6.1.1) were measured according to the methods described by Bergmeyer and Harder [[#bib32|[32]]] . Urea, uric acid, and creatinine levels in rat sera were determined as outlined by Fawcett and Scott [[#bib33|[33]]] , Barham and Trinder [[#bib34|[34]]] , and Bartles et al [[#bib35|[35]]] , respectively.      

===2.8. Statistical analysis===

Data are expressed as mean ± standard deviation. Data were statistically analyzed in completely randomized design in factorial arrangement according to the procedures outlined by Gomez and Gomez [[#bib36|[36]]] , and the treatment means were compared by least significant differences and Duncan multiple range using Excel (Microsoft Office 2007) and SPSS Version 18.0 (SPSS Inc., Chicago, IL, USA). Data are presented in text and tables as the means of five determinations.      

==3. Results and discussion==

[[#tbl2|Table 2]]  shows initial and final body weights, body weight gain, and feed intake of the rats. The weight gain and food intake of the rats fed the HCD were significantly higher than the rest of the groups ([[#tbl2|Table 2]] ). Rats fed a diet rich in cholesterol are often used as an experimental model for dietary hyperlipidemia; these rats exhibit increased plasma TG, TC, and LDL-C; decreased circulation of HDL-C; and an overweight state [[#bib37|[37]]] . Addition of ''kishk''  to the HCD caused significant (''p''  < 0.05) reductions in body weight gain. This reduction increased gradually and significantly with increasing levels of ''kishk''  incorporated in the diet. This finding suggests that daily consumption of dried fermented milk/wheat mixture is effective in reducing weight gain. Several works have reported that the reduction in weight gain is associated with the consumption of fiber-rich foods, particularly whole-grain products, as part of a healthy lifestyle  [[#bib38|[38]]]  and [[#bib39|[39]]] . Dietary fiber may exhibit its impacts on food intake and body weight gain through different mechanisms. The satiating effect elicited by both soluble dietary fiber and insoluble dietary fiber is not only a consequence of mechanical intestinal distension by the bulking action of nondigested and unabsorbed food components and water; delayed starch digestion and glucose absorption also reduce glycemic and insulin responses, prolonging satiety and reducing energy consumption [[#bib40|[40]]] . Dietary fiber can also affect food intake by modulating the production of gut hormones involved in appetite regulation such as ghrelin (an orexigenic peptide) or anorexigenic hormones such as glucagon-like peptide-1, peptide YY, or the adipocyte-produced leptin; these hormones would also affect lipid metabolism and energy expenditure, thus influencing body weight  [[#bib41|[41]]]  and [[#bib42|[42]]] . The total feed intake was similar in all groups throughout the experimental period. There were no statistically significant differences (''p''  > 0.05) between control diet, HCD diet, and HCD diet supplemented with different levels of ''kishk'' . This finding indicates that addition of ''kishk''  into the diet did not affect the approximate feed intake in all groups ( [[#tbl2|Table 2]] ).

<span id='tbl2'></span>

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|+

Table 2.

Body weight gain and average food intake of experimental rats.

|-

! Groups
! Initial body weight (g)
! Final body weight (g)
! Weight gain (g)
! Average food intake (g/d)
|-

| Control
| 195.5<sup>a</sup>  ± 18.1                                                    
| 264.7<sup>a</sup>  ± 19.5                                                    
| 69.2<sup>b</sup>  ± 4.61                                                    
| 13.9<sup>a</sup>  ± 0.90                                                    
|-

| HCD
| 187.9<sup>a</sup>  ± 14.3                                                    
| 271.6<sup>a</sup>  ± 21.2                                                    
|  83.7 <sup>a</sup>  ± 6.20                                                    
| 16.6<sup>a</sup>  ± 1.45                                                    
|-

| DK10
| 179.7<sup>a</sup>  ± 17.6                                                    
| 242.5<sup>a</sup>  ± 17.3                                                    
| 62.8<sup>b,c</sup>  ± 5.70                                                    
| 14.7<sup>a</sup>  ± 1.30                                                    
|-

| DK20
| 198.1<sup>a</sup>  ± 16.2                                                    
| 258.2<sup>a</sup>  ± 21.4                                                    
| 60.1<sup>b,c</sup>  ± 4.21                                                    
| 14.9<sup>a</sup>  ± 1.62                                                    
|-

| DK30
| 190.1<sup>a</sup>  ± 13.4                                                    
| 244.5<sup>a</sup>  ± 14.8                                                    
| 54.4<sup>c</sup>  ± 7.33                                                    
| 15.1<sup>a</sup>  ± 1.25                                                    
|-

| LSD at 0.05
| 29.65
| 34.57
| 10.41
| 2.41
|}

Data are expressed as mean ± SD.

Values given represent means of five determinations.

Values followed by the same letter are not significantly different (''p''  < 0.05).                      

Control represents rats fed the basal diet HCD.

DK10, high-cholesterol diet supplemented with 10% of ''kishk'' ; DK20, high-cholesterol diet supplemented with 20% of ''kishk'' ; DK30, high-cholesterol diet supplemented with 30% of kishk; HCD, high-cholesterol diet; SD, standard deviation.                      

Least significant difference at ''p''  ≤ .05 according to Duncans multiple-range test.

===3.1. Relative organ weight (g) of experimental rats===

Weighing of organs of treated animals may reveal specific organ changes related to the treatment [[#bib43|[43]]] . There were no significant differences in the heart and kidney/body weight ratios in all groups under study. On the contrary, the liver/body weight ratio significantly increased in rats fed a cholesterol-rich diet (HCD) compared with the control rats. These increases could be attributed to the accumulation of cholesterol in the liver [[#bib44|[44]]] . The highest change in the liver/body weight ratio was observed for rats receiving an HCD ([[#tbl3|Table 3]] ). This finding is similar to those reported by Jemai et al [[#bib42|[42]]]  and Osfor et al [[#bib45|[45]]] , who found that the liver/body weight ratio increased in rats fed a cholesterol-rich diet compared with those fed the control diet. Incorporation of ''kishk''  into the HCD diet at levels of 10%, 20%, and 30% could significantly (''p''  ≤ 0.05) decrease the change of liver/body weight ratio by 14.46%, 17.51%, and 18.78%, respectively, when compared with the liver/body weight ratio of the HCD group. Higher dietary fiber content may have increased fecal lipid excretion and reduced serum TC level  [[#bib46|[46]]] . These findings revealed that high-fiber diets and probiotic bacteria of dairy products may have synergistic effects and reduce the accumulation of cholesterol in the liver.

<span id='tbl3'></span>

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|+

Table 3.

Relative weight (%) of organs of experimental rats.

|-

! Groups
! Liver
! Kidney
! Heart
|-

| Control
| 3.16<sup>b</sup>  ± 0.45                                                    
| 0.71<sup>a</sup>  ± 0.12                                                    
| 0.30<sup>a</sup>  ± 0.02                                                    
|-

| HCD
| 3.94<sup>a</sup>  ± 0.06                                                    
| 0.86<sup>a</sup>  ± 0.09                                                    
| 0.33<sup>a</sup>  ± 0.01                                                    
|-

| DK10
| 3.37<sup>b</sup>  ± 0.29                                                    
| 0.72<sup>a</sup>  ± 0.10                                                    
| 0.31<sup>a</sup>  ± 0.03                                                    
|-

| DK20
| 3.25<sup>b</sup>  ± 0.18                                                    
| 0.76<sup>a</sup>  ± 0.03                                                    
| 0.33<sup>a</sup>  ± 0.02                                                    
|-

| DK30
| 3.20<sup>b</sup>  ± 0.21                                                    
| 0.70<sup>a</sup>  ± 0.08                                                    
| 0.30<sup>a</sup>  ± 0.01                                                    
|-

| LSD at 0.05
| 0.366
| 0.124
| 0.037
|}

Data are expressed as mean ± SD.

Values given represent means of five determinations.

Values followed by the same letter are not significantly different (''p''  < 0.05).                      

Control represents rats fed the basal diet HCD.

DK10, high-cholesterol diet supplemented with 10% of ''kishk'' ; DK20, high-cholesterol diet supplemented with 20% of ''kishk'' ; DK30, high-cholesterol diet supplemented with 30% of kishk; HCD, high-cholesterol diet; SD, standard deviation.                      

Least significant difference at ''p''  ≤ .05 according to Duncans multiple-range test.

===3.2. Effect of tested diets on lipid profile===

Total cholesterol, LDL-C, HDL-C, triacylglycerol concentrations (mg/dL), and AIs of rats fed HCD and HCD supplemented with different levels of ''kishk''  are presented in  [[#tbl4|Table 4]] . Generally, the final values of serum cholesterol and LDL-C of rats fed either HCD or HCD supplemented with different levels of ''kishk''  were significantly (''p''  ≤ 0.05) higher than those of rats fed the negative control diet (NCD). Numerous studies showed that the increased levels of TC and LDL-C raise the risk of developing atherosclerosis and coronary heart disease  [[#bib47|[47]]] , [[#bib48|[48]]]  and [[#bib49|[49]]] . Consumption of the cholesterol-rich diet resulted in significant (''p''  ≤ 0.05) increases of TC, with values approximately 2.59 times higher than those in NCD rats ( [[#tbl4|Table 4]] ). However, when ''kishk''  was incorporated into the HCD, significant reductions of 17.22%, 19.23%, and 26.06% in the TC levels were observed in comparison with the HCD group that consumed cellulose as a source of fiber. There were significant (''p''  ≤ 0.05) differences in LDL-C values between rats fed NCD and experimental groups. Rats fed HCD had the highest (126 ± 3.92 mg/dL) concentration of LDL-C (''p''  ≤ 0.05). Inclusion of different levels of ''kishk''  into the HCD caused a significant (''p''  ≤ 0.05) decline in the concentration of LDL-C. The concentration of LDL-C of rats fed diets supplemented with 10%, 20%, and 30% of ''kishk''  were, respectively, about 1.23, 1.46, and 1.87 times as low as those of rats fed the HCD. Wheat fiber reduced serum cholesterol level by increasing the surface area for bile binding or altering the quantity of short-chain fatty acids  [[#bib50|[50]]] . Several studies have shown that propionate generated from dietary fibers containing polysaccharides exhibits hypocholesterolemic effects and offsets acetate generation, which tends to increase serum cholesterol via a mechanism probably involving liver lipogenesis  [[#bib51|[51]]] , [[#bib52|[52]]] , [[#bib53|[53]]]  and [[#bib54|[54]]] . The reduction of cholesterol is possibly a sum of several effects; the most accepted one is decreased absorption of bile acids that causes removal of steroids from the body by fecal excretion, resulting in increased catabolism of cholesterol, an increase in the secretion of bile acids, a decrease in lipoprotein cholesterol secretion, and a reduction in the total body pool of cholesterol [[#bib55|[55]]] .

<span id='tbl4'></span>

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Table 4.

TC, LDL-C, HDL-C, and TG concentrations (mg/dL); atherogenic index; and cardiac risk factor in rats fed experimental diets for 8 weeks.

|-

! Parameters
! Control
! HCD
! DK10
! DK20
! DK30
! LSD at 0.05
|-

| TC
| 90.28<sup>d</sup>  ± 2.59                                                    
| 234<sup>a</sup>   ±  6.31                                                    
| 193.7<sup>b</sup>  ± 14.09                                                    
| 189<sup>bc</sup>   ±  11.0                                                    
| 173<sup>c</sup>   ±  6.9                                                    
| 16.55
|-

| LDL
| 43.3<sup>e</sup>   ±  1.21                                                    
| 156<sup>a</sup>   ±  3.92                                                    
| 112<sup>b</sup>  ± 12.9                                                    
| 86.1<sup>c</sup>   ±  7.0                                                    
| 67.3<sup>d</sup>  ± 2.9                                                    
| 12.62
|-

| HDL
| 41.14<sup>a</sup>   ±  1.32                                                    
| 26.6<sup>d</sup>  ± 0.96                                                    
| 32.25<sup>c</sup>   ±  1.34                                                    
| 36.0<sup>b</sup>  ± 1.52                                                    
| 40.16<sup>a</sup>   ±  0.98                                                    
| 2.26
|-

| TG
| 71.01<sup>b</sup>  ± 4.84                                                    
| 97.67<sup>a</sup>   ±  4.99                                                    
| 81.60<sup>b</sup>  ± 5.12                                                    
| 81.22<sup>b</sup>  ± 4.16                                                    
| 73.24<sup>b</sup>  ± 5.26                                                    
| 8.89
|-

| AI
| 1.19<sup>e</sup>   ±  0.01                                                    
| 7.79<sup>a</sup>   ±  0.08                                                    
| 5.00<sup>b</sup>  ± 0.18                                                    
| 4.25<sup>c</sup>   ±  0.08                                                    
| 3.31<sup>d</sup>  ± 0.07                                                    
| 0.18
|-

| Cardiac risk factor
| 2.19<sup>e</sup>   ±  0.01                                                    
| 8.92<sup>a</sup>   ±  0.08                                                    
| 6.76<sup>b</sup>  ± 0.18                                                    
| 5.38<sup>c</sup>   ±  0.08                                                    
| 4.31<sup>d</sup>  ± 0.07                                                    
| 0.18
|}

Data are expressed as mean ± SD. Values given represent means of five determinations.

Values followed by the same letter are not significantly different (''p''  < 0.05).                      

Control represents rats fed the basal diet HCD.

AI, atherogenic index; DK10, high-cholesterol diet supplemented with 10% of ''kishk'' ; DK20, high-cholesterol diet supplemented with 20% of ''kishk'' ; DK30, high-cholesterol diet supplemented with 30% of kishk; HCD, high-cholesterol diet; HDL, high-density lipoprotein; HDL-C, high-density lipoprotein cholesterol; LDL, low-density lipoprotein; LDL-C, low-density lipoprotein cholesterol; SD, standard deviation; TC, serum total cholesterol; TG, triglycerides.                      

Least significant difference at ''p''  ≤ .05 according to Duncans multiple-range test.

In this regard, ''kishk''  is typically prepared by adding strained yoghurt to parboiled wheat and allowing the mix to ferment at an ambient temperature for different periods of time  [[#bib20|[20]]] , [[#bib21|[21]]]  and [[#bib22|[22]]] . From this viewpoint, probiotic lactobacilli play a vital role in lowering cholesterol levels  [[#bib56|[56]]]  and [[#bib57|[57]]] . During anaerobic growth, ''Lactobacillus acidophilus''  removed cholesterol from the laboratory media supplemented with cholesterol and bile salts  [[#bib58|[58]]] . Several studies showed that ''L. acidophilus''  or related lactobacilli controlled serum cholesterol levels  [[#bib59|[59]]] , [[#bib60|[60]]] , [[#bib61|[61]]]  and [[#bib62|[62]]] . ''L. acidophilus''  interferes with the enterohepatic circulation of bile acids, which in itself could be an important factor in reducing serum cholesterol levels. ''L. acidophilus''  can deconjugate bile acids  [[#bib63|[63]]] . Bile, a water-soluble end product of cholesterol in the liver, is stored and concentrated in the gallbladder and released into the duodenum upon ingestion of food [[#bib64|[64]]] . It consists of cholesterol, phospholipids, conjugated bile acids, bile pigments, and electrolytes. Once deconjugated, bile acids are less soluble and absorbed by the intestines, leading to their elimination in the feces. Cholesterol is used to synthesize new bile acids in a homeostatic response, resulting in lowering of serum cholesterol [[#bib64|[64]]] . The hypocholesterolemic effect of the probiotics has also been attributed to their ability to bind cholesterol in the small intestines [[#bib65|[65]]] . Compared with the NCD group, there were significant (''p''  ≤ 0.05) reductions in the concentration of HDL-C in HCD groups. HDL-C decreased significantly from 41.14 mg/dL in rats fed NCD to 26.6 mg/dL in rats fed HCD. Moreover, the concentration of HDL-C of rats fed HCD supplemented with various levels of ''kishk''  increased significantly (''p''  < 0.05) compared with those of rats in the HCD group. Indeed, significant increases (21.24%, 35.33%, and 50.97%) in the level of HDL-C were observed for rats fed on HCD containing 10%, 20%, and 30% of ''kishk'' , respectively, in comparison with the HCD group. These increases in HDL-C levels of rats fed HCD supplemented with various levels of ''kishk''  may be due to the reduction in TC considering that dietary fiber increases the enzymatic activity of cholesterol-7 alpha-hydroxylase, the key regulatory enzyme in the hepatic conversion of cholesterol to bile acids, thereby contributing to higher depletion of hepatic cholesterol  [[#bib66|[66]]] . Several studies have demonstrated that high levels of HDL-C are associated with a lower incidence of CVDs due to the inhibition of LDL oxidation and protection of endothelial cells from the cytotoxic effects of oxidized LDL  [[#bib67|[67]]]  and [[#bib68|[68]]] . The increased HDL facilitates the transport of cholesterol from the serum to the liver, where it is catabolized and excreted from the body.      

Addition of different levels of ''kishk''  into the HCD was able to attenuate both TC and LDL-C, and elevate the level of HDL-C in serum of rats.      

Rats fed HCD had significantly higher TG values compared with those fed NCD. Addition of different levels of ''kishk''  into the HCD caused significant (''p''  ≤ 0.05) reductions in TG levels. TG level in rats fed HCD was 97.67 mg/dL, which is 16.45%, 16.86%, and 25.01% higher than in rats fed HCD supplemented with 10%, 20%, and 30% of ''kishk'' , respectively ( [[#tbl4|Table 4]] ). These reductions in TG levels may be due to the formation of a protective layer by the dietary fiber around the cholesterol-containing lipid drops, which prohibits absorption in the intestine  [[#bib69|[69]]]  and [[#bib70|[70]]] . Dietary fibers increase the viscosity of the aqueous solution surrounding the lipid droplets, which may alter the efficiency of droplet disruption and coalescence in the stomach and small intestine [[#bib17|[17]]] . This finding complies with the findings that fibers of fruits cereals and vegetables reduced the levels of triacylglycerols in rodents  [[#bib70|[70]]] , [[#bib71|[71]]]  and [[#bib72|[72]]] . AI is considered to be an important parameter of atherosclerosis. AI indicates the risk of deposition of foam cells, plaque, and fatty infiltration or lipids in the heart, coronaries, aorta, liver, and kidney [[#bib73|[73]]] . The abovementioned data reflected in the values of AI and the cardiac risk factor. Generally, the values of AI and the cardiac risk factor of rats fed either HCD or HCD supplemented with different levels of ''kishk''  were significantly (''p''  ≤ 0.05) higher than in rats fed the NCD. The highest value of AI (7.79) was recorded for rats fed HCD. Administration of different levels of dried ''kishk''  led to a significant (''p''  < 0.05) reduction in the AI value. AI values of rats fed HCD supplemented with 10%, 20%, and 30% of ''kishk''  were, respectively, about 1.55, 1.83, and 2.36 times as low as those of rats fed HCD.      

The same trend was observed for cardiac risk factor ([[#tbl4|Table 4]] ). Hypercholesterol is a risk factor for CVD, which is a leading cause of death in many countries [[#bib74|[74]]] . A 1% reduction in serum cholesterol is estimated to result in 2–3% reduction in the risk of coronary artery disease [[#bib75|[75]]] . It is suggested that intestinal lactobacilli may reduce serum cholesterol level through bacterial assimilation in the intestine [[#bib58|[58]]]  and deconjugation of bile salts [[#bib76|[76]]]  and [[#bib77|[77]]] . Short-chain fatty acids produced by lactobacilli may also inhibit hepatic cholesterol synthesis and distribution of cholesterol in the plasma and liver [[#bib78|[78]]] . Numerous studies showed that the increased levels of TC and LDL-C raise the risk of developing atherosclerosis and coronary heart disease [[#bib47|[47]]] , [[#bib48|[48]]]  and [[#bib49|[49]]] . On the contrary, a raised level of HDL-C was associated with a reduced risk of atherosclerosis, since HDL in serum is thought to facilitate the translocation of excess cholesterol from the peripheral tissue to the liver for further catabolism [[#bib79|[79]]] .      

===3.3. Effect of tested diets on some liver and kidney functions===

[[#tbl5|Table 5]]  summarizes the effect of HCD and HCD supplemented with different levels of ''kishk''  on some liver and kidney functions. Assessment of the levels of AST and ALT provides a good and simple tool to measure the protective activity of the target drug against the hepatic damage of the target compounds  [[#bib80|[80]]] . ALT and AST enzyme activities of rats fed HCD and HCD supplemented with different levels of ''kishk''  for 8 weeks compared with those of the rats of the NCD group are presented in  [[#tbl5|Table 5]] . Rats fed with HCD for 8 weeks had significant increases in the activities of AST and ALT enzymes (35.34% and 46.58%, respectively; ''p''  < 0.05) compared with the NCD group. Consumption of HCD induces damage to the rat liver due to oxidative stress caused by the excess of cholesterol in the diet. This oxidative stress damages cells due to an increase in free radicals and consequent lipid peroxidation  [[#bib81|[81]]] , [[#bib82|[82]]]  and [[#bib83|[83]]] . However, administration of various levels of ''kishk''  reversed the injuries induced by HCD to normal values ( [[#tbl5|Table 5]] ). No significant (''p''  ≥ 0.05) differences in the activities of serum ALT and AST were shown for rats administrated HCD incorporating 30% of ''kishk''  and for the control group. These findings are consistent with another result suggesting a protective role of dietary fibers  [[#bib84|[84]]]  and [[#bib85|[85]]] . The mean values of creatinine, urea, and uric acid in rats fed HCD were significantly higher than those in rats of the NC group ([[#tbl5|Table 5]] ). Hypercholesterolemia is considered a factor that contributes to renal dysfunction [[#bib86|[86]]] . The hypercholesterolemia leads to reduced renal blood flow and increased renal vascular resistance [[#bib87|[87]]] , which are factors directly related to the impairment of renal function. Significant (''p''  ≤ 0.05) reductions were seen in the levels of creatinine, urea, and uric acid for rats fed HCD supplemented with various levels of ''kishk'' . In the same time, no significant (''p''  ≥ 0.05) changes in the levels of creatinine, urea, and uric acid were shown for rats administrated HCD supplemented with various levels of ''kishk''  and for the control group. Such findings coincided with those of El Rabey et al  [[#bib85|[85]]] , who showed that dietary fiber significantly improves the level of kidney function.

<span id='tbl5'></span>

{| class="wikitable" style="min-width: 60%;margin-left: auto; margin-right: auto;"
|+

Table 5.

Effect of HCD and HCD supplemented with different levels of ''kishk''  on some liver and kidney functions.                  

|-

! rowspan="2" | Group
! colspan="2" | Liver functions
! colspan="3" | Kidney functions
|-

! Aspartate aminotransferase (AST) (IU/L)
! Alanine aminotransferase (ALT) (IU/L)
! Urea (mg/dL)
! Creatinine (mg/dL)
! Uric acid (mg/dL)
|-

| Control
| 36.50<sup>d</sup>  ± 1.60                                                    
| 24.60<sup>d</sup>  ± 0.73                                                    
| 26.60<sup>b</sup>  ± 4.10                                                    
| 0.66<sup>b</sup>  ± 0.09                                                    
| 1.94<sup>b</sup>  ± 0.19                                                    
|-

| HCD
| 49.40<sup>a</sup>   ±  4.02                                                    
| 36.06<sup>a</sup>   ±  1.19                                                    
| 34.20<sup>a</sup>   ±  1.60                                                    
| 1.40<sup>a</sup>   ±  0.13                                                    
| 2.60<sup>a</sup>   ±  0.23                                                    
|-

| DK10
| 45.30<sup>b</sup>  ± 1.20                                                    
| 33.20<sup>b</sup>  ± 1.35                                                    
| 31.40<sup>ab</sup>  ± 1.20                                                    
| 0.78<sup>b</sup>  ± 0.10                                                    
| 2.10<sup>b</sup>  ± 0.16                                                    
|-

| DK20
| 41.70<sup>bc</sup>  ± 0.96                                                    
| 29.10<sup>c</sup>  ± 1.89                                                    
| 29.00<sup>b</sup>  ± 0.96                                                    
| 0.73<sup>b</sup>  ± 0.09                                                    
| 1.98<sup>b</sup>  ± 0.18                                                    
|-

| DK30
| 38.10<sup>cd</sup>  ± 2.00                                                    
| 25.42<sup>d</sup>  ± 1.36                                                    
| 27.10<sup>b</sup>  ± 1.10                                                    
| 0.71<sup>b</sup>  ± 0.11                                                    
| 1.96<sup>b</sup>  ± 0.17                                                    
|-

| LSD at 0.05%
| 4.07
| 2.47
| 3.89
| 0.19
| 0.34
|}

Data are expressed as mean ± SD. Values given represent means of five determinations.

Values followed by the same letter are not significantly different (''p''  < 0.05).                      

Control represents rats fed the basal diet HCD.

ALT, alanine transferase; AST, aspartate transferase; DK10, high-cholesterol diet supplemented with 10% of ''kishk'' ; DK20, high-cholesterol diet supplemented with 20% of ''kishk'' ; DK30, high-cholesterol diet supplemented with 30% of kishk; HCD, high-cholesterol diet; SD, standard deviation.                      

Least significant difference at ''p''  ≤ .05 according to Duncans multiple-range test.

==Conflicts of interest==

The author has no conflicts of interest to declare.

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