Kefir Fermented Milk Ameliorates Serum Indicators of Hyperglycemia, Dyslipidemia, Pro-Inflammatory Cytokines and Hepatic Histology in Streptozotocin-Diabetic Rats

Diabetes is a systemic, long-term endocrine, metabolic condition distinguished by persistent high blood sugar levels via a deficit in insulin action and/or secretion. It is a multifactorial illness. Diabetic prevalence, considered a world epidemic, is increasing significantly and affecting approximately 5-10% of adults globally. According to WHO reports, diabetes become the 7th leading cause of death [1]. In Saudi Arabia, the incidence of diabetes is increasing due to socioeconomic changes affecting the lifestyle, including altering physical activity and diet [2]. Around 13 % of Saudis are diagnosed with diabetes relative to 2.8-4.4% global prevalence [3]. One in 10 are prediabetes (at risk of developing diabetes) of the remaining Saudi population [4]. Elevation diabetes prevalence is proportionally correlated with the high prevalence of cardiovascular diseases and premature death, accounting for 42% in Saudi Arabia [4].

Rationale and Knowledge Gap

Studies investigating the human microbiome’s role in health and diseases have attracted more attention in the past decade. The human microbiome is essential for digestion, nutrition metabolism and immune system regulation [5]. It is vital in illness pathogenesis, including liver, diabetes, obesity and cancer [6]. The restoration of homeostasis of gut microbiota via consumption of probiotics and prebiotics resulted in preventing as well as attenuating numerous metabolic complications and cardiovascular diseases [7].

Kefir grains are symbiotic associated with various microorganisms. It is made primarily of yeasts as well as lactic and acetic acid bacteria in a natural polysaccharide and protein matrix. Kefir-fermented dairy products are made through the fermentation via yeasts and bacteria naturally present in grains of kefir. Kefir milk (KM) has beneficial properties [8]. It contains many bioactive peptides with antibacterial, antihypertensive, immunomodulatory and antioxidative properties [9].

Previous animal studies have shown that administration of KM improved specific oxidative stress markers by restoring the ROS/NO imbalance, as well as showed hypoglycemic, hypolipidemic and pro-inflammatory cytokine expression [10]. Moreover, it has anticarcinogenic and anti-inflammatory activities [11,12].

Objective

As a result of the increasing prevalence of diabetes globally, especially in Saudi Arabia and given that fermented milk is one of the most popular consumed beverages among Saudis. Therefore, the objective of this research is to assess the effect of KM as anti-hyperlipidemic and hypoglycemic on streptozotocin-diabetic rats.

Preparation of KM

Organic kefir grains were kindly provided from Honest Com., Jeddah, Saudi Arabian (SA). Fresh whole cow’s milk (Almarai) with 3% fat, homogenized and UHT treated was obtained from a local market in Jeddah, SA.

The kefir fermented milk (KM) was prepared following a previously established protocol [13]. Kefir grains (10% w/v) were added to glass containers with cow's milk (3% fat) and kept at 25°C in a dark environment for 24 hours. After fermentation, the grains were separated from the fermented milk using a 1-mm² mesh sieve. The resulting liquid was referred to as kefir beverage. A portion of this liquid, termed cell-free supernatant (CFS), was centrifuged and filtered through membranes with a pore size of 0.22 μm, then stored at -80°C for later use. The kefir grains were reused for subsequent fermentations, stored at 4°C between cycles and inoculated in fresh milk for up to seven fermentation rounds.

As stated in reference [13], the microbial composition in KM primarily consists of lactic acid bacteria (LAB), acetic acid bacteria (AAB) and yeasts. The dominant LAB species identified was Lactococcus lactis, while a single AAB species, Acetobacter orientalis, was also present in KM. Additionally, various yeast species, including Pichia kudriavzevii, Geotrichum candidum, Geotrichum bryndzae, Saccharomyces cerevisiae and Kazachstania unispora.

Standardization of KM preparation involves maintaining consistent conditions to ensure uniformity across batches. Key factors include using the same microbial composition of kefir grains, controlling fermentation conditions such as temperature (25°C) and duration (24 hours) and using the same type and fat content of milk (3% fat). Additionally, standardized processes like filtering kefir grains, maintaining consistent storage conditions (4°C) and monitoring parameters such as pH and acidity ensure that each batch has the same characteristics, which is crucial for reproducibility in both research and product quality.

The use of UHT milk in kefir fermentation supports better growth of L. acidophilus colonies by providing a sterile environment free from competing microbes. UHT-fermented kefir meets the minimum colony-forming units (CFUs) required for L. acidophilus to remain effective, ensuring consistent probiotic benefits. This makes UHT milk a reliable option for producing kefir with adequate probiotic content [14].

Animals

Thirty-two adult male Albino rats (190-210 g) were obtained from the Pharmacy College, KAU, SA. The sample size was calculated according to a crude method “resource equation” based on the law of diminishing return [15]. Animals were housed in cages and adapted to lab setting at a temperature of 24±2°C and a humidity of 60±10% during a 12:12 h dark/light cycle. All rats were fed a standard diet containing protein, soybean oil, sucrose, fiber, mineral mixture, vitamin mixture, choline chloride (14, 5, 10, 5, 3.5, 1 and 0.25, respectively) and the remainder was corn starch up to 100% [16].

Induction of Diabetes

Streptozotocin (STZ) was purchased from Sigma-Aldrich (St. Louis, MO, USA), it is used to induce diabetes in the animal. STZ solution prepared freshly in 0.01 citrate buffer (pH = 4.5). After fasting overnight, rats were injected intraperitoneally with STZ (60 mg/kg), while control rats were injected with the citrate buffer only. As the number of early deaths was less than 20%, rats were allowed to drink glucose solution (5%) within 6 hours after STZ injection to overcome death from rapid and massive β-cell necrosis that results in the release of large quantities of insulin, causing fatal hypoglycemia [17]. Diabetes was confirmed after 72 h. of STZ injection. Glycemia was measured by glucometer; blood was drawn from the tail vein. Rats with fasting blood glucose (FBG) levels exceeding 250 mg/dL were classified as diabetes [18]. The success rate of diabetes induction was 80 (24 rats out of 30).

Study Protocol

This study is an experimental study. Thirty-two rats were randomly assigned into two main groups: a control group consisting of 8 rats and a diabetes group with 24 rats. The diabetes group was then further randomly subdivided into three groups, each with 8 rats: a diabetic group, a diabetic group treated with KM (10 ml/kg) by gavage and diabetic group treated with KM (20 ml/kg) by gavage. The control and diabetic untreated groups received saline via oral gavage in the same manner as in diabetic treated groups to ensure consistency across all groups. The dose 10 ml/kg/day was chosen based on prior study according to [19]. All groups were fed a standard diet for a long experiment period (35 days).

Analytical Measurements

Serum samples obtained from animals after an overnight fast of 12 h were used to determine biochemical parameters. The glycemia and lipid profile were measured using colorimetric enzymatic kits (BioMerieux, France) followed the standard procedures as described in kits’ protocol. The insulin, glycosylated hemoglobin (HbA1c) and proinflammatory cytokines (IL-6 and TNF-α) were measured using a fully automated enzyme-linked immunosorbent assay ELISA kits (Abcam, UK). A monoclonal antibody specifically targeting rat insulin, HbA1c, IL-6 and TNF-α were employed. All parameter concentrations were quantified by comparing them to a standard curve.

Histopathology Examination

Hepatic sections of all groups underwent standard histopathology procedures including trimming of the samples, fixation within neural buffer formalin, clearance by xylene and embedding in paraffin wax. Samples were section by microtome at 5 μm thickness before being stained by the hematoxylin and eosin (H and E) stain. For identification of the histological alterations in each group, the stained hepatic slides were examined under a microscope.

Quantitative scoring of the hepatic lesions was done through blind examination of the slides of the different groups based on the hepatic degeneration, necrosis and inflammation As stated in reference [20].

Statistical Analysis

The normality of the distributions was evaluated using the Kolmogorov-Smirnov normality test for all measured parameters. The one-way analysis of variance (ANOVA) test followed by Tukey’s multiple comparisons test was used to compare the results in normally distributed data. The Kruskal-Wallis test followed by Dunn’s multiple comparisons test was used to compare the hepatic lesions score (no normal distribution) of all groups. The research utilized statistical analysis through the Statistical Package (SPSS) (Version 27) to analyze the results. The main data analyses consisted of descriptive statistics in the form of means, SD (standard deviations) and confidence interval (CI, 95%) . Data with the p-value <0.05 were regarded as significant. The data was blinded collected and statistically analyzed to reduce the bias.

Impact of Kefir Milk (KM) on Glucose, Insulin and HbA1c in Diabetic Rats

Table 1 represents the impact of KM on glucose, insulin and HbA1c measured in diabetic rats. Serum glucose and HbA1c concentrations of STZ-induced hyperglycemic rats were significantly elevated, whereas serum insulin was significantly reduced relative to the control rats. Diabetic animals received KM significantly improved glucose, insulin and HbA1c concentrations toward normal levels. The KM treatment was significantly effective in lowering the blood glucose and HbA1c, concurrent with significant elevate insulin concentration relative to the diabetic untreated group. The effectiveness of KM was dependent on the dose administered. This was evident by the significant differences in blood glucose levels, HbA1c and insulin concentrations between the 10 and 20 ml/kg KM groups.

Impact of Kefir Milk (KM) on Lipid Profile Indices in Diabetic Rats

In diabetic untreated rats, the indices of hyperlipidemia (TC, TG, VLDL-C and LDL-C) concentrations were significantly elevated. In contrast, serum HDL-C was significantly reduced relative to the control rats. Diabetic rats treated with KM exhibited significant improvement in lipid profile indicators toward normal levels. The KM treatment was significantly effective in lowering the TC, TG, LDL-C and VLDL-C, concurrent with significantly elevating HDL-C levels relative to the diabetic untreated group. The effectiveness of KM was dose-dependent, as evidenced by significant differences between KM at a low dose (10 ml/kg) and high dose (20 ml/kg) in TC, TG, VLDL-C and LDL-C, as well as HDL-C concentrations (Figure 1).

Table 1: Impact of KM on glucose, insulin and HbA1c in diabetic rats

All values are offered as Mean±SD (n = 8). CI: Confidence interval, ^a: Significant versus the control, ^b: Significant versus the diabetic, ^c: Significant versus the diabetic +KM (10 ml/kg) group, (*p<0.05, ^p<0.01, ^#p<0.001).

Figure 1: Impact of KM on markers of dyslipidemia (TC, TG, HDL-C, LDL-C and VLDL-C) measured in STZ-induced hyperglycemic rats. All values are offered as Mean±SD (n = 8). ^a: Significant versus the control, ^b: Significant versus the diabetic, ^c: Significant versus the diabetic +KM (10 ml/kg) group, (*p<0.05, ^p< 0.01, ^#p< 0.001). 95% CI (confidence interval) for TC [118.52-130.48; 232.88 -275.37; 154.71- 185.54; and 121.20- 141.30]; TG [68.79- 74.71; 99.09- 128.15; 76.86-90.64; and 69.19-75.81]; HDL-C [42.25-48.00; 26.45-30.55; 38.11-45.14; and 42.41-49.09]; LDL-C[72.93-85.82- 205.71-245.79; 103.08-151.92; and 78.16-92.84]; and VLDL-C [13.76-14.94; 19.82-25.63; 15.37-18.13; and 13.84-15.16] for control; diabetic; diabetic+ KM (10 ml/kg); and diabetic+ KM (20 ml/kg), respectively].

Impact of Kefir Milk (KM) on Pro-Inflammatory Biomarkers in Diabetic Rats

Diabetic rats exhibit significant inflammation, as evidenced by significantly elevated IL-6 and TNF-α relative to the control rats. Diabetic rats treated with KM exhibited significant improvement in pro-inflammatory cytokines (IL-6 and TNF-α) toward normal concentrations. The KM treatment was significant and effective in lowering the IL-6 and TNF-α levels relative to the untreated diabetic group. The effectiveness of KM was dependent on the dose administered. This was evident by the significant differences in IL-6 and TNF-α concentrations between the 10 and 20 ml/kg KM groups (Table 2).

Impact of Kefir Milk (KM) on Hepatic Histopathology in Diabetic Rats

Figure 2 represents the histopathological findings within the different groups. Control rats showed normal hepatic histological appearance with normal hepatocytes and normal biliary and vascular tissues (Figure 2A). Diabetic rats’ hepatic tissue sections revealed the centrolobular and periportal hepatic necrosis associated with severe degree of hepatic degeneration, mononuclear inflammatory cells including monocytes and macrophages, as well as giant cells (Figures 2B-D). Diabetic rats treated with KM (10 ml/kg) showed a remarkable decrease in the hepatic necrosis and inflammation within the hepatic tissues (Figure 2E). Diabetic rats treated with KM (20 ml/kg) showed marked improvement of hepatocytes consistent with the normal control unless mild congestion of the central vein and blood sinusoids (Figure 2F).

Table 2: Impact of KM on pro-inflammatory indices (cytokines IL-6 and TNF-α) in diabetic rats

All values are offered as Mean±SD (n = 8). CI: Confidence interval, ^a: Significant versus the control, ^b: Significant versus the diabetic, ^c: Significant versus the diabetic + KM (10 ml/kg) group, (*p<0.05, ^#p<0.001)