This study hypothesizes that a combination of kefir and spirulina could serve as a novel functional food in DM management through controlling glycemic status and antioxidant capacity in a diabetic rat model.

This research obtained ethical approval with Ethical Clearance 152/EC/H/KEPK/FK-UNDIP/XII/2019 Faculty of Medicine, Diponegoro University, Semarang, Indonesia.

All the chemicals used were of the highest purity grade. Streptozotocin (STZ) and nicotinamide (NA) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Chloric acid, sulphuric acid, potassium sulfate, copper sulfate, ethanol, methanol, demineralized water, potassium hydroxide, and acetone were purchased from Merck. A kit for the glucose assay (GOD-PAP) was procured from DiaSys, Holzheim, Germany. A kit for estimation of SOD activity was procured from BioVision, Inc.

Spirulina was obtained from PT Neoalgae Sukoharjo, Central Java. Spirulina generally contains 60 – 70% total protein, including essential amino acids such as leucine, isoleucine and, valine. It has 4 – 7% lipids, essential fatty acids such as linoleic and γ-linolenic acid, and ω-3 and ω-6 polyunsaturated fatty acids. It also contains provitamin A, vitamin B12, and β-carotene (Koru, 2012).

Kefir-spirulina was made by combining 1% cow's milk with either 1% (w/v) or 2% (w/v) spirulina, followed by pasteurization. Kefir grain (5%) was added to the solution after it cooled down, followed by incubation at room temperature (25 – 37 °C) for 24 hours (Figure 1). After separating the grain from the solution, the kefir-spirulina mixture was analyzed for its nutritional content, ethanol content, and antioxidant capacity.

Nutrient analysis was performed according to the standard official methods of the Association of Official Analytical Chemists procedures 2005 (AOAC, 2005). Nutritional parameters such as moisture, protein, fat, ash, carbohydrate, and fiber were determined.

The sample used in this study was a male Sprague Dawley Rattus novegicus aged 8 weeks, weighing approximately 150 – 200 g. The animals were acclimatized for one week in a temperature-controlled room (20 ‒ 25 °C) and maintained on a 12 h light/12 h dark cycle. They were allowed free access to a standard commercial diet (Comfeed II, Japfa) and tap water. A total of 30 male Sprague Dawley rats were divided into five groups: normal control (K1), diabetic control (K2), pioglitazone treatment (K3), kefir combined with 1% spirulina treatment (P1), and kefir combined with 2% spirulina treatment (P2). All rats were induced by streptozotocin-nicotinamide (STZ- NA), except for the normal control. Nicotinamide (NA) was dissolved in saline solution and given intraperitoneally (i.p.) at a dose of 110 mg.kg^-1 body weight. After 15 minutes, the rats were induced by 45 mg.kg^-1 body weight of streptozotocin (STZ) in citrate buffer (i.p.). The rats considered having diabetes if three days after induction the fasting blood glucose was more than 126 mg.dL^-1 and plasma glucose were more than 200 mg.dL^-1. Groups P1 and P2 received kefir-spirulina via gavage with a total dose of 3 mL.200g^-1 body weight per day orally for 28 days. Group K3 received the diabetes drug pioglitazone at a dose of 0.27 mg.200g^-1 body weight per day orally for 28 days.

Before and after the 28 days of intervention, blood samples were taken through the retro-orbital sinus and were analyzed for fasting plasma glucose, postprandial glucose, and SOD activity.

Statistical analyses were performed using SPSS version 24 (IBM, 2016). The obtained data were analyzed using Shapiro-Wilk since the data were normally distributed. The paired t-test and one-way ANOVA were used to determine the effect of experimental treatments. Differences were considered to be significant if p-values less than 0.05 (p <0.05). In the case of variance homogeneity p >0.05, post-hoc LSD was used; otherwise, post-hoc Tamhane was used.

Table 1 showed the nutrient content of the two formulations of kefir-spirulina. The water content was 93.82% and 92.67% for formulas A and B, respectively. The high water content in the two formulations was due to the clear part of the kefir used in the formulation, while the curd was separated. Our result is in line with several other studies that showed that kefir generally contained 89 – 90% water (Sarkar, 2007; Plessas et al., 2017).

The ash content for the kefir formulation was similar, at 0.76% (formula A) and 0.72% (formula B). Our results were comparable with several other studies that found ash content was around 0.7 – 1.1% (Arslan, 2014; Plessas et al., 2017; Rosa et al., 2017).

The two kefir formulations prepared in this study contained 4.02% and 5.53% protein, almost twice the minimum amount suggested by CODEX STAN 234-2003 (FAO, 2010). It has been shown previously that kefir usually contains 3% of protein (Sarkar, 2007; Plessas et al., 2017; Magalhães et al., 2011). The improvement of the protein content of the kefir prepared in this study is likely due to the addition of spirulina, which is rich in protein (circa 60 – 70% of its dry weight) (Wu et al., 2005). The addition of 2% spirulina to the kefir showed higher protein content than the addition of 1% spirulina.

The carbohydrates content of kefir-spirulina for formula A and B were 1.56% and 1.12%, respectively. These results are lower than previous studies indicating that kefir contains carbohydrates between 3.8% and 4.7% (Arslan, 2014). The fiber content in this study's kefir formulations was 2.49% (formula A) and 6.40% (formula B). Fiber is a nonstarch polysaccharide with high nutritional value; the high fiber content in spirulina will slow down the glucose absorption in the ileum, thereby lowering postprandial glucose (Hernández-Alonso et al., 2017; Chandalia et al., 2000).

Fat content in the kefir formulations was 1.04% (formula A) and 0.59% (formula B). The low level of fat found in our kefir formulations was presumably due to lipase activity, which was produced by lactic acid bacteria (BAL). This study's fat content is relatively higher compared to the 0.2% that was shown in previous studies (Sarkar, 2007; Plessas et al., 2017). Fat content in the kefir could vary depending on the type of milk used in the fermentation process (Otles and Cagindi, 2003).

One of the main products of kefir fermentation was ethanol. The kefir-spirulina prepared in this study has a relatively low ethanol content (Table 2), which was 0.59% (formula A) and 0.65% (formula B). Ethanol in the kefir is usually in the range of 0.5 – 2.0% (Rosa et al., 2017; Karaçalı, Özdemİr and Çon, 2018). Foods or beverages with low ethanol content are beneficial, especially in a country with religious restrictions regarding foods and beverages containing alcohol. The lactose fermentation of kefir generated ethanol, which contributed to the kefir's characteristic aroma (Ertekin and Guzel- Seydim, 2010). Generally, the level of ethanol in kefir is influenced by yeast metabolism and heterofermentative bacteria that produce ethanol (Farnworth, 2008).

The antioxidant activity of kefir-spirulina in this study was determined based on DPPH radical scavenging activity and presented as half-maximal inhibitory concentration IC₅₀. The average total antioxidant activity of kefir-spirulina showed relatively strong activity, with IC₅₀ of 43.65 ppm and 42.00 ppm, for formulas A and B, respectively (Table 2). An antioxidant is considered to be very strong if the IC₅₀ is less than 50 ppm, strong if IC₅₀ is 50 ppm – 100 ppm, weak if IC₅₀ is 150 ppm – 200 ppm, and very weak if IC₅₀ is more than 200 ppm (Molyneux, 2004; Badarinath et al., 2010).

Kefir has been known to contain various antioxidant compounds capable of binding free radicals, decreasing the malondialdehyde concentration, and increasing glutathione peroxidase, thus regulating the level of oxidative stress (Rosa et al., 2017). Antioxidant activity in kefir-spirulina increased during the fermentation process. The proton of the acids produced by lactic acid bacteria during fermentation can be donated to the free radicals, increasing the primary antioxidant capacity (Waris and Ahsan, 2006).

The rats' body weight during the whole experiment (before and after treatment) is shown in Table 3. After 7 days of acclimatization, all rats except for the normal control (K1) were induced by STZ-NA. After 3 days, all diabetic rats received treatments for 28 days, except for the negative control (K2). Rats in diabetic control groups (K2) experienced a decrease in their body weight (p <0.05). This result is not surprising since STZ-NA induction may cause physiological changes and alteration in energy metabolism, affecting body weight. STZ induction may damage the pancreas. The pancreas' function is to synthesize and secrete insulin, which is vital in glucose utilization and, eventually, energy production. A disruption in insulin production causes a disturbance in energy homeostasis. As compensation, the body will shift towards fat and protein metabolism and may cause weight loss (Szkudelski, 2001). When diabetic rats were treated with the diabetic drug (K3) or kefir-spirulina (P1 and P2), body weight was compensated. We found no significant body weight changes (p >0.05) in diabetic rats treated with kefir-spirulina (P1 and P2) or with diabetic drugs (K3). Kefir has an immunomodulatory effect capable of improving intestinal microflora and absorption, therefore improving body weight (Judiono, Djokomoeljanto and Hadisaputro, 2010).

Table 4 compares the changes in fasting plasma glucose levels in the five groups. The rats' blood glucose level in the normal control (K1) remained relatively stable at a normal level. In contrast, diabetic rats with no treatment (K2) suffered from consistently high glucose levels. The effect of 28 days of administration of kefir-spirulina (P1and P2) or the diabetic drug pioglitazone (K3) on the diabetic rats significantly decreased the glucose level (p <0.001). Pioglitazone reduced blood glucose by improving glucose uptake in peripheral tissue and eventually enhanced insulin sensitivity (Waugh et al., 2006). On the other hand, kefir's ability to control plasma glucose levels has been shown through several studies (Nurliyani, Harmayani and Sunarti, 2015; Bellikci- Koyu et al., 2019). Kefir contains bioactive components such as peptides, short-chain fatty acids (SCFA), and exopolysaccharides (EPSs) that could affect the plasma glucose level. EPSs can increase cAMP concentration on Langerhans' islands so that the pancreas can increase insulin secretion (Al-Shemmari, Kassim Altaee and Hassan, 2018). It also activates glucagon-like peptide 1 (GLP 1) and gastric inhibitory peptide (GIP), which subsequently enhances insulin secretion (Al-Shemmari, Kassim Altaee and Hassan, 2018; Hadisaputro et al., 2012; Judiono, Djokomoeljanto and Hadisaputro, 2011; El-Bashiti, Zabut and Abu Safia, 2019). Furthermore, EPSs have also been proved to have antioxidant properties through free radical scavenging activity (Mao et al., 2014), which gives protection from oxidative damage that occurred on pancreatic cells. The high protein content in the kefir has an indirect role in regulating glucose levels through the regeneration and maintenance of pancreatic β cells (Hulston, Churnside and Venables, 2015). SCFAs, on the other hand, are produced by the activities of probiotic microorganisms present in the kefir grain (Simova et al., 2002). SCFA were thought capable of protecting pancreatic β-cells, increasing enzymatic antioxidant activity, and improving insulin resistance through regulating the expression of tumor necrosis factor-alpha (alpha TNFα) and nuclear factor-kappa B (NF-kB) (Wang et al., 2017).

As expected, combining spirulina and kefir improved the ability to control glucose levels. Increasing the spirulina composition by a factor of two (formula B on group P2) improved the ability of kefir-spirulina to lower the glucose level (glucose changes of P1 vs P2 were 112.47 mg.dL^-1 vs 154.12 mg.dL^-1; post-hoc test (p <0.05). This result indicated the synergistic effect of bioactive compounds present in the kefir-spirulina formulation. The synergistic effect of modified kefir has also been shown in several other studies (Nurliyani, Harmayani and Sunarti, 2015; Judiono, Djokomoeljanto and Hadisaputro, 2011). Spirulina consists of several bioactive compounds such as phycocyanin, carotenoids, vitamin E, chlorophyll, flavonoids, saponins, and phenolic compounds (Okechukwu et al., 2019). The C-phycocyanin (C-PC) peptide activates the insulin signaling pathway, regulates glucokinase expression in the pancreas and liver, and protects pancreatic β cells (Li et al., 2020). Previous studies showed that spirulina consumption could reduce blood glucose levels (Huang et al., 2018; Aissaoui et al., 2017; Jarouliya et al., 2015). Spirulina reduced blood glucose levels by stimulating β cell activity and increasing glucose transport to peripheral tissues (Layam and Reddy, 2007). Moreover, it has high fiber content, which slows down macronutrients' digestive process (Hernández-Alonso et al., 2017), thus maintaining the plasma glucose level.

We also analyzed postprandial glucose levels. Compared with fasting glucose, postprandial glucose level was thought to be better associated with the risk factor of diabetes and the level of HbA1c (Haddadinezhad and Ghazaleh, 2010), which reflects the glycemic control in diabetes patients over a certain period. According to the American Diabetes Association (ADA), the recommended postprandial glucose level for diabetes patients is below 180 mg.dL^-1 (ADA, 2018). The postprandial glucose levels of the five groups are presented in Table 5. In general, the changes in postprandial glucose of all groups are in agreement with the results obtained in fasting glucose levels. As can be seen in Table 5, treatment with pioglitazone (K3) and kefir-spirulina (P1 and P2) successfully reduced (p <0.001) the postprandial glucose concentration to less than 180 mg.dL^-1 (K3 = 110 mg.dL^-1, P1 = 170 mg.dL^-1, and P2 = 126 mg.dL^-1). The ability of kefir to reduce postprandial glucose has also been shown in other studies (Alsayadi et al., 2014). The biggest changes in postprandial glucose levels in this study were observed in group P2, which was treated with kefir combined with 2% spirulina (formula B).

Table 6 shows the changes in the SOD activity of the experimental groups. Administration of kefir-spirulina in groups P1 and P2 significantly increased SOD levels (p <0.001). The increase in SOD activity was 48.47 U.mL- 1, 41.86 U.mL^-1, and 25.86 U.mL^-1 for groups K3, P2, and P1, respectively. There is some background increase (6.39 U.mL^-1) in the negative control group (K2), while the normal control group (K1) showed a relatively constant SOD activity (p >0.05). A study by Omayma et al. (2013) showed that fermented soy could normalize the SOD and glutathione peroxidase (GPx) activities in rat tumor tissue. Kefir has a better ability to donate protons than nonfermented milk. Therefore, kefir can give protection against free radicals and thus increase SOD activity (Niccolai et al., 2019; Bellikci-Koyu et al., 2019; Zhang, Wu and Fei, 2016).

Oxidative stress is closely related to various diseases, including diabetes. The body will naturally develop enzymatic and nonenzymatic antioxidant defenses to balance oxidative stress. However, this endogenous protection might fail to prevent the overpopulated free radicals that are generated as the disease progresses. Therefore, exogenous antioxidant-rich foods such as kefir and spirulina are beneficial to back up the role of endogenous antioxidant systems.