MATERIALS AND METHODS

The Nono used in this study was obtained from a single local producer in rural Nigeria. This was to ensure reasonable consistency of the product. The LAB population of each sample purchased was assayed for by the pour plate method employing MRS agar with incubation in a microaerophilic condition at 37°C.

Weaned albino rats were obtained from the Department of Biochemistry, University of Jos, Nigeria. The rats were fed commercial feed and water ad libitum. Thirty-two rats with average body weight of 100 g were sorted at random into 4 groups. The four groups were treated as follows: Diabetes induced given normal saline; Diabetes induced given Nono; diabetes not induced given normal saline and diabetes not induced given Nono.

Diabetes was induced in the rats by intraperitonial injection of alloxan monohydrate dissolved in distilled water at a concentration of 100 mg kg^-1 body weight. Diabetic situation was confirmed established before experimentation progressed. Administration of Nono was by oral intubation at 1 mL per rat thrice daily for 4 weeks. Groups not receiving Nono received 1 mL of normal saline per rat to equilibrate the stress of the treatment.

The method of Usman and Hosono (2000) was employed to collect blood samples from the tip of the tail of each rat which was used to determine the plasma glucose on a weekly basis. Plasma glucose concentration was determined by the method of Trinder (1969). At the end of the fourth week of nono administration the rats were sacrificed and the organs (kidney, heart and liver) were removed and preserved for the determination of total protein, lipid and cholesterol concentrations. Total protein in the serum and tissue homogenates was determined by the method of Mao and Tucci (1991). Total lipid and cholesterol in plasma and tissue homogenates were determined enzymatically with a commercial kit (Biosystem, SA.) The standard error of means of the different values was determined.

RESULTS AND DISCUSSION

Successful establishment of diabetes raised plasma glucose concentration to over 250 mg dL^-1 from 100 mg dL^-1 after induction by alloxan in rats (Fig. 1). This is expected (Mitchell, 2005). Diabetic rats fed with Nono had their plasma glucose concentration restored to the level of the control rats in which diabetes were not induced by the fourth week of feeding with Nono; while the plasma glucose concentration in non-diabetic rats suffered a slight reduction when fed with Nono (Fig. 1). This confirms the work of Tabuchi et al. (2003b), which reported hypoglycemic effects in rats fed with cells of lactic acid bacteria. It is thus conceivable that the Nono effect may be a function of its lactic acid bacteria component which was recorded at an average of 2.04x10⁶ mL^-1 (range = 1.16-4.0x10⁶).

The diabetic rats had significantly lower tissue protein and a concomitant raised plasma protein level relative to the non diabetic rats. This again is expected (William, 1999). Nono by the 4th week of feeding restored the protein level to the level of non diabetic rats (Table 1).

Hyperlipidemia and hypercholesterolemia are characteristic features of diabetes (Baggio and Drucker, 2006). Lipids and cholesterol have been reported to also accumulate in the tissues of the heart, liver and kidney. Table 2 and 3 show increased concentration of lipids and cholesterol in the organ tissues and the plasma of the diabetic animals. However, administration of Nono had significant hypolipidemic and hypocholesterolemic effect in both the plasma and the organs of diabetic rats by the 4th week of feeding.

Fig. 1:	Plasma glucose concentration in diabetic rats fed with Nono

Table 1:	Total protein concentration (mg g-1) in selected organs and plasma (mg dL-1) in diabetic rats administered fermented milk (Nono) at 4 weeks of feeding
Each value is the mean of determinations from the eight rats in each treatment group±SEM. Values with different notations are significantly different (p≤0.05)

Table 2:	Total lipid concentration (mg g-1) in selected organs and plasma (mg dL-1) of alloxan-induced diabetic rats administered fermented milk (Nono) at 4 weeks of feeding
Each value is the mean of determinations from the eight rats in each treatment group±SEM. Values with different notations are significantly different (p≤0.05)

Table 3:	Cholesterol concentration (mg g-1) in selected organs and plasma (mg dL-1) of alloxan-induced diabetic rats administered fermented milk (Nono) at 4 weeks of feeding
Each value is the mean of determination from the eight rats in each treatment group±SEM. Values with different notations are significantly different (p≤0.05)

The alterations in the protein, lipid and cholesterol concentrations in the organ tissues and the plasma are consequential on plasma hyperglycemic situation of diabetes. A control of the blood sugar is likely to result in normalization of the concentration of these other compounds. This apparently is what happened with the significant reduction in the plasma glucose concentration of the diabetic rats fed with Nono. It has been suggested that the chronic presence of LAB in the intestine through daily consumption of bacteria as in this study is needed for a long term effect on metabolism (St-Onge et al., 2000). Increase in the population of LAB in the intestine as a result of the administration of Nono may result in increased demands for glucose for normal energy requirement of these organisms to run their metabolic activities. This may lead to a decrease in the glucose concentration released into the serum and the various organs of the animals.

The hypocholesterolemic effect observed may also be as a result of the increased excretion of bile acids due to deconjugation activities of the LAB in Nono to produce free bile acids. Xiao et al. (2003) explained that serum cholesterol level decreases when a component suppresses resorption of bile acid. Nono is produced through natural fermentation without use of pure starter culture except fortuitous inoculation from previous fermentations (Akinyanju, 1989; Savadogo et al., 2004). Thus, this research indicates that wild strains of LAB in fermented milk may be capable of reversing diabetic situation. Further work is required to elucidate the mechanism of diabetic control by lactic acid bacteria containing foods.

ACKNOWLEDGMENTS

This research was supported by a research grant of Adekunle Ajasin University, Akungba-Akoko, Nigeria.