Detarium microcarpium Bread Meal: It’s Physiological Effects on the Postprandial Blood Glucose and Insulin Levels of Healthy Non Diabetics Subjects

This work studied the effect of Detarium bread meal on the postprandial plasma glucose and insulin levels of healthy participants. The subjects of the study were ten healthy non diabetic male subjects who were fed two meals, an experimental bread meal containing detarium flour and a control bread meal made from wheat flour only. The test meals consisted of two small bread rolls, 38g of apricot jam (Robinson’s) and water to make a total meal weight of 400g. The available carbohydrate portion of the meal was 75g. The bread rolls contain 50g carbohydrate mostly in the form of starch. The jam provided 25g of available carbohydrate in the form of sucrose. The experimental breads rolls provided 5g of s-NSP as calculated from the nutrient analysis plus s-NSP from the brown flour. The subjects visited the metabolic kitchen twice a week after an overnight fast. All the subjects ate the two types of meals detarium and control bread meal in random order. The subjects were weighed and their heights were taken. A three days food record was kept to ensure adequate carbohydrate intake. Fasting blood samples were taken. Postprandial blood samples were taken from the subjects at 30, 60, 120, and 150 minutes from the commencement of the meal. The blood samples were analyzed for glucose and insulin levels. Data obtained were analyzed using ANOVA. The results of the incremental plasma glucose level showed a significant bread meal effect (Wilks’ Lambda 11.1 df 6 and 18; p=0.0049) and a significant time effect at (p=0.0129). The result showed a significant difference between the effect of the detarium bread compared to the control bread meal at (p=0.0008). The result also showed there was a significant difference for Detarium bread meal at 90, 120 and 150 minutes when compared to the control bread meal. ANOVA also showed a significant meal effect on the incremental insulin levels (Wilks’ Lambda 16.0; df 2, 18; p=0.0016) and a significant time effect (p=0.0230). There was a significant difference on the plasma insulin levels between the control and the Detarium bread meals (p=0.0022). Detarium bread showed a significant difference on the plasma insulin levels at 30, 60 and 150 minutes.


Introduction
Incorporation of Detarium flour into traditional Nigerian soup which was fed to healthy subjects resulted in significant lower post-prandial plasma glucose levels compared to the control meal (Onyechi, 2009). Lower plasma insulin levels were also seen after consumption Detarium soup meal compared to the control meal, though the result was not significant (Onyechi, 2009). However, the consumption of this type of traditional soup using thickeners like Detarium has been on the decline in most urban areas. These soup meals are consumed more in the rural areas while urban meals are getting very westernized (Onyechi, 1995). An attempt was therefore made to find an acceptable, palatable food vehicle for Detarium flour.
Studies have been done to introduce soluble non starch polysaccharide (s-NSP) into a variety of different foods (Fuessl et al., 1986;Peterson et al., 1987;Fairchild et al., 1990). A wide variety of foods have been developed like crisp bread (Jenkins et al., 1978a); biscuits (Ellis et al., 1988); pasta (Tognarelli et al., 1986); wheat bread (Apling and Ellis 1982;Peterson et al., 1987) and breakfast cereals (Fairchild et al., 1996). The most commonly used s-NSP has been guar gum, a galactomannan extract of the Indian cluster bean cyamopsis tetragonoloba which is normally produced as flour for commercial use. Guar containing wheat bread was one of the early food products to be investigated in acute and long term studies (Apling and Ellis, 1982). Most clinical studies in the literature have indicated that wheat bread containing guar gum improves carbohydrate tolerance and causes a reduction of plasma cholesterol levels in healthy and diabetic subjects (Ellis et al., 1991;Peterson et al., 1987). Wheat bread was considered to be a suitable medium for incorporating guar gum because it could be used as a part of any meal. Bread has been classified as a high glycaemic index food (BNF, 1990). However adding polysaccharide gum to wheat bread transforms the bread into a food with low or medium glycaemic index food similar to legumes or pasta (Ellis, 1994).
On the basis of these results detarium flour was incorporated into wheat flour to produce bread rolls. The physiological effect of Detarium bread meal was examined to determine the effect on postprandial blood glucose and insulin levels in healthy subjects. This novel approach could break the monotony of using detarium flour soups only.
The aim of the study was to provide variety in the use of locally available foods that is high in s-NSP in Nigeria. Detarium microcarpium locally known as "offor" is leguminous crop seed belonging to the Subdivision Caesalpinoideae (Balogun and Fetuga, 1986). Preliminary studies (Onyechi et al., 2007a) on the composition and nutrient analysis of Detarium showed that detarium was high in s-NSP

Bio-Research
Published December 2010 ISSN 1596-7409 (55.9g/100g). Detarium has been shown to have similar rheological properties as medium grade guar gum (M90) (Onyechi, 1995). Both detarium and guar gum had the same physiological effect on rats as both plants significantly reduced the fasting plasma cholesterol level of rats (Onyechi et al., 2007b). Detarium soup meal also had significant reduction on the incremental plasma glucose level in healthy non-diabetic subjects (Onyechi, 2009). These results indicated a positive physiological effect of detarium. Detarium flour was therefore incorporated into wheat flour to produce bread rolls. These were fed to healthy non-diabetic subjects. The physiological effects on postprandial incremental blood glucose and insulin levels were determined and compared to control bread meal effect.

Materials and Methods
Subjects: Ten healthy non-diabetic male subjects from King's College, London, participated in the study. Written information was given to each subject and consent forms were signed. The General practitioners (GP) of the participants were written to ascertain their health status with respect to the study. The protocol was approved by the King's College Research and ethical committee.

Preparation of detarium bread:
The composition and quantity of the ingredients used in the preparation of the bread is shown in Table 1. Chorleywood bread process (Apling and Ellis, 1982) was used in the preparation of the bread rolls. Ploughman's brown flour (Sovereign, Allied Mills, London) was the type of flour and flora brand (Unilever, UK) was the hydrogenated vegetable fat used. Each batch of the bread rolls contained variable amounts of water depending on the viscosity of the flour. Detarium flour was incorporated into the bread as a replacement for wheat flour. The weight of the dough was calculated such that a total of 50g carbohydrate was contained in the two bread rolls. Each detarium bread roll contained 2.5g s-NSP. Two hours after baking the bread rolls were frozen in self sealed freezer bags at -20 o C until required for experimental use.  (Onyechi, 1995) Glucose analysis: The glucose and insulin increments (changes relative to fasting values) were determined at 30, 60, 90, 120, and 150 minutes after the subjects had consumed detarium and control bread rolls. The plasma glucose was measured by standard glucose oxidase method (Werner et al., 1970) using a Bushrangers Mannhein kit (Boehringer Mannheim House, Bell Lane, Lewes BN7 1LG). The frozen deproteinized plasma was allowed to thaw and mixed in a rotamixer for 2 minutes. A 100 ul of the supernatant was mixed with 5 ml of the reagent which contains buffer, enzymes and chromogen. The sample was mixed in a rotamixer and incubated in a water bath at 20-25 o C for 40 minutes avoiding direct exposure to sunlight. The absorbance of the sample and the standard were measured against a blank in a spectrophotometer at 610 nm.

Insulin analysis:
The Boehringer Mannhein diagnostic kit based on enzyme immunological reactions was used for the quantitative determination of human insulin in-vitro. The ES 22 combi step analyzer program B auto machine was used. Precipath IM was the quality control serum used to run each analysis and values were within the stipulated range. Five standards were used which ran in duplicates along with duplicate samples of the control and test sera. The machine automatically dispensed and washed out the tubes with reaction solutions. The tubes were automatically read after the incubation period by passing along a conveyer into the spectrophotometer, where the solution is aspirated out and absorbance plotted. A computer program was used to read the blank, standard, control serum and the test sample for each run. The absorbance readings were calculated in the calculation mode and the standard curve plotted. The concentration of plasma insulin in the test sample was calculated from the standard curve.

Statistical analysis:
The difference between the effects of the control and detarium bread meals on the blood glucose and insulin were analysed by repeated measure of analysis of variance, ANOVA, SAS Statistical package, (SAS Institute Inc., 1985). Significance difference between the control and the detarium bread meals were accepted at p<0.05.

Results
Subjects: Ten subjects between the ages of 21 and 39 years participated in the study. The body weight varied between 57 and 94 kg and calculation of the BMI showed that all but three were within desirable range of weight for height. Each subject consumed two small bread roll of control and detaruim that weighed 98g and 125g each respectively.

Nutrient composition of the bread rolls by calculation:
The nutrient composition of the bread rolls indicated that the detarium bread had more moisture and fat than the control bread, while the control bread had more protein and carbohydrate than the detarium bread ( Table 2). Weight of cooked dough to provide 50g carbohydrate was lower than the raw dough, 98g and 125g respectively for control and detarium bread rolls. The proximate analysis indicated that the control Effects of detarium bread meal on blood glucose and insulin levels of healthy non diabetics subjects 662 bread rolls had higher protein (7.8g) than the detarium bread (6.8g). With regards to energy, the control bread had higher energy profile than the detarium bread (Table 2).

Effect of the bread meals on the plasma glucose levels of the subjects:
The result showed that fasting blood glucose levels were found to be within the normal range for non-diabetic subjects. The pooled mean of the fasting blood glucose level for the subjects was 4.30mmol/L. The post-prandial rise in blood glucose levels was expressed as incremental blood glucose levels which were calculated relative to the fasting values. The mean incremental blood glucose levels were shown in Table 3. Analysis of the data using ANOVA showed a significant bread meal effect (Wilks' Lambda 11.1; df 6 and 18; (p = 0.0049) and a significant time effect at (p = 0.0129). Comparison of the mean incremental blood glucose rise after the consumption of the control bread and the detarium bread showed a significant difference between the detaruim and control meals at (p = 0.0008). When the difference between the bread meals was analyzed at each time interval, the result showed that there was a significant difference for detarium bread at 90, 120 and 150 minutes when compared with the control.

Effect of the bread meal on the plasma insulin levels:
The result showed that the pooled mean fasting insulin levels was 15.07Uu/ml and within the normal range for non diabetics. Incremental plasma insulin levels were calculated relative to the fasting levels at all postprandial times and shown in Table  4. ANOVA showed that there was significant bread meal effect (Wilks' lambda 16.0; df 2,18 p = 0.0016). There was also a significant time effect (p = 0.0230), when the control bread was compared to the detarium bread.

Discussion
The result of this study showed that when healthy subjects were fed bread meals containing 75g carbohydrate and 6g s-NSP from detarium flour, there was a significant main meal effect (p=0.0049) compared to the low fibre control bread. When the incremental plasma glucose level of detarium was compared to the control bread, a significant lowering effect was found after the consumption of detarium bread (p=0.0008) at 90,120 and 150 minutes post-prandially. Comparison of the effect of the control bread and the detarium bread on insulin level also showed a significant meal effect between the detarium bread and the control bread (p=0.0016). The detarium bread showed a significant lowering effect (p=0.0016) at 90 and 120 minutes. The detarium bread meal proved to be significantly effective in lowering plasma glucose and insulin levels. Jenkins et al., (1980) listed the mechanisms by which guar gum modulates postprandial hyperglycaemia and hormone responses. These mechanisms may be similar to the mechanism by which detarium lowered postprandial glucose and insulin levels. Detarium and guar gum are strikingly similar. Both are legumes and contain s-NSP. Both have similar rheological properties and detarium has a molecular weight similar to medium grade guar gum (Onyechi, 1995). These mechanisms of action include viscosity effect. Jenkins et al., (1978a) showed a positive correlation between the peak rise of blood glucose and 2-hour post-prandial glucose level with the viscosity of four viscous NSP, guar, tragacanth, pectin and methyl cellulose in healthy subjects. Reduction of insulin response - Jenkins et al., 1976 showed that fibre enriched meal produced a marked flattening of the postprandial glycaemia and insulin response. Other studies in the literature have observed similar effects with high NSP meals (Jenkins et al., 1977;Wolever et al., 1979;Morgan et al., 1979). Jenkins et al., 1980 indicated that there was overall flattening of the endocrine response induced by viscous NSP as shown by Morgan et al., (1979) in which GIP response was flattened after guar supplementation. Slow absorption - Jenkins et al., (1978b) indicated that the depressed postprandial glycaemic response with s-NSP was due to slow absorption rather than malabsorption. These results were later confirmed by Leeds et al., (1978) who showed that guar did not cause carbohydrate malabsorption due to the absence of hydrogen breath in the subjects. Gastric emptying small intestine absorption, Jenkins et al., (1980) pointed out that delayed mouth to caecum transit times are associated with ingestion of the viscous NSP and showed a significant relationship between mouth-to-caecum transit with viscosity (Jenkins et al., 1978). Holt et al., (1979) suggested that delayed gastric emptying may be of great importance in flattening post-prandial glycaemia. Jenkins et al., (1978b) explained that viscous NSP prevents gastrointestinal hurry and slows the rate of nutrient absorption resulting in flatter post-prandial levels of glucose, metabolites and hormones.
It is possible that the positive physiological effect of detarium in modulating postprandial glucose and insulin profile could be attributed to the above mentioned mechanisms listed by Jenkins et al., (1980). Detarium in addition to having the same physiological properties as guar gum both s-NSP had the same effect on the rats. Both detarium and guar gum covariates such as weight gain, food intake, faecal output and they both lowered the plasma cholesterol levels of rats Onyechi et al., (2007b). The similarity of effects of detarium to guar gum suggests this indigenous food could be a useful adjunct to the treatment of diabetes mellitus in Nigeria and detarium bread rolls may provide a variety to the diet of the diabetics that resides in the urban areas.

Conclusion:
The result of the study showed a significant lowering effect on the post-prandial glucose and insulin levels of healthy non diabetic subjects. On the basis of this result it was decided that detarium could be studied further with noninsulin diabetic subjects. This to ascertain if there would be any improvement in the postprandial glucose and insulin profiles of the diabetics. colleagues at King's College, London, Mrs Rose Colokasia and David Lincoin for their expert technical assistance; Professor Simon Ross-Murphy for his helpful discussion on the physicochemical properties of the polysaccharides; Mr Peter Milligan for advice on statistics. The author also is grateful to Dr. Hans Englyst (MRC Dunn Clinical Nutrition Centre, Cambridge) for his help with the analysis on the NSP and starch of the foods. This work could not have been possible without the support of Association of Commonwealth Universities that granted a scholarship to the author.