White yam (Discorea rotundata) was obtained from National Root Crop Research Institute, Umudike, Abia State, Nigeria; while white Bambara groundnut was obtained from a local market in Lagos, Nigeria. Common salt and granulated sugar were also obtained from a local market in Lagos, Nigeria.

A laboratory sized Single screw extruder (Komet Extruder, 1993 Model Made in Germany) was used in this study requiring a minimum input of 0.2 kg as feed raw materials for extrusion cooking.

Yam grits (<750 µm) obtained by processing white yam through various operations of washing peeling, pregelatinizing, drying and milling into grits and Bambara groundnut flour obtained by processing White Bambara groundnuts through processing operations of manual destining, cleaning, milling into flour (<250 µm) were co-extruded in a single screw extruder at ratio 4:1 yam grits and bambara flour respectively with added flavouring agents of salt (<1%) and sugar (2% - 3%) respectively. The extrusion cooking process occurred at the screw speed of 70 rpm, barrel temperature of 145˚C and 17.5% feed moisture. The resulting extrudates were allowed to cool after exiting the extruder die, packaged (0.002 µm gauge size) in high density polyethylene bag and stored at room temperature (28˚C ± 2˚C) and at refrigeration temperature (9˚C ± 2˚C) for storage and nutritional evaluation studies.

The total plate count of the flavoured extrudates was determined using established procedure of Harrigan and Mclance [11].

1 gm of each sample already milled into flour was weighed into 9 ml of sterile normal saline solution. The mixture was thoroughly homogenized and diluted serially from 10⁻¹ to 10⁻⁵. About 0.1 ml of 10⁻³ diluted sample was spread evenly with sterile horse stick on each media used, namely Malt extract (MA), Maconkey agar (MAC) and Nutrient agar (NA) and each incubated at 37˚C for a period of 24 hr. The colonies that grew on each plate were then counted manually. The extrudates were stored at room temperature (28˚C ± 2˚C) and in a refrigerator (9˚C ± 2˚C) and the microbiological test was done on each sample monthly for a period of 4 - 5 months.

The chemical composition of the extrudates determined includes moisture content, crude protein, crude fibre, ash, and carbohydrate by difference. These were carried out using established procedures of AOAC [12]. The energy content of the flavoured extrudates (DF₁ and DF₂) as well as the protein free diet was determined by calculation using Egan et al. [13] and James et al. [14] method respectively.

Total mineral content in the extrudates and faeces were determined by ashing in a furnace at 550˚C and individual minerals (calcium, iron and phosphorous) were determined in the ash solution on 10% HNO₃ as follows: Phosphorous by colorimetry using the ammonium phosphate vanadium molybdate method. Calcium and Iron by the ortho-pheno-throline method of Pearson [15].

This was determined in the extrudates using established procedure of Kakade et al., [16]. One gram of sample was weighed and 50 ml of 0.01 N NaOH was added to each sample. The pH was adjusted to 8.8 using 1 NHCl to reduce pH to the required level. The sample was allowed to stay for 3hours and stirred continuously to maintain the sample in suspension. One ml extract, 2 ml of the diluted sample was taken and poured into three (3) test tubes while no trypsin solution was added to the third test tube. Also, 2 ml of distilled water was withdrawn in three (3) test tubes and 2 ml of trypsin solution was added to the test tube and was not added to the test tube, the sample in the test tube were returned to the water bath and warmed for 10 minutes. After 10 min, 5 ml of BAPA solution (Benzoyl-DL-arginine-p-nitroanalide hydrochloride) was added to all the test tubes, vortexed and warmed again for 10 min. After 10 min, 1 ml of glacial acetic acid was added to all the test tube except the third test tube that did not contain trypsin initially. The samples were filtered using Whatman No. 2 filter paper and the absorbance read at 420 nm using a spectrophotometer.

The rat diets casein control and protein free diet were formulated according to method of Lape [17] and Fasuyi et al. [18]. The various ingredients used include some amino acids such as L-Threorine, DL-Methionine, LCystine and Lysine, mineral mix, soya oil, potassium carbonate, magnesium citrate, zinc sulphate, copper sulphate, vitamin mix and starch. The detail of the formulation is as shown in Table 1.

Weanling albino rats of the Wister strain were used. Five rats (3 males and 2 females) were assigned to the casein-based diet and each of the yam-bambara extrudates/diet under investigation. Rats were divided into groups with similar average weights (70 g ± 1 g) and growth rates and were housed in individual polyethylene metabolic cages in a room at about 28˚C and 60% relative humidity and with a 12 hour light/dark cycle.

Rats were fed with the diets (i.e. flavoured extrudates DF₁, DF₂ casein diet and protein free diet) for 7 days (adaptation period) and then for further 10 days (experimental period) during which feed intake were measured. They also received distilled water ad libitum and were weighed every three (3) days of the experimental period to determine average daily weight gain or loss.

During the last five (5) days of the experimental period, daily collections of the total urine and faeces of each rat were made separately, posted together and frozen until analysis. A 0.5 ml sample of 4.8 M HCI was added to recipients before urine collection to prevent loss in the form of ammonia. The care for all the animals was in accordance with the National Law on Animal care and use [20,21].

Means, standard deviation of the data were calculated using Microsoft Excel package while the test and separation of means among the samples were carried out using Analysis of variance (ANOVA) and Duncan’s New Multiple Range Tests (DNMRT) with SPSS package version 15.

Figure 1 shows the microbiological changes as measured by the total plate count in the flavoured extrudates stored at different conditions of temperature. The microbiological load as measured by the total plate count per gram of sample was generally low in all the samples stored under refrigeration conditions. This shows that temperature has a significant effect on the growth of micro-organisms. Generally as the storage time increased, the measured total plate count at room temperature steadily increased while for samples stored in the refrigerator, there was a much lower rate of increase in the total plate count. The total plate count of the extrudates on week 0 showed that the extrudates had an initial total plate count of 0.2 × 10⁴

cfu/g. On storage in high density polyethylene bag (gauge size 0.03 µm), there was a steady increase in the total plate count among the extrudates both at room temperature and at refrigeration temperatures. As the storage time further increases to 8 weeks, there was a continuous increase in the total plate count particularly at the room temperature. A similar trend was observed when the extrudates were stored at 12, 16 and at 20 weeks.

Based on the above result (Figure 1), extrudate DF₃ was discarded because of the relatively higher microbial load when stored under both conditions investigated in this study as compared with the other extrudates (i.e. DF₁ and DF₂).

The maximum permissible level of total aerobic colony of ready-to-eat foods as given by Fylde Borough Council extracted from manual of PHLSG [22] was 10⁴ to less than 10⁶ cfu/g of ready-to-eat food products. This therefore shows that the extrudates were stable under the different storage conditions investigated in this study. The microbial loads of the extrudates stored under refrigeration conditions were generally low which showed that temperature had a significant effect on the growth of micro-organisms [23].

Also micro-organisms have been reported to be inhibited by the inclusion of salt, sugar and spices [24] in food.

The microbial load of any food material is however a useful index of quality in the extrudate as well as revealing the potential safety status of the extruded food products from human consumption point of view and storage of the products. Total counts have been reported to be

useful monitor in processing of food products and may therefore reflect poor handling or storage at retail level [25]. Bacillus cereus and coliforms have however been identified as the major organisms causing spoilage in extruded food products and the total plate count must also be very low in order to rule out the possibility of formation of preformed toxins in the extruded food products [22].

The chemical composition of the flavoured extrudates ((DF₁ and DF₂) as shown in Table 2 revealed that they contained 13.31% and 14.20% crude protein respectively. The starch content were 60.83%, 58.95% and 98% in DF₁, DF₂ and PF diets respectively while the fat content was 1% and 1.28% in DF₁ and DF₂ respectively. The crude fibre was 2.04% in the flavoured extrudates, DF₁ and 1.09% in the flavoured extrudate DF₂, 2.63% and 2.90% ash in DF₁ and DF₂ respectively, while the energy value was 1707.21 KJ/100g in the flavoured extrudate DF₁, 1724.24 KJ/100g in the flavoured extrudate, DF₂ and1671.4 KJ/100g in the protein free diet. The high values of crude protein in the flavoured extrudates were expected as Bambara groundnut have been  reported to be a very good source of protein which could be as high as 22% - 25% in the raw beans [26].

Also the starch content values in extrudates DF₁ and DF₂ were also expected as both raw yam and bambara groundnut contain appreciable level of starch [9,26].

Both flavoured extrudates contained appreciable levels of calcium, iron and phosphorous as shown in Figure 2 below. Raw yam and bambara groundnut have been reported to contain appreciable levels of calcium, Iron and phosphorous [7,24]. Both crops contain appreciable levels of calcium, Iron and Phosphorous.

The potential value of a food for supplying a particular nutrient can be determined by chemical analysis but the actual value of the food to the animal can be arrived at only after making allowances for the inevitable losses that occur during digestion, absorption and metabolism [18,27].

The digestibility of a food is defined as that portion of the food which is not excreted in the faeces and which is therefore assumed to be absorbed by the animal.

The rat feeding experiment conducted in this study showed that there was significant difference among the experimental diets at (p ≤ 0.05) in terms of the protein concentration in the different diets fed to the experimental animals with the casein diet (CD) containing the highest value of protein followed by the extrudate/diet DF₁ as shown in Table 3.

Table 2. Chemical composition of flavoured extrudates.

Values are mean of three determinations ± SD values.

Also there were significant differences among the yam-bambara extrudates in terms of the protein concentration in faeces of rate fed with different flavoured extrudates (p ≤ 0.05) with the casein diet recording the highest value protein. This was followed by the protein concentration in the faeces of rats fed with extrudate DF₂ (0.38 g·g^–1) followed by extrudate DF₁ (0.34 g·g^–1) and lastly by the protein free diet, PF which had a protein concentration in the faeces of the rats of 0.17 g·g^–1.

The study also revealed that in terms of the total protein intake by each experimental rat, there were significant differences (p ≤ 0.05) between the protein intake in group DF_1, DF₂ and casein diet. However, there were significant differences (p ≤ 0.05) among the total protein intake by each rat fed with extrudate/diet DF_1, DF₂ CD and PF, which was supposed to be an almost protein free diet. However, the casein diet remained the best diet in terms of the total protein intake by the rats as it had the highest mean protein intake value of DF₁ with a mean protein intake value of 7.26 g in the fed rats. A mean protein intake of 3.99 g was obtained for extrudates/diet DF₂ while the least protein intake of 0.11 was obtained free diet. A maximum protein intake value of 8.97 g was recorded in the casein diet used in this study.

Generally, there were significant differences (p ≤ 0.05) among the extrudate and diet fed to the experimental animals in term of feed consumed, faeces excreted, urine excreted and weight gain in the rats. Flavoured extrudate DF₁ was better consumed by the animals than flavoured extrudate DF₂.

In terms of faeces excreted, rats fed with extrudate/diet DF₂ had the highest excreted faeces, followed by extrudate DF1. In terms of urine excreted, rats fed with extrudate DF₂ produced the highest level of urine followed by the rats fed with flavoured extrudate DF₁. It appears that flavoured extrudates/diet (DF₁) is better than flavoured extrudates/diet (DF₂) from the standpoint of feed consumed, faeces excreted and weight gained by the laboratory animals during the study as shown in Table 3.

The protein efficiency ratio (PER) normally uses growth of the rat as a measure of the nutritive value of dietary proteins. It is defined as the weight gain per unit weight of protein eaten. The extrudates produced supported the growth of the laboratory animals used with flavoured extrudate DF₁ giving a better growth in the experimental rats used then the flavoured extrudate or diet DF₂.

Biological value shows a direct measure of the food protein which can be utilized by the animal for synthesizing body tissue and compounds and may be defined as the percentage of the nitrogen absorbed which is retained by the animal. The biological value in both flavoured extrudates was very high showing that most of the nitrogen was absorbed by the animals. Part of the nitrogen of the faeces, the metabolic faecal nitrogen, is not directly derived from food. Urinary nitrogen also contains a proportion of nitrogen known as endogenous urinary nitrogen which is not directly produced from the ingested food by the test animals.

Both extrudates (DF₁ and DF₂) produced moderate quantities of urinary protein and faecal protein. Urinary nitrogen/protein and faecal nitrogen protein have been reported to be totally directly produced from ingested food nitrogen by the animals, but it is nitrogen derived from expendable tissues and secretions together with waste nitrogen arising during normal catabolism and re-synthesis of tissue proteins.

The existence in both faeces and urine of nitrogen fractions whose excretion is independent of food nitrogen is most convincingly demonstrated by the fact that some nitrogen is excreted when the animal is given a nitrogen free diet [22,28] as revealed by the urinary nitrogen/ protein and faecal nitrogen/protein produced by the experimental animals fed with non-protein diet in this study.

The study also indicated that extrudate DF₁ was better

Table 3. Nutritional qualities of yam-bambara extrudates.

consumed than extrudate DF₂ by the experimental animals employed in this study. This might be due to the salt to sugar ratio in the feed blend. Sugar and salt have been reported to enhance flavour and sensory acceptability of certain foods [4,6].

Rats fed with the flavoured extrudate DF₂, however, had the highest excreted faeces lower levels of protein in urine excreted than the flavoured extrudate DF₁ implying that rats were fed with the flavoured extrudate DF₂ did not make use of the extrudate properly in the digestive system thereby getting rid of them as faeces. This, in turn, affected the weight gain of the experimental rats fed with extrudate/diet DF₂ resulting into lower weight gained by the rats than in the rats fed with extrudate/diet DF₁.

Similar studies on extruded products have shown similar observations [29-31]. The study has shown that extrudate DF₁ was better than extrudate DF₂ based on the positive feed consumption, faeces excreted and weight gain pattern obtained in this study. High protein efficiency ratios of 0.74 and 0.79 have been reported in the extrudates DF₁ and DF₂ with high biological values of 0.99 and 0.96, respectively. A similar observation in extruded corn-soya mixtures had been documented [29]. It has been reported that extrusion cooking improves the protein digestibility of extruded food products [28,30, 31].

The challenging performance of rats fed with extruded diets to those fed casein diet was attributed to the effect of extrusion cooking as a form of heat treatment to the mixtures. Heat is known to improve the availability of some nutrients, inactivate enzymes that speed up nutrient damage and destroy undesirable microorganisms and food contaminant [23,28,29,31]. It also favourably changes the physical attributes of food such as colour, texture and flavour [32] and improves palatability and digestibility due possibly to higher starch damage [28, 29,32,33].

The urinary output of rats fed with yam-bamabara extrudates DF₁ and DF₂ were higher than those from casein diets. Rats with lower urinary nitrogen output showed higher nitrogen retention [27]. Low urinary and faecal nitrogen indicate high protein quality of fed diets/extrudates [31]. Also the biological values of 0.99 and 0.96 obtained for extrudates DF₁ and DF₂ were however comparable to that obtained in extruded Bread fruit-soya blend of 0.94 as reported by Nwabueze [28] while the standard casein diet had a biological value of 1.0, lower biological values of diets fed to rats may be due to lower nitrogen intake by the fed rats and higher urinary output, which could be indicative of poor utilization of digested/retained nitrogen [30].

The protein efficiency ratio (PER), is an index of protein quality in the extrudates which shows the relationship between weight gain by the test animals and the corresponding protein as nitrogen intake [18,23,25].