Investigation into the Coagulating Properties of Acid and Enzyme Coagulated Soy Protein Precipitate

Precipitation is widely used in processing of biological products, such as proteins. The coagulating properties of Sodom apple ju ice, vinegar, lemon juice and rennet as coagulants of soy protein precipitate from soymilk at different temperature were investigated. Soymilk was produced using the hot grind method. The studied coagulants were used to precipitate soy protein from the produced milk at d ifferent temperature respectively. The coagulation time was observed to decrease with increasing temperature, with 100°C giv ing the best time. The pH of the coagulated proteins was observed to decrease with increasing temperature with the optimal pH observed at 70°C for all the coagulants. No significant difference was observed on the effect of coagulating temperatures on the yield of coagulated soy protein precipitate. Results from this study indicate that optimum coagulating activ ities can be achieved between 7080°C . However, the yields by the studied coagulants are not affected by temperature. Thus, implying that temperature modulate the coagulating activit ies, pH and not yields of the coagulants. Therefore, production at 70 – 80°C will g ive the best soy protein quality.


Introduction
Protein deficiency and malnutrition is a well documented cause of ill health and death in developing countries, and plays a major role in the disease, kwashiorkor [1]. Its deficiency can also lead to several ailments including mental retardation. Food-based strategies favouring local mu lti-nutrient food materials have been reported to be the best suitable and sustainable approach for combating protein malnutrition [2]. A mongst such food is the soybean which contains high quality protein and readily available in most developing countries. Over the years, they have been processed and used to fortify cereal flours and other food products. The use of high-protein soy products to combat malnutrit ion in developing countries is now being emp loyed by several hunger-fighting organizations [3].
Soy protein is a popular food ingredient used throughout the world for its nutritional and functional p roperties [4]. It has gained considerable attention due to its potential role in improving risk factors for card iovascular disease (CVD) especially after approval by FDA [5]. Their approval was based on clinical studies showing that at least 25 g of soy protein per day lowered total and LDL cholesterol [6]. So me clin ical studies suggest that soy protein and its isoflavones protect bone density, but this has not been firmly established. Soybean protein is regarded as complete protein since it provides all of the essential amino acids for human nutrition [7]. However, some scientific sources disagree on this as the best measure of complete protein [8]. Soybean protein is essentially identical to that of other legume pulses and is one of the least expensive sources of dietary protein [9]. These claims have led to the development of new soy foods and new technologies as well as imp rovement/ modification of old methods of their preparation.
Among such methods of preparation is the coagulation of soy protein precipitate employed in the production of tofu fro m soy milk. Tofu is one of the most important tradit ional soy foods in the eastern world, with a limited shelf life [4]. Tofu is lo w in calories, contains a relat ively large amount of iron, and contains little fat [10]. Depending on the coagulant used in manufacturing, the tofu may also be high in calciu m and/or magnesium. Coagulation of the protein and oil (emulsion) suspended in the boiled soy milk is the most important step in the production of tofu. This process is accomplished with the aid of coagulants. Coagulation could be achieved by increase in the acid ity, extended heating or enzy me activ ity. Rennet ext ract fro m the abomasums of calves has been the traditional coagulant, but it is now substituted with rennet extracted fro m some bacteria and mould [11]. Acid ic coagulation is often achieved by the addition of vinegar or vegetable extracts.This paper aims at investigating the coagulating properties of Sodom apple juice, vinegar, lemon juice and rennet as coagulants of soy protein precipitate fro m soymilk at d ifferent temperature.

Plant material
Soybeans (Glycine max) of yello w variety tax 306-036C were obtained fro m the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. Lemon fruits and leaves of Sodom apple (Calotropis procera) were obtained from Ketu fruit market, Lagos, Nigeria. Rennet and vinegar were obtained from a food grade chemical store and supermarket respectively at Lagos, Nigeria.

Extraction of lemon juice
The lemon fru its were washed and cut into halves. The juice was extracted using juice extractor after which it was filtered through a sieve to remove the seeds, packed in air tight containers and refrigerated until further analysis.
Extraction of Sodom apple juice 45 g of the leaves of Sodom apple juice was homogenized with 300ml distilled water and then sieved to remove solid particles. It was centrifuged at 10,000 rp m for 20 minutes in an ultracentrifuge at a temperature of < 2℃. The supernatant was decanted and stored at < 4℃ for subsequent analysis.
Production of soy milk This was carried out to according to the Illinois method as described by Nelson et al. [12].

Determination of the p hysicochemical properties of soymilk
This was carried out according to AOAC methods [13].

Coagulation and precipitation of soy protein with various coagulants
100ml of the soymilk samp le was heated to different temperature of 50℃, 60℃, 70℃, 80℃, 90℃ and 100℃ in separate boiling tubes. 5ml of each coagulant (Sodo m apple juice, rennet, vinegar, and lemon ju ice) was added separately to each of the boiling tube at different temperatures. The time required for coagulation to take place was noted and recorded. The pH of each sample and coagulant were noted. The percentage yield for each coagulant at different temperature was calculated as: % Yield = weight of soy protein precipitate x 100 (w/v) Vo lu me of milk Statistical Analysis Data were reported as mean + standard deviation. Statistical analyses were carried out using SPSS for Windows, version 14.0 (SPSS Inc. Ch icago, IL.USA).

Results and Discussion
Precip itation is widely used in downstream processing of biological products, such as proteins [14]. Th is serves to concentrate and fractionate the target product fro m various contaminants. This paper reports the coagulating properties of different natural coagulants in soy protein precipitation fro m soymilk at different temperatures.

Physicochemical properties of soymilk
The physicochemical propert ies of soymilk are presented on table 1. A very high content of crude protein was observed. This is far h igher than results observed by Salim-u r-Reh man et al. [15] on soymilk and cow milk. The observed high protein content is very encouraging as protein deficiency is a serious cause of ill health and death in developing countries and plays a part in the disease kwashiorkor [16]. Thus, correlating with previous reports on the choice of soybean in combating protein malnutrition [2]. The fat and ash contents were observed to be much higher than those of previous studies [15,17]. Th is may be as result of the method of preparation.  Figure 2 shows coagulation time at different temperatures. The coagulation time was observed to decrease with increase in temperature (50 -100℃). 100℃ was the best coagulation time with vinegar having the lowest coagulation time (30 seconds) and rennet having the highest time (4 minutes). However rennet was observed to have a lower coagulation time (3 minutes) at 80℃, which increased thereafter. 80℃ may thus be said to be the optimu m temperature for rennet. En zy mes are denatured with increasing temperature; therefo re, at temperature above 80℃ rennet may be inactivated. This dependant increase in coagulation time on high temperature corresponds to previous reports by Dybowska and Fujio [18]; and Naz et al. [19]. The pH of coagulant has been reported to greatly affect coagulating efficiency [20]. Furthermore, Naz et al. [19] reported that coagulation is related to the effect of pH on the enzy matic and aggregation phases of milk coagulation. Figure 3 shows the pH of soymilk and the coagulants. The pH of the coagulated proteins by the different coagulants was observed to decrease with increasing temperature (50-100℃) (figure 4). The optimal pH was observed at 70℃ for all coagulants.  The effect of coagulation temperature on yield coagulated soy protein precipitate is presented on Figure 5. No significant difference was observed on the effect of coagulating temperatures on the yield o f coagulated soy protein precipitate.

Conclusions
Results fro m this study indicate that optimu m coagulating activities can be achieved between 70-80℃. Ho wever, the yields by the studied coagulants are not affected by temperature. Thus, imp lying that temperature modulate the coagulating activities, pH and not yields of the coagulants. Therefore, production at 70 -80℃ will give the best soy protein quality.