(166m) Canola Protein Production Using Different Extraction and Concentration Methods: In Vitro protein Digestibility, Functional Properties and Process Yields

Proteins are important macronutrients in human nutrition and their consumption is relevant to sustain proper growth and maintenance. However, in developing countries protein malnutrition or Kwashiorkor is still one of the chief causes of death among preschool-aged children (Kalu and Etim, 2018). Unfortunately, there are still around 690 to 815 million undernourished people worldwide. The high levels of malnutrition and elevated cost of animal proteins have prompted research into the generation of novel plant-based alternatives that are produced massively. Among the best options, there are raw materials and coproducts generated by the oilseed industry. After oil extraction of most oilseeds, the crushing industries generate defatted protein meals with high protein that are normally channeled to the production of feedstocks.

Canola is the second-largest oilseed crop grown worldwide (USDA, 2020). The canola protein meal (CPM) is the primary byproduct of the oil recovery process containing around 40% protein (Jia et al., 2021). This oilseed was obtained after a long-lasting breeding work or regular rapeseed in Canada with the aim to obtain food-grade oil, namely with low erucic acid (<2%) and glycosylates (<30 µmol/g) contents (Canola Council of Canada, 1990).

Several studies on canola protein reveals the great potential for food application due to the well-balanced essential amino acid profile and the functional properties of the proteins (Gerzhova et al., 2015, Jia et al., 2021, Yang et al., 2021, Witczak, 2021).

Despite a large amount of CPM generated worldwide, its usage as a food ingredient is still limited due to the challenge that exists in separating the protein from the other chemical components (fiber, phenols, glucosinolates, and others) with adequate yields and the generation of desired functional properties for the various sectors of the food industry.

The present investigation aimed to evaluate first different pretreatments to prepare CPM for the further extraction of proteins with/without alcohol-based pretreatment, alkaline extraction and protein separation by isoelectric precipitation and/or, ultrafiltration. Treatments were evaluated in terms of extraction yield, physicochemical and functional properties of extracted proteins and in vitro protein digestibility.

To achieve the objectives of this research, four treatments were evaluated: 1) Control: extraction by alkaline process and precipitation with acetic acid (Alk-Ac); 2) pretreatment with ethanol, extraction by alkaline process and precipitation with acetic acid (EtOH-Alk-Ac); 3) alkaline extraction and concentration by ultrafiltration, 5 kDa (Alk-UF); 4) pretreatment with ethanol, extraction by alkaline process and concentration by ultrafiltration, 5 kDa (EtOH-Alk-UF).

The CPM utilized for the different protocols was supplied by AGYDSA Aceites, Grasas y Derivados, S.A. The CPM was ground into a fine meal and chemically characterized by triplicate (moisture, crude protein (Nitrogen *6.25), and crude fat contents according to approved AACC methods 44-15.02, 46-13.01, and 30-25.01, respectively, AACC International, 1999).

The solid extraction yield was calculated by relating total solids recuperated after protein extraction divided by the initial CPM solids. On the other hand, the protein extraction yield was estimated as the total protein obtained from the process in relationship with the initial total protein of the CPM.

All resulting protein pellets or protein concentrates were lyophilized and assayed in terms of moisture and crude protein according to approved AACC methods 44-15.02 and 46-13.01 respectively (AACC International, 1999). The free alpha-amino nitrogen was also determined by the ninhydrin-colorimetric method of Haque and Kito (1989).

Functional properties were also determined. The water absorption and water solubility indexes were assessed according to the centrifugation method of Cheftel et al. (1989). The fat absorption capacity (FAC) and emulsion stability were calculated according to Ahn et al., (2005) and Haque and Kito (1989), respectively. Foaming activity, foam stability, and foam density were determined with 3% (w/w) protein water dispersions according to Haque and Kito (1989).

Protein digestibilities of protein extracts were assayed by the enzymatic in vitro method suggested by Hsu et al. (1977), using protease from Streptomyces griseus (Type XIV, >3.5 units/mg solids) instead of discontinued porcine intestinal peptidase.

Statistical analyses were performed using variance procedures and means were compared using Tukey´s tests (p< 0.05) using the Minitab 16 Statistical Software.

Experimental results indicated that the alkaline extraction and ultrafiltration protein concentration generated yield values of protein and solids with 34.95% and 14.38% respectively. Nevertheless, treatments that used acid precipitation comparatively yielded less protein and solids, 18.12-20.08% and 7.18-8.53% respectively.

The functional properties of proteins obtained with the treatment EtOH-Alk-UF showed the best values in terms of water solubility (39.17%) whereas the Alk-Ac had the best water absorption value with 3.86%.

In terms of foaming properties, the proteins obtained from the EtOH-Alk-UF treatment showed the best values, with 505% of foaming activity and 34.37% in foam density. Nevertheless, there were not significant differences among treatments in terms of foam stability.

Regarding protein digestibility in vitro, the higher value was obtained by EtOH-Alk-Ac with 95.5% and the lowest value were obtained by treatments that used ultrafiltration for protein concentration (89.9-88.1%).

The extracted canola proteins had acceptable properties for the food industry. The functional properties varied according to the pretreatment, extraction, and concentration processes. The best extraction process was the treatment of ground CPM first with ethanol pretreatment followed by alkaline extraction and ultrafiltration. Treatments precipitated with acetic acid yielded lesser amounts of concentrates and these had comparatively worse functional properties.

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