PROTEIN HYDROLYSATE FROM CANARY (CANARIUM INDICUM L.) SEEDS: PREPARATION AND CHARACTERIZATION AS MATERIAL FOR NUTRACEUTICAL PRODUCTS WITH ANTIOXIDANT ACTIVITY

• Main Author: Nurul Apsari, Cintya
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/70966
• Institution: Institut Teknologi Bandung

Canary (Canarium indicum L.) is an indigenous Indonesian plant. Its growth is widely spread in eastern Indonesia, with distribution centers including Kangean Island, Bawean Island, Nusa Tenggara, Sulawesi, and Maluku. Canary production annually is relatively high, reaching 4-7 tonnes of NIT (nut in testa)/ha/year. Even though the production has been high until now, in Indonesia, canaries are not considered a leading commodity, and their use still needs to be improved. It has been reported that canaries contain high-fat levels, reaching ±75% (w/w). Currently, canary oil has been commercialized under the name "Nangai oil" by SeneGence International, with distribution areas in America, Australia, and Canada. The cake left over from pressing canary kernel oil (defatted canary seeds) still contains nutrients that can be utilized further. During this time, defatted canary seeds are usually used as animal feed. Research on canary seed protein and its utilization has not been widely carried out. This study aims to examine the protein content of canary (Canarium indicum L.) seeds to develop the utilization of canary seed protein as a protein hydrolysate with antioxidant activity. NIT (nut in testa) canary seeds were pretreated in roasting, peeling, mechanical pressing, and flouring to obtain defatted canary seeds in powder. The powder was further characterized by proximate analysis by determining protein content using the Kjeldahl method. The analysis showed that the defatted canary seeds had a protein content of 36.039 ± 0.003 % of the total sample weight (w/w). Furthermore, defatted canary seeds were extracted with phosphate buffer and produced defatted canary seed protein extract. The results of protein content analysis using the Lowry method showed that the defatted canary seed protein extracts had a protein content of 43.246 ± 3.064 mg/mL. This protein extract was then hydrolyzed using proteases, namely papain, flavourzyme, and pepsin. Process optimization was carried out using the response surface methodology (RSM) to determine the best hydrolysis conditions. The optimization involved three independent variables, namely substrate concentration [E/S] (%), hydrolysis time (hours), and hydrolysis temperature (°C). Parameters observed from the various treatments included protein content (Lowry method), degree of hydrolysis (Ninhydrin method), and antioxidant activity (DPPH method), as well as additional observations were made of the SDS-PAGE profile of each treatment. The highest antioxidant activity is the determining parameter for the most optimum hydrolysis conditions. The results showed that protein hydrolysate produced using pepsin at a concentration of 1% (w/v) and under hydrolysis conditions at 39.5°C for 5 hours produced the highest antioxidant activity. This protein hydrolysate has IC50 = 0.195 ± 0.001 mg/mL or equivalent with ascorbic acid IC50 = 10.635 ± 0.252 ?g/mL. This hydrolysate had a protein content of 0.871 ± 0.084 mg/mL and a degree of hydrolysis of 2.830 ± 0.057%. Defatted canary seed protein hydrolysate with the highest antioxidant activity was further characterized. Characterization was carried out to determine the content that affects the antioxidant activity. The characterization carried out included analysis of amino acids (LCMS/MS method), identification of peptides through a proteomic approach (UHPLC-HRMS method), identification of other compounds (non-peptides) (UHPL-HRMS method), and bioinformatics/in silico studies. The results of amino acid analysis using the LCMS/MS method showed that the highest amino acid content in protein hydrolysates was Arg (505.920 mg/kg), Leu (152.189 mg/kg), Ile (43.383 mg/kg), Val (26.119 mg /kg), His (6.592 mg/kg), and Cys (3.968 mg/kg). This composition revealed that the defatted canary seed protein hydrolysate contained more essential amino acids and branched-chain amino acids (BCAA), namely Leu, Ile, and Val. The peptide identification results using the UHPLC-HRMS method showed that there were 22 peptides contained in the defatted canary seed protein hydrolysate. The peptide consists of 6-20 amino acids. The amino acid sequence of the peptide also contains BCAA-type amino acids. Moreover, it is dominated by leucine (L). Apart from being an antioxidant, this protein hydrolyzate can be used for supplementation in patients with hepatic encephalopathy and rehabilitation of poststroke patients, especially those with symptoms of sarcopenia. This is based on the amino acid composition of BCAA as the majority of peptide constituents and can open up more significant opportunities for the potential utilization of canary seed protein hydrolysate as a nutraceutical. The results of metabolite analysis using the UHPLC-HRMS method identified 11 compounds in defatted canary protein hydrolysates. Most of these compounds are dominated by fatty acids and their derivatives. Compounds from amino acid groups were also identified, namely phenylalanine and tryptophan. The results of an in silico study on antioxidant prediction found that the EYKLTYYTPEYPTK peptide is a bioactive peptide that influences antioxidant activity. This peptide acts as a radical scavenger. Toxicity prediction results showed that all peptides contained in defatted canary seed protein hydrolysate were non-toxic. These results support the potential for defatted canary seed protein hydrolysate to be utilized as a nutraceutical. However, the allergenicity prediction results showed that nine peptides might be allergens. This data is essential to know, bearing in mind the use of canary seed protein hydrolysate as a nutraceutical, so further studies are needed to confirm the allergenicity prediction results. Overall, the protein hydrolysate of defatted canary seeds has the potential to be developed as a raw material for nutraceutical products with antioxidant activity, which can be produced as a health supplement in the prevention of various degenerative diseases.