Nghiên cứu xây dựng quy trình công nghệ thu nhận isoflavone từ một số nguồn thực vật và ứng dụng sản xuất thực phẩm chức năng tt

TRÍCH YẾU LUẬN ÁN TIẾN SĨ: 1. Lý do chọn đề tài Isoflavone là một hợp chất thuộc nhóm flavonoid, thường có trong các cây họ đậu, có hoạt tính estrogen giúp chống lại các triệu chứng như loãng xương, trầm cảm, ngăn ngừa các bệnh tim mạch, ung thư vú, v.v…thường xuất hiện ở phụ tiền mãn kinh. Do đó, isoflavone được giới khoa học trên thế giới quan tâm nhiều thập kỷ qua và được sử dụng chế biến các sản phẩm thực phẩm chức năng, dược phẩm, mỹ phẩm, nước giải khát và đồ uống trên trong nước và trên thế giới. Tuy nhiên, các nghiên cứu trong nước về isoflavone còn ít, các sản phẩm thực phẩm bổ sung isoflavone hầu hết nhập ngoại có giá thành cao. Vì vậy cần xây dựng quy trình thu nhận isoflavone từ nguồn nguyên liệu dồi dào trong nước và ứng dụng vào sản xuất thực phẩm bảo vệ sức khỏe bổ sung isoflavone làm đa dạng hóa sản phẩm trong nước, giảm giá thành sản phẩm so với sản phẩm ngoại nhập, phát triển nền nông nghiệp và công nghiệp chiết xuất các hợp chất isoflavone. Do đó đề tài “Nghiên cứu xây dựng quy trình công nghệ thu nhận isoflavone từ một số nguồn thực vật và ứng dụng sản xuất thực phẩm chức năng” đã được lựa chọn. 2. Mục tiêu nghiên cứu Xây dựng quy trình công nghệ thu nhận isoflavone từ nguồn nguyên liệu cung cấp isoflavone sẵn có tại khu vực miền Trung-Tây Nguyên bao gồm các quá trình trích ly, tinh chế và nâng cao hàm lượng hoạt tính sinh học cao isoflavone; Kiểm tra an toàn thực phẩm và hoạt tính sinh học của cao isoflavone; Ứng dụng sản xuất thực phẩm bảo vệ sức khỏe bổ sung isoflavone. 3. Đối tượng và phạm vi nghiên cứu Nguyên liệu thực vật dùng để nghiên cứu trích ly isoflavone: Hạt đậu nành (Glycine max (L.) Merr), hạt đậu xanh (Vigna radiata L. Wilczek), củ sắn dây (Pueraria montana var. lobata (Wild) Sanjappa& Pradeep) và bã đậu nành. Phạm vi nghiên cứu: Đánh giá thành phần và hàm lượng isoflavone ở một số nguyên liệu thực vật tại khu vực miền Trung-Tây Nguyên; Khảo sát các phương pháp trích ly, tinh chế và nâng cao hoạt tính cao isoflavone tinh chế; Kiểm tra chất lượng an toàn thực phẩm và hoạt tính sinh học của cao isoflavone; Ứng dụng cao isoflavone để sản xuất thực phẩm bảo vệ sức khỏe; Khảo sát hàm lượng isoflavone trên 6 hợp chất: daidzein, genistein, glycitein, daidzin, genistin và glycitin. 4. Phương pháp nghiên cứu Phương pháp nghiên cứu trong luận án là phương pháp nghiên cứu thực nghiệm kết hợp với phân tích lý thuyết và tổng hợp tài liệu. Các phương pháp phân tích được sử dụng: Phân tích isoflavone theo phân tích sắc ký lỏng cao áp HPLC (AOAC 2008.83) và các chỉ tiêu hóa lý, vi sinh được thực hiện theo tiêu chuẩn Việt Nam. Phương pháp đánh giá cảm quan: phép thử mô tả đặc tính của sản phẩm, phép thử cho điểm thị hiếu, phép thử so hàng thị hiếu, phép thử tam giác được thực hiện. Các thí nghiệm đều được thực hiện ít nhất 2 lần, kết quả được trình bày dưới dạng giá trị trung bình ± độ lệch chuẩn, so sánh sự khác nhau có nghĩa ở mức =0,05. Tính toán quy hoạch thực nghiệm toàn phần 3 yếu tố 2 mức (TYT) 23 và phân tích ANOVA thực hiện trên phần mềm Minitab 18. 5- Bố cục của luận án Luận án gồm 137 trang, 50 Bảng biểu, 62 Hình ảnh. Nội dung của luận án gồm: Mở đầu (6 trang); Chương 1:Tổng quan (18 trang); Chương 2: Nguyên liệu và phương pháp nghiên cứu (17 trang); Chương 3. Kết quả và thảo luận (92 trang); Kết luận và kiến nghị (4 trang). 6- Kết quả của luận án (1) Từ 4 loại nguyên liệu thực vật có sản lượng lớn tại khu vực miền Trung-Tây Nguyên, nghiên cứu đã chọn hạt đậu nành và bã đậu nành làm nguyên liệu để thu nhận isoflavone. (2) Quá trình trích ly isoflavone từ hạt và bã đậu nành đã được nghiên cứu bằng phương pháp chiết khuấy. Ảnh hưởng của nhiệt độ, thời gian chiết và tỷ lệ dung môi / nguyên liệu đến hàm lượng isoflavone chiết được nghiên cứu, được tiến hành quy hoạch thực nghiệm toàn phần với 2 mức 3 yếu tố và tối ưu hóa với mô hình Box-Wilson. Kết quả của việc tối ưu hóa cho thấy điều kiện tối ưu của chiết xuất isoflavone từ đậu nành là nhiệt độ 72,5oC, thời gian chiết là 67,5 phút, và tỷ lệ dung môi/đậu nành là 26,5/1(ml/g). Tương tự, điều kiện tối ưu của chiết xuất isoflavone từ okara là nhiệt độ 74,5oC, thời gian chiết 79,0 phút, và tỷ lệ dung môi/bã đậu nành là 26,5/1 (ml/g). Lượng isoflavone tổng thu được từ đậu nành và bã đậu nành lần lượt là là 1932,4µg / g chất khô và 160,0µg / g chất khô. (3) Luận án đã nghiên cứu cụ thể sự hấp phụ và giải hấp phụ tĩnh học của các hợp chất isoflavone thu nhận từ đậu tương và đậu bắp lên nhựa macroporous D101. Sự hấp phụ đã được chứng minh là phù hợp với các mô hình đẳng nhiệt Langmuir và Freundlich. Trong nghiên cứu hiện tại này, các điều kiện hấp phụ động cho quá trình tinh chế isoflavone trên cột đóng gói nhựa D101 đã được lựa chọn ở thể tích cột (BV) là 200 mL, thể tích nạp cột là 3,75 BV và tốc độ dòng 1,5 BV/h. Quá trình giải hấp động được thực hiện với dung dịch rửa giải 70% etanol, thể tích rửa giải 2,5 BV, và tốc độ dòng 1 BV / h. Hàm lượng isoflavone đạt được ở cao tinh chế đã cao gấp 8,7 lần so với cao thôđậu nành và hiệu suất thu hồi gần 80%. Sử dụng nhựa của macroporous D101 cho kết quả: chiết xuất tinh khiết / chiết xuất thô (mg / g chiết xuất) là 144,664 / 16,624. Kết quả cho thấy khả năng cao cho việc sản xuất isoflavone quy mô lớn để ứng dụng hơn nữa trên các sản phẩm thực phẩm chức năng hoặc dược phẩm. (4) Đề tài đã nâng cao hàm lượng aglycone trong dịch chiết tinh khiết nhờ sự thủy phân hoàn toàn liên kết -1-4-glycosidic giữa gốc glucose và aglycone của hợp chất glycoside bằng enzyme thương mại Celluclast 1,5 L có hoạt tính endoglucane. Trong thời gian thủy phân 360 phút, tỷ lệ thu hồi của daidzein, glycitein và genistein lần lượt là 91,8%, 71,6% và 84,3% và không có sự khác biệt có ý nghĩa (p> 0,05) so với 420 phút và 480 phút. Kết quả thí nghiệm cho thấy nhóm -glycoside đã được thủy phân hoàn toàn, tỷ lệ aglycone chiếm hơn 95% trong tổng lượng isoflavone. (5) Các chiết xuất isoflavone khô được đánh giá về hoạt tính sinh học (hoạt tính estrogen in vitro và in vivo, hoạt tính bảo vệ gan ngoại vi, hoạt tính chống viêm, lipid peroxide bằng MDA-test) và an toàn thực phẩm (hàm lượng kim loại nặng, độc tính cấp, ...). Các chiết xuất isoflavone thô và tinh khiết cho thấy có hoạt tính sinh học cao và an toàn thực phẩm. (6) Từ dịch chiết isoflavone thô và tinh khiết, luận án đã nghiên cứu xây dựng hai quy trình sản xuất sản phẩm thực phẩm bảo vệ sức khỏe giàu isoflavone đảm bảo tiêu chuẩn thực phẩm, bảo vệ sức khỏe, được Hội đồng cho điểm thị hiếu trên 4 chỉ tiêu cơ bản (trạng thái, màu sắc, mùi, vị) đạt điểm trung bình> 7 trên thang điểm 9. Đóng góp của luận án • Quá trình trích ly isoflavone từ hạt đậu nành và bã đậu nành bằng chiết khuấy đạt được thông số trích ly tối ưu, với việc tăng nhiệt độ trích ly và lượng dung môi sử dụng nên rút ngắn thời gian trích ly. • Tinh chế isoflavone từ đậu nành và bã đậu nành bằng sắc ký cột D101 là đề xuất mới cho quy trình tinh chế isoflavone quy mô công nghiệp, đáp ứng “SẢN XUẤT XANH”, thân thiện với môi trường. • Thử nghiệm in vitro đánh giá hoạt tính sinh học của cao thô và cao tinh isoflavone trên dòng tế bào MCF-7 (estrogen), dòng tế bào HepG2 (bảo vệ gan), hoạt tính chống peroxy hóa lipid màng tế bào trên dịch đồng thể gan được thực hiện đầu tiên tại Việt Nam. • Đề xuất 2 quy trình sản xuất sữa chua đậu nành - nước cốt dừa và nectar chanh dây đáp ứng các tiêu chuẩn của thực phẩm bảo vệ sức khỏe

THE UNIVERSITY OF DA NANG UNIVERSITY OF SCIENCE AND TECHNOLOGY TRAN THI NGOC THU A STUDY ON BUILDING UP THE TECHNOLOGICAL PROCESS FOR OBTAINING ISOFLAVONES FROM BOTANICAL SOURCES TO PRODUCE FUNCTIONAL FOOD Major: Food Technology Code: 54 01 01 SUMMARY OF DOCTORAL THESIS Da Nang, 2022 THE UNIVERSITY OF DA NANG UNIVERSITY OF SCIENCE AND TECHNOLOGY Supervisor: Assoc Prof., PhD Truong Thi Minh Hanh PhD Bui Xuan Vung Reviewer 1: ……………………………………… Reviewer 2: ……………………………………… Reviewer 3: ……………………………………… The dissertation will be defensed at the doctoral thesis review meeting held at University of Science and Technology on …/……./……… INTRODUCTION Rationale Isoflavone composes of structure close to estrogen, a female hormone that plays an important role for menopausal women, supporting to fight symptoms related to sudden decrease of estrogen such as osteoporosis, depression, prevention of cardiovascular diseases, breast cancer, etc., and of premenopausal women Therefore, isoflavones have been concerning for decades and are used for the processing of functional food products, pharmaceuticals, cosmetics, soft drinks, and beverages in the country and around the world However, there are few studies on isoflavones in Vietnam and isoflavone supplement products are in high cost Consequently it is necessary to build up a technological process for obtaining isoflavones from abundant raw materials in local and use them to produce health supplement products, aiming at not only reducing costs, but also developing agriculture and isoflavones extraction industry.Thus, the study chosen for the thesis was “A study on building up the technological process for obtaining isoflavones from botanical sources to produce functional food” Objectives Building up the technological process obtaining isoflavones using materials from Central-Highland Central Vietnam, including extraction, purification, and Quantitative enhancement of isoflavones in extracts; Assessing food quality and bioactivity of isoflavones extracts; Applying production of health supplement with isoflavone Contents Evaluating the composition and isoflavone content of plant materials in Central Highland-Central Vietnam; Investigating methods of isoflavones extraction, purification and enhancing the aglycone content of extracts; Assessing food quality and bioactivity of isoflavones extracts; Building up a technological process for obtaining isoflavones; Proposing solutions for utilising by-product after extracting isoflavone Methods The method in the thesis is empirical and combined with theoretical computing and documentary analysis Scientific and practical significance The thesis used 23 full factorial designs and the Box-Wilson model for optimizing the conditions of isoflavones extraction from soybean seeds and okara The thesis has studied the static adsorption and desorption of isoflavone compounds extracted from soybean and okara onto D101 macroporous resin in a specific way The adsorption has been demonstrated to be suitable to the Langmuir and Freundlich isothermal models The dynamic adsorption and desorption of isoflavone extracts have been implemented on column chromatography D101, from there, it will provide a model of a largescale purification The thesis has completed a process of isoflavones collected from soybean and okara with some modifications, specifically: stirring extraction, purification by D101 resin, and enhancing aglycones amount using Celluclast®1.5 L The proposed process of isoflavones collection is easy in operation, low-cost, safe and minimizes environmental pollution due to the utilization of ethanol and water solvents The crude and purified isoflavone extracts have shown to be high biological activities and food safe The isoflavone extracts were used to develop health supplement products, satisfying the Vietnamese Standards Thesis structure The thesis has 137 pages, 50 Tables, 62 Figures Introduction (6 pages), Chapter Overview (18 pages) , Chapter Materials and Methods (17 pages); Chapter 3: Results and discussion (92 pages), Conclution (4 pages) CHAPTER OVERVIEW Presenting international and national researches on: 1.1 Introduction of isoflavones Isoflavone are a sub of the flavonoid group, found in legumes (leguminosae) The botany resources with isoflavones were researched such as soybeans, red clover, alfalfa, , kudzu root, … While aglycone form (daidzein, glycitein, genistein) is with high bioavailability, β-glycoside form (daidzin, glycitin, genistin)accouns for a large proportion of the total isoflavones content So, they are both most interested in extraction and application 1.2 Literature review in Vietnam and international researches on technology of extraction, purification and health supplement with isoflavones In the world, in response to the added content of isoflavones in health food products, many studies on extraction and purification methods have been published and applied to the industrial production in large-scale The process of isoflavones collection accords the "green manufacturing", based on factors of raw materials and "green" extraction techniques Although Vietnam has a richness of agricultural commodities containing isoflavone, studies on isoflavones extraction and the development of healthy supplements with isoflavone are still limited CHAPTER MATERIALS AND METHODS 2.1 Materials and chemicals 2.1.1 Materials Soybean seed (Glycine max (L.) Merr), mung bean seed (Vigna radiata L Wilczek) was collected at Dai Loc-Quang Nam, kudzu roof (Pueraria montana var lobata (Wild) Sanjappa& Pradeep) was obtained at Thieu Hoa-Thanh Hoa, and okara was obtained at Da Nang Raw materials were cleaned, dried, mixed, milled, and stored at -20±2oC 2.1.2 Chemicals Isoflavone standards, analysis grade chemicals were purchased from Merck (Germany), Sigma (USA), D101, AB8 macroporous (Anhui Sanxing Resin Technology Co., Ltd, China), Diaion HP20 (Mitsubishi Chemical Corporation, Japan), and XAD4 (Acros, France); Comercial starter culture Yoflex Express 1.0 (Chr Hansen Denmark); Celluclast1.5L (Novozymes, Denmark) 2.2 Experiments 2.2.1 Evaluating the composition and content of isoflavones in plant materials Investigating the content of isoflavones and other chemical components of materials: soybean seed, mung bean seed, kudzu root, and okara They were assessed with their isoflavone contents and other chemical components 2.2.2 Assessing of methods for isoflavone extraction from soybean seed and okara Assessing of isoflavone extraction conditions on selected materials by two methods: stirring and using ultrasound waves; optimizing extraction conditions of total isoflavone by the experimental design of levels of factors (temperature, extraction time, and ratio of solvent/material) and Box-Wilson model 2.2.3 Investigation of isoflavone purification method and enhancement of aglycone content in purified isoflavones Selecting the isoflavone purification method between solidliquid extraction and column chromatography (stationary phase particles uses macroporous resins);Enhancing of aglycone content by isoflavone glycoside hydrolysis using Celluclast1.5L 2.2.4 Evaluating for biological activity and food safety of dry isoflavone extracts from soybean seed and okara Dry isoflavones extracts are assessed about bio-activity (estrogenic activity in vitro and in vivo, ex vivo hepatoprotective activity, anti inflammatory activity, lipid peroxidation by MDA-test) and food safety (heavy metal content, acute toxicity, ) 2.2.5 Proposing to build up a technological process for obtaining isoflavones from soybean seed and okara Proposing to build up a process of isoflavones obtainment with alternative modifications 2.2.6 Producing health supplements with dry isoflavone extracts Developping productions are soy-coconut yogurt and passion fruit nectar, fortified isoflavones from dry extracts, quality of health supplements according to Vietnamese standards 2.2.7 Proposing solutions for utilising by-products after extracting isoflavone Soybean flour after isoflavone extraction was assessed chemical components and proposed to process food productions 2.3 Analysis method Determination of isoflavone from soybean using HPLC (AOAC 2008.83) Physic-chemical and microorganism properties of samples were analyzed according to Vietnamese standards Sensory evaluation methods: product characteristics test, hedonic test (9-scale), ranking test, triangle test is performed The experiments were performed at least times, the results are presented as mean± standard deviation, compared with the significant difference at the level = 0.05 Calculated experimental design of factors level and ANOVA analysis were performed on Minitab ver 18 software CHATER RESULS AND DISCUSSIONS 3.1 Evaluation of several isoflavone-rich sources The total isoflavone content in soybean found to be significantly higher than from okara and kudzu root by 14.30 times and 4.25 times, respectively Mung beans only contain genistein in very low level Soybean is a short-term crop and intensively cultivated with 23 crops yearly, large yield, and low expenditure while kudzu root crop only harvests once a year The utilization of okara for isoflavones extraction plays an important role in exploring remaining nutritional value and solving environmental pollution issue Figure 3.1 and 3.3 demontrated the HPLC chromatograms of isoflavones in soybean seed and okara Thus, soybean seed and okara were chosen to investigate the condition of isoflavone extraction 3.2 Effects of extraction methods on isoflavone extraction effectiveness from soybean 3.2.1 Stirring method Effects of stir-assisted extraction method on isoflavone extraction were examined by using independent variables Among obtained extracts under different pH conditions (pH 2.5, pH 7.1, and pH 9) the extract of pH condition showed highest isoflavone content In the findings, ethanol concentration, solid/liquid ratio, extraction temperature, and extraction time had significant impact on extracted isoflavone content (figure 3.5 and 3.7) 3.2.2.Ultrasound-assisted extraction method Using the two-way analysis of variance (ANOVA), the highest isoflavone content with 1773.0 ± 21.7g/g dry basis obtained at 15 and the power ultrasound (US) 70% (figure 3.11) 3.2.3 Efficiency evaluation of stirring extraction and ultrasoundassisted extraction The ultrasound assisted extraction at the power of ultrasound 70% maintained antioxidant activity of isoflavone compounds and decrease of extraction time However, this process is not feasibility to apply in the industrial scale The stirring extraction shown in higher extraction efficiency, this method is simple and easy in operation, lowers energy costs compared to ultrasound- assisted extraction (figure 3.12) Therefore, stirring extraction was chosen as the appropriate extraction method to extract isoflavone in the following experiments 3.3 Optimization of isoflavones extraction from soybean seed and okara using stirring method The application of 23 full factorial design was conducted to evaluate the simultaneous influence of factors such as temperature (x1), time (x2), and solvent/material ratio (x3) on isoflavone extraction Through calculation and analysis, the regression equation has been built (eq 3.1) Ỹsoybean= 1803,63+65,00x1+24,96x2+68,99x3 - 44,98x1x3 (eq 3.1) According to the regression equation, the variables of temperature, time, the ratio of solvent/material were in a proportional effect on the total extracted amount of isoflavones Meanwhile, the interaction between temperature and the ratio of solvent/material had a reversed effect on the target function Ỹ Figure 3.13 also presents 11 Amberlite®XAD4 >D101>AB-8>HP-20 (figure 3.19) The desorption capacities of daidzin, genistin, and total isoflavones on D101 resin were 1.45 mg/g, 2.21 mg/g, and 4.48 mg/g, respectively, and there were insignificantly different (p>0.05) from Amberlite® XAD4 As compared to high-priced Amberlite®XAD4 resin, in this study, D101 resin, which has a cheaper price, fairly possessed the same adsorption and desorption performances Therefore, D101 resin was chosen for isoflavone purification from the crude extract 3.4.3 Purification of isoflavones from crude soybean extract using D101 macroporous resins Figure 3.20 showed that the adsorption isotherms data of total isoflavones, daidzin, and genistin on the D101 resin fitted well with the Langmuir and Freundlich model The dynamic adsorption conditions for the purification process of isoflavones on the D101 resins packed column were selected at 12 the bed volume (BV) of 200 mL, feed volume of 3.75 BV, and flow rate of 1.5 BV/h (figure 3.22) The dynamic desorption was carried out with the elution solution of 70% ethanol, elution volume of 2.5 BV, and flow rate of BV/h (figure 3.23) The total isoflavones content in the purified isoflavones solid extract was enriched to 144.664 ± 1.210 (mg/g), being more than 8.7 folds higher than that in the crude soybean extract with a recovery yield of nearly 80%, higher than the solid-liquid method, using solvent dichlormethane/ ethanol (1/3, v/v) 3.4.4 Enhancement of aglycones content in purified soybean extract by enzyme cellulase This study was aimed at increasing aglycone content in purified soybean extracts by enzymatic glycoside hydrolysis mehod using Celluclast 1.5 L (cellulase) The investigation results showed the efficiency of the glycoside conversion into corresponding aglycones was affected by enzyme concentration, substrate concentration, and hydrolysis time During the hydrolysis time (from 60-480 minutes), the hydrolysis ratio of daidzin, glycitin, and genistin reached over 96% Figure 3.27 have shown increasing the aglycones and the peaks of glycosides almost disappeared after hydrolysis For the hydrolysis time of 360 minutes the recovery ratio of daidzein, glycitein, and genistein were 91.80.2%, 71.65.8% 13 84.30.2%, respectively and there was no significant difference (p>0.05) compared to those of 420 minutes and 480 minutes Particularly the experimental results revealed that the hydrolysis of -glycoside group in isoflavone glycosides took place completely when using enzyme Celluclast 1.5 L concentration of 0.16% for isoflavone substrate concentration of 17.5 mg/ml in the hydrolysis time of 360 minutes so as to obtain aglycone percentage more than 95% in total isoflavone amount (figure 3.27) 3.4.5 Purification of isoflavones from crude soybean extract using D101 macroporous resins The adsorption isotherms data of total isoflavones, daidzin, and genistin on the D101 resin also fitted well with the Langmuir and Freundlich model (figure 3.30) The total isoflavones content in the purified isoflavones dry extract was enriched to 36.22 ± (mg/g), being more than 7.9 folds higher than that in the crude okara extract with a recovery yield of nearly 75% Meanwhile, the use of adsorption processes using macroporous resins presents advantages such as selective adsorption properties and low operating costs, the usage of non-toxic solvents (distilled water, ethanol), and often times reusing plastic beads 14 3.5 Examination of the biological activity and food safety of dried isoflavone extracts 3.5.1 Examination of the biological activity of dried isoflavone extracts According to the E-Screen assay, the proliferation of MCF7 cells under the impact of dry isoflavone extracts enables the estrogen activity of the research samples to be evaluated The results indicated that the proliferation of purified extracts from soybean and okara reached the ratio 7.4% and 14.9%, respectively as compared to the negative control, that is only cultured in a non-steroid media Estrogen in-vivo assay was tested in a VCD-induced ovarian mouse model that closely simulates human menopause The results showed that the mass of the induced ovarian mouse using the crude extract and purified extract was without differences compared to pathological control Figure 3.33 showed that the uterine mass per the weight of mice from the pathological control lot was lower than the physiological lot without injecting VCD Furthermore, since uptaking the crude extract and purified extract, both follicles and estradiol content in the serum of mouse were higher than the negative control Thus, isoflavone extracts have somewhat demonstrated the similar function of estrogen, improving the physiology of rats with an impaired ovarian function 15 The determination of lipid peroxidation ex vivo on mouse liver homogenate illustrated that the target samples of crude and purified extracts were capable of inhibiting the lipid peroxidation, in which IC50 of purified soybean and okara extracts was 360.65 ± 17.49 (µg/ml) and 418.13±9.19 (g/ml), respectively Besides, crude and purified isoflavone extracts have shown the capacity of HepG2 cell protection with the presence of CCl4 The efficiency of free radicals DPPH·of isoflavone extracts was proportional to the amount of Isoflavones The IC50 value of the isoflavones-inducing purified soybean and okara extracts were 335.1(µg/ml) and 853.6 (µg/ml), respectively 3.5.2 Examination of the food safety of dry isoflavone extracts Results of acute toxicity activity for four dried extracts did not show the ability to cause acute toxicity to white mice of oral BALB/c in the highest dose of 10 grams/kg of oral trial, ensuring food safety Test results of heavy metal concentrations such as Pb, Cd, As and Hg in purified soybean and okara extracts were below the quantitative limit of the analytical method (AOAC 999.10) In summary, the analysis showed that isoflavones in soybean seed and okara both ensure food safety and show biological activities to protect the health of consumers 3.6 Proposing to build up the technological process for obtaining isoflavones from soybean seed and okara The isoflavone obtainment process from soybean seed and okara, including extraction, purification, and enhancement of isoflavone bioavailability was presented in figure 3.39 Three dry extract products were crude, purified isoflavones and high-aglycones extracts 16 3.7 Application of isoflavone extract in producing health foods 3.7.1 Production trial of soy-coconut yogurt supplemented with isoflavones extracted from soybean The study has selected a combination formula of soy-coconut yogurt (SoCY) with a percentage of 15% coconut milk compared to soy suspension, which is suitable for use in the production of nutritious health foods with isoflavone supplement The soy-coconut yogurt product with a high dose of crude isoflavones (CIs) supplemented with a proportion of g CIs per 100g of SoCY has been tested to ensure physico-chemical and microbiological criteria 17 according to QCVN 5- 5:2010/BYT-National Technical Regulation fermented dairy Vietnam for products, Standard (TCVN 7030:2016 Fermented milk) The isoflavones 21.15mg/100g content of is product, which is 15% more than the minimum recommended daily dose of 30-90 mg/day Figure 3.41 indicated the increase in isoflavone content at SoCY with isoflavones supplemented compared with the one free CIs (control sample) via isoflavones’HPLC chromatograms SoCY supplemented with isoflavones was awarded a pass score on the criteria above points by the Sensory Panel, significantly higher than other yogurt samples and no difference compared to samples of soy-coconut yogurt without isoflavones added (p>0.05) The thesis has proposed the process of producing healthprotecting products of SoCY described in detail in figure 3.45 The SoCY product has been built "The announcement of a health supplement for soy-coconut yogurt with isoflavones", meeting the necessary conditions for health supplement 18 3.7.2 Production trial of passion fruit nectar enriched with purified isoflavones from okara The content of isoflavone extract refined from soybean residues and added to the passion fruit nectar product is 0.5g/100ml to ensure the total isoflavone of 17.87mg/100ml Passion fruit (table nectar 3.41) products supplemented with isoflavones ensure the physicochemical and microbiological quality criteria according to TCVN 7946:2008 ‘Fruit 19 juice and nectar - Technical regulations and QCVN 2:2010/BYT – National Technical Regulations of Non-Alcoholic Beverages At the same time, 200ml of passion fruit nectar product will provide nearly 40mg of isoflavones, which is in line with the recommended isoflavone dosage for consumers The product achieved a passing score on the criteria above 7.5 points by the Taste and Sensory Panels (100 members) for sensory criteria, and there was no significant difference compared to the control product without isoflavones added (p>0.05) This proves that isoflavone supplements not affect the organoleptic properties and physico-chemical parameters of passion fruit nectar products 20 The process for producing health care products of passion fruit nectar supplemented with isoflavones was proposed in figure 3.53 With the results of this research, a passion fruit nectar product has been developed " The announcement of a health supplement for passion fruit nectar with isoflavones", meeting the necessary conditions for health supplement 3.8 Proposing solutions for utilising by-products after extracting isoflavone As compared with the raw soybean, by-products after being isoflavone extraction retained most of the chemical components such as total protein (32,692g/100g), lipid (17,810g/100g), total fiber (12,897g/ 100g) and minerals (Ca, P, K, Mg) The content of linoleic acid accounts for approximately 50%, followed by oleic acid and palmitic acid Thus, soybean flour extracted for collecting isoflavones is a rich source of protein and lipid components, suggesting an application of producing soy/oil–protein extract CONCLUSIONS AND RECOMMENDATIONS A Conclusions (1) From types of plant materials with large output of the Central-Central Highlands region, the study selected soybean seeds and soybean residues as raw materials to obtain isoflavones The content of this compound (mg/g dry matter) in soybean seeds is 1.579 and in okara contains 0.111 (2) The study researched using extraction methods: stirring extraction and ultrasound to obtain the isoflavones present in the crude extract as follows:  Extraction stirring: 21 Using the ratio of ethanol solvent 80%: material (ml/g) is 26.5:1 • The crude extract from soybeans contains 1.932 (mg/g dry matter) isoflavones when extracted at 72.5°C for 67.5 minutes • From okara, the crude extract contained 0.160 (mg/g dry matter) isoflavones when extracted at 74.5oC, 79.0 minutes The parameters used in the method have been verified by full experimental planning with levels of factors and optimized according to the Box-Wilson model  Ultrasound The resulting crude extract contained 1.773 (mg/g dry matter) isoflavones (at 75% of the power ultrasound for 15 mins) With the results obtained from the two extraction methods, it is enough grounds to choose the heated stirring extraction method that has high efficiency of isoflavone extraction, is cheap and easy to use (3) With the goal of purifying isoflavones in the crude extract as well as enriching aglycone components, the study conducted:  Purification of crude soflavones extract from soybean seed Using resins of macroporous D101 resulted in: purified extract/crude extract (mg/g extract) of 144.664/16.624  Enrichment of aglycone Using Celluclast1.5L resulted in: aglycone contents for >95% of total isoflavones, 4.57 times higher than aglycone in the purified extract 22 (4) Evaluation of the quality of crude and purified extract containing isoflavones obtained from soybean seed and okara  Estrogen hormone activity: MCF-7 cell proliferation rate of purified/crude extract (%) was 14.94/3.90 from soybean seeds and 7.41/1.07 from the okara By in vivo method on white female mice: • When vinylclohexene diepoxide (VCD) was used, the uterine mass of the rat was reduced from 29.8 to 20.7 (mg/10g rat body weight) • Addition of isoflavones [crude extract (M1)/purified (M3)] at a dose of 2000/1000 (mg/kg body weight of rat) resulted in a significant recovery in rat uterine mass (mg/10g body weight) from 20.7 to 27.3, from 20.7 to 26.6  Biological activity: The hepatoprotective activity of HepG2 from crude/purified extract (%) reached from soybean seeds, soybean residues to15.39/2.32; 22.17/4.55, respectively Using the MDA-test method to evaluate the antioxidant capacity of isoflavones in cell membrane lipid peroxidation through IC50 (g/ml): 360.65 from soybean seeds and 418.13 from okara (5) Proposing to improve some stages in the isoflavones collection line from soybean seeds and okara These stages are:  Extraction by stirring: 23 Condition: • The temperature for processing from soybeans and soybean residues, respectively: 72.5oC and 74.5oC • The time for processing from soybeans and okara is 67.5 and 79.0 minutes, respectively • Ratio of ethanol 80%/material (ml/g): 26.5/1  Purification of isoflavones onto resin D101 The efficiency of isoflavone collection in the purified extract is 8.7 times higher than that of crude extract from seeds; and 7.09 times from soybean seeds  Enhancing the content of aglycone - a component with the high biological activity by Celluclast1.5L Condition: pH 5; temperature at 50 oC; time h (6) Proposing processes of the production of isoflavone health supplements  Soy-coconut yogurt  Passion fruit nectar The two proposed processes above have been developed a declaration of healthy food products according to Decree 15/2018 ND-CP These food products also have been assessed with four basic properties (state, color, smell, taste) by the Sensory Panel, reaching an average score of >7 on a 9-point scale (7) Proposing the use of by-products from soybean after extracting isoflavone for the following products:  Soybean oil  Soy protein extract ... solvent/material=26.5/1 reached the maximum isoflavone content of 160.2 (g/g) Ỹ okara= 133,52+5,99x1+5,10x2+5,28x3 (eq 3.2) Thus, isoflavones extraction from soybean and okara were fitted with full factorial design,... soybeans contains 1.932 (mg/g dry matter) isoflavones when extracted at 72.5°C for 67.5 minutes • From okara, the crude extract contained 0.160 (mg/g dry matter) isoflavones when extracted at 74.5oC,... Assessing food quality and bioactivity of isoflavones extracts; Applying production of health supplement with isoflavone Contents Evaluating the composition and isoflavone content of plant materials