RESEARCH ON PHLOROTANNIN COLLECTION FROM BROWN ALGAE (SARGASSUM SERRATUM) IN NHA TRANG AND ASSAY APPLY TO ANTIOXIDANT BEVERAGE

MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY Dang Xuan Cuong RESEARCH ON PHLOROTANNIN COLLECTION FROM BROWN ALGAE (SARGASSUM SERRATUM) IN NHA TRANG AND ASSAY APPLY TO ANTIOXIDANT BEVERAGE SPECIALITY: Technology of fisheries processing CODE: 62540105 ABSTRACT OF TECHNICAL DOCTOR THESIS Khanh Hoa - 2015 Thesis was completed in: Nha Trang university and Insititute of Technology Application and Research Science instructor: Dr Vu Ngoc Boi Nha Trang university Ass Pro Dr Tran Thi Thanh Van Insititute of Technology Application and Research Reviewer 1: Prof Sci Dr Nguyen Trong Can Reviewer 2: Assoc Dr Phung Thi Thanh Tu Reviewer 3: Assoc Dr Nguyen Anh Tuan Thesis will be defened at the doctor thesis assessment committee of Nha Trang university …………………………………………………………………………… At …………hours ……… Date …… ……… ……… The thesis can be found in: The library of Nha Trang university or National library INTRODUCTION Necessity of thesis Brown algae (Phaeophyta) is bioactive-rich algae such as lamilaran, fucoidan, alginate, phlorotannin, … The substances have many bioactives, inside, antioxidant, antibacterial, antifungal,… is being interested Brown algae phylum (Phaeophyta) has many families as: Alariaceae, Fucaceae and Sargassaceae In Vietnam, brown algae genus (Sargassum) belong to brown algae family (Sargassaceae) The genus has large output and high value on medicine because they consists of many bioactive substances as fucoidan, laminaran, alginate, phlorotannin,… Inside, a noteworthy biocompound is phlorotannin – a compound of secondary metabolism belong to phenolic group Phlorotannin has different bioactives such as: antioxidant, antibacterial, antifungal, antitumor, anticancer,… Antioxidant activity of phlorotannin is one of the most extensively studied activities and phlorotannin was known as non-toxic and safe antioxidants for human healthy Thus, phlorotannin can be exploited and used to support free radical removal and health improvement for human body Thence, phlorotannin of brown algae was interested by researchers Nha Trang sea has abundant and diverse resource of brown algae Sargassum in numerous and category Alternatively, brown algae in Nha Trang sea contains a high content of bioactive substances as fucoidan, phlorotannin, alginate,… Therefore, a study of “Research on phlorotannin collection from brown algae (Sargassum serratum) in Nhatrang and assay apply to antioxidant beverage” is necessary The target of the thesis: - Research on antioxidant phlorotannin collection from brown algae (Sargassum serratum) collected in Nhatrang sea, Khanhhoa - Assay apply phlorotannin into the antioxidant beverage The object and scope of research 3.1 The object of research Brown algae (Sargassum serratum) growed in Nhatrang sea was collected, cleaned by using salt water, dried until the humidity of 19%, storaged in the room temperature and used for the research of phlorotannin collection 3.2 The scope of research: contain parts 1) Extraction and optimization study on phlorotannin collection from brown algae (Sargassum serratum) grown in Nhatrang sea, Khanhhoa 2) Initial fraction and purification of phlorotannin collected from brown algae (Sargassum serratum) 3) Study on a spray drying of brown algae S Serratum extract to receive phlorotannin powder 4) Evaluation of a toxic of phlorotannin obtained from brown algae S serratum 5) Assay apply phlorotannin to antioxidant beverage The method of research The standard methods of the world and Vietnam used for the study of phlorotannin obtainment from brown algae (Sargassum serratum) were utilized in the thesis The mathematic methods were also exploited to discovery new rules, natures, and a relation between the quantity and hyppothesis verification in the thesis The new contribution to the science - This is the first time the comprehensive research on phlorotannin of brown algae (Sargassum serratum) harvested at Nhatrang sea, Khanhhoa was done The research was from a optimization of the extraction step to purification, assay of the toxic on mice, spray drying to create high antioxidant phlorotannin powder and test a production of antioxidant phlorotannin beverage Thence, the research results of thesis have high significance in science These results contribute to affirm the value of brown algae resource of Vietnam Alternatively, the result of thesis will be the reference to serve the study and teach-in the field of active element of marine seaweed - The thesis results will be the basis for development and commercialization of some products originated from brown algae (Sargassum serratum) of Nhatrang sea at company of food production The structure of thesis The thesis contains 171 pages, inside, a review of 34 pagas, a study method of 22 pages, a result of 89 pages, a conclusion of pages, a references of 24 pages and a appendix CHAPTER I OVERVIEW 1.1 INTRODUCTION OF BROWN ALGAE Brown algae is a bioactive abundant and bioactive element – rich resource, as antioxidant, antifungal, antibacterial, anticoagulant and antiradiation, healing effect and cell regeneration (Kang et al., 2003) In fact, Khanhhoa province has 21 brown algae species, inside, Sargassum serratum species is large distribution, great reserves and new species, they simultaneously contains many bioactive compounds such as fucoidan, phlorotannin,… but they have never been fully studied 1.2 INTRODUCTION OF PHLOROTANNIN Phlorotannin is one of strong antioxidant compounds originated from brown algae (Ahn et al (2007); Kang et al (2003); LIM (2002); Kuda (2007) and Shibata (2008)) Phlorotannin contains basis units of phloroglucinol (1,3,5-trihydroxybenzene), and their molecular size oscillate between 126 Da and 650 kDa (Singh et al (2006)), and characteristic linkages of phlorotannin structure are the linkage of ether, phenyl, ether and phenyl, dibenzodioxin (La Barre et al (2010)) It is very interesting, a content, structure and antioxidant mechanism of polyphenol/ phlorotannin in brown algae closely related to antioxidant activity Phlorotannins is a phenolic compound and their nature is polymer of phloroglucinol They exist in many families of brown algae phylum, such as: Alariaceae, Fucaceae and Sargassaceae Thus, phlorotannin is worth bioactive agent to study 1.3 SITUATION OF PHLOROTANNIN STUDY IN THE WORLD AND VIETNAM * Situation of phlorotannin study in the world Many studies showed that, phenolic compound in brown algae is phlorotannin (Jormalainen et al (2004); Koivikko et al (2007); Swanson et al (2002)) Siti A Budhiyanti et al (2012) published free radical scavenging (DPPH) oscillated from 14.61 to 48.71% and 0.17 to 44.05%, respectively, when extract of cell membrane and cytoplasm has a concentration of 0.45 mg/ml; reducing power activity vacillated conforming 2.05 to 12.51% and 26.77 to 68.80%, when extract of cell membrane and cytoplasm of some Sargassum species harvested in Indonesia sea has the concentration of 0.45 mg/ml, all extract consist of phlorotannin EC50 oxidant index performed DPPH free radical scavenging activity of species S ramifolium and S pteropleuron was from 6.64 to 7.14 mg/ml (Mayalen Zubia et al., 2007) Free radical scavenging activity of species S thunbergii and S fulvellum was 97.41% and 84.66%, respectively, was simultaneously showed by Soo Jin Heo et al (2005) Strong antioxidant activity of purify phlorotannin originated from some brown algae species has closely relation to skeleton molecular (Ahn et al 2007) A activity and vitality of phlorotannin is diverse in different species of Sargassum in the worldwide, therefore All over the study and a announcement of Barry Halliwell (2001) performed, phlorotannin has a minimizing power of cell damage under a impact of free radicals such as: superoxide, hydroxyl, peroxyl, alkoxyl, hydroperoxyl, nitric oxide and nitrogen dioxide The thing was also evidenced by Naja et al., 2012, (.OH) and (O2-) radical scavenging ability of S ringgoldianum species got 78% and 88.4%, respectively, and free radical scavenging activity (DPPH) of S vulgare species was 0.2% ÷ 20.3% Total antioxidant activity of S pallidum species also oscillated from 0.04 ± 0.02 to 52.08 ± 0.02µmol FeSO4/mg (Hong Ye et al., 2009) * Situation of phlorotannin study in Vietnam Early studies of antioxidant phlorotannin extracted from Vienamese brown algae were announced in the years 2009 However, first publications about antioxidant activity of algae species grown in Nhatrang sea Khanhhoa, Vietnam were announced in the years 2010 and the publications were noticed by the group of author 1.4 APPLY OF SPRAY DRYING TO PREPARE PHLOROTANNIN POWDER In fact a technique of spray drying will help to transform active agents from a liquid state into a powder state, remove almost solvent, cost-saving of shipping, lengthen a shelf-life of active element and a formation of microcapsule (the most important thing), stable improvement of active element through linkage and packaging capacity of drying aid added in a process of spray drying Accordingly, the technique of spray drying is very suitable to prepare antioxidant phlorotannin powder originated from marine algae Over analysis shows that it have not any publication of extraction, optimization, spray drying of powder preparation, application, determination of phlorotannin structure or screening and evaluation of phlorotannin content and reserve according to a crop and growing time of each parts of algae or storage time of brown algae Sargassum serratum grown in Nhatrang sea, Khanhhoa, Vietnam Therefore, the thesis focus on over researchs CHAPTER II MATERIAL AND METHOD OF RESEARCH 2.1 Material Brown algae (Sargassum serratum) belong to: phylum (Phaeophyta), class (Phaeophyceae), order (Fucales), family (Sargassaceae), genus (Sargassum) Brown algae (Sargassum serratum) grown in Nhatrang sea - Khanhhoa was collected, cleaned by using a salt water, dried until the humidity of 19% and storaged in a room temperature to use in the research 2.2 The method of research 2.2.1 The analysis method 2.2.1.1 The quantification of phlorotannin: determined according to Swanson et al 2002 Phloroglucinol was used as a standard 2.2.1.2 Determination of antioxidant activity + Determination of total antioxidant activity (TA): quantified in accordance with Prieto et al (1999) Acid ascorbic was used as the standard + Reducing power activity (RP): evaluated by the method of Zhu et al (2002) The standard of FeSO4 + (DPPH) free radical scavenging activity: measured on Blois M S (1958) Absorbant value was measured by using UV-Vis Spectrophotometer JenWay 6400/6405 machine 2.2.1.3 Determine of colour degree of beverage: determined by the method of UF treatment of Neslihan et al (2005) 2.2.1.4 Quantification of other compositions - Carbohydrate content: quantified by the method of AOAC (1990) - Pb content: measured on the Vietnamese standard of 7602:2007-Food - Citric acid content: determined according to AOAC (1932) - Sucrose sugar: explained by AOAC (2000) 2.2.2 The method of purification Each fraction is analysed by thin layer chromatography and the solvent system of elution is chloroform: methanol: acid formic (90: 9: 1) Phlorotannin have been detected with 5% FeCl3 reagent (5% FeCl3 in 96% ethanol) The purification of sample on thin layer chromatography was tested by LC/MS and phlorotannin analysis according to the method of Swanson et al The most purificatory fraction of phlorotannin was loaded onto the Sephadex LH 20 column and the solvent system of chloroform : methanol : acid formic was used to elute, the ratio of 90 : 9: 1, respectively The fraction of purificatory phlorotannin got from LH 20 column was analysed by 1H and 13 C NMR spectroscopy (mobile phase contains: A phase of H2O and 0.1% formic acid, B phase of acetonitril and 0.1% formic acid) The analysis method of HPLC-MS, recognition of element by using a m/z score - A gradient programme run LC/MS as follow: Table 2.1 Gradient programme on LC/MS Time Phase B (%) 10 10 10 40 90 60 90 61 10 70 10 24 solvent-to-material score of 27:1 (v/w) The maceration method was used to extract + Filter A filtrate was collected by filtering of ethanol extract through the filter paper The filtrate of phlorotannin was stored in a dark bottle to limit the impact of light destroyed phlorotannin + Concentration A jelly was received by the concentration of filtrate at the temperature of 400C The jelly was continuously dissolved and removed a sediment by using 99.5% ethanol Non-sediment solution was continuously concentrated to 1/3 volumn of non-sediment solution and stored in a dark bottle The solution (A) will be fracted by different solvents (non-polarization to strong polarization) at the continuous steps + 1st fraction Substances dissolved in n-hexan were admited by using n-hexan to fract A and the solution of substances was kept in a dark bottle + 2nd fraction The residual solution fracted by n-hexan was continuously fracted by chloroform Solution of substances dissolved in chloroform was kept in a dark bottle + 3rd fraction The residual solution fracted by n-hexan and chloroform was continuously fracted by ethyl acetate Solution of substances dissolved in ethyl acetate was kept in a dark bottle and continuously purified through the column chromatography of sephadex LH 20 + 4th fraction The residual solution fracted by n-hexan, chloroform, ethyl acetate was continuously fracted by n-butanol The residual solution fracted by nbutanol and solution of substances dissolved in n-butanol was stored in 25 different dark bottles + Gel chromatography on Sephadex LH 20 Ethyl acetate fraction is the most purity and has largest phlorotannin content, so they were continuously loaded onto the gel chromatography of sephadex LH 20 to collect pure phlorotannin The eluent system of chloroform : methanol : acid formic (90:9:1) was used for pure phlorotannin receiving The purity of phlorotannin of n-hexan/ chloroform/ ethyl acetate/ nbutanol fractions was 29.8%, 20.6%, 81.8% and 51.94%, respectively, compared to initial ethanol extract The purity of phlorotannin sau sắc ký cột 92% Epicatechin was estimated from phloroglucinol deviration collected after the column, and m/z of epicatechin determined by the spectroscophy results was about 301 Phlorotannin need to study deeper to service a medicine and food 3.5 PREPARING ASSAY OF PHLOROTANNIN POWDER FROM BROWN ALGAE S SERRATUM BY USING THE TECHNIQUE OF SPRAY DRYING 3.5.1 Determine of drying aids The testing results of various drying aids (maltodextrin, glucose, saccharose) in the preparation of phlorotannin powder S serratum announced that antioxidant Phc of phlorotannin powder decreased in the following order: maltodextrin/ glucose/ saccharose Thus, maltodextrin aids was collected to prepare the phlorotannin powder from brown algae S serratum 3.5.2 Determine of the maltodextrin ratio ANOVA and regression analysis showed that a close correlation between a ratio of maltodextrin and phlorotannin content, suggesting that a antioxidant activity is due to the phlorotannin of powder (R2 > 0.81); 2- 26 level model of non-linear functions were found on all target functions The results also noticed that 10% maltodextrin was suitable to prepare the powder of phlorotannin from brown algae S serratum 3.5.3 Determine of pump presure The results of ANOVA analysis performed phlorotannin content and antioxidant activity was closely related to the pump presure (R2 > 0.75) when phlorotannin extract from brown algae S serratum was dried spray, and the pump presure of 0.8bar was choiced as the suitable factor for spray drying of phlorotannin extract from brown algae S serratum harverst in Khanhoa 3.5.4 Determine of pump speed The correlating analysis of factors using ANOVA noticed that a close relation between the pump speed and Phc of powder (R2 > 0.92; F > Fcrit) The pump speed of 10 rpm was used as the suitable factor for the spray drying of phlorotannin extract from brown algae S serratum 3.5.5 Determine of inlet temperature of spray drying The analysing results of ANOVA and regression showed a significant difference between phlorotannin content and antioxidant activity of powder in various inlet drying temperature was exhibited The inlet temperature of 1100C is suitable for the formation of phlorotannin powder originated from the extract of brown algae S serratum through the method of spray drying 3.5.6 Promotion of preparing process of phlorotannin powder by using the technique of spray drying From over analysis, the collecting process of phlorotannin powder using the technique of spray drying was suggested and performed in fig 3.14 * Explanation of process: + Phlorotannin extract: phlorotannin from brown algae S serratum was extracted according to the method of maceration After extract was condensed until 500ml extract contain 3g of phlorotannin and antioxidant of extract got 6.7g ascorbic acid equivalent 27 + Assimilation: Add 10% maltodextrin into the condensed extract The purpose of aids addition is the formation of phlorotannin microcapsules and limit the changes of phlorotannin in the drying process After aids was added, the assimialtion was realized to ensure the homogenous solution Phlorotannin extract Maltodextrin 10% - Pump presure of 0.8bar - Pump speed 10 rpm - Inlet temperature of 1100C Assimilation Spray drying Phlorotannin powder Package Storage Sản phẩm Fig 3.14 The process of spray drying for the preparation of phlorotannin powder from the extract of brown algae S serratum + Spray drying: Process of spray drying for the phlorotannin extract was realized on the machine of spray drying SD 05 and factors of drying process was as follow: The pump pressure of 0.8 bar, pump speed of 10 rpm (350ml/h), inlet temperature of 1100C, maltodextrin was used as the aids 28 + Collection of phlorotannin powder: phlorotannin powder was collected and contained in a dark vessel Because, phlorotannin will be destroyed and antioxidant activity was decreased under the impact of light + Package: the powder was packed by the bag of LDPE, PP or compounds film of layers The bags will limit the destroying of powder under the impact of humidity and air The packaged product was contained in the paper box + Storage: the product packaged by the bag of LDPE, PP or compounds film of layers was storaged under the condition of drying, cleanness, and non direct-radiated light + Product: the product prepared according to the over technique process got the standards of food hygienic safety, and Phc and antioxidant activity 3.6 EVALUATION OF TOXIC OF PHLOROTANNIN PRODUCT The toxic of powder was evaluated on white mice The mice were separated into different groups, group 1: the mice were drunk biosalt water, group 2: the mice were drunk the solution of maltodextrin and biosalt water and group 3: the mice were drunk the solution of phlorotannin powder and biosalt water The results was shown in table 3.4 Table 3.4 Weight of white mice in various conditions after test days (use of biosalt water, maltodextrin dissolved into biosalt water, and antioxidant phlorotannin powder dissolved into biosalt water) STT Code of mice Average weight of mice drunk biosalt water Code of mice Average weight of controled mice Code of mice Average weight of mice drunk the sample LV 26.450±2.940 GV 26.888±2.000 MV 28.900±4.280 LVPSV 25.988±2.770 GVPTV 21.275±0.610 MVPTV 24.525±2.400 LVTTV 25.900±2.050 GVPSV 26.213±3.510 MVPSV 27.575±3.380 29 LVTSV 25.025±2.550 GVTTV 25.913±2.250 MVTTV 25.375±1.660 LV2PV 27.488±2.720 GVTSV 24.850±2.580 MVTSV 23.175±0.980 LV2TV 26.975±3.710 GVE0PV 23.375±2.250 MVE0PV 23.063±1.060 LVPTV 28.525±3.980 GVE0TV 23.825±2.020 MVE0TV 26.475±1.930 LVE0PV 25.325±2.680 GV2PV 23.900±2.930 MV2PV 23.750±2.150 LVE0TV 25.725±3.250 GV2E0V 23.900±1.190 MV2E0V 25.613±1.720 10 LV2E0V 26.150±3.830 GV2TV 25.588±2.610 MV2TV 24.750±2.880 26.360 ± 1.050 g/ body TB 24.570 ± 1.660 g/body 25.320 ± 1.900 g/body * Note: NMSL: biosalt water The results showed white mice developt very good, their weight increase (table 3.4) The results of weight evaluation and statistic analysis showed that the average weight of mice group 1, 2, and got 26.360 ± 1.050g/ body, 24.570 ± 1.660g/ body and 25.320 ± 1.900g/ body after the assay of days The results of ANOVA analysis showed a non-significant difference between the weight and the growth ratio of the studied mice groups (p = 0.055; F = 3.22 < Fcrit = 3.35) In addition, all studied mice are strong Thus, phlorotannin powder originated from brown algae S serratum is non-toxic 3.7 ASSAY APPLY PHLOROTANNIN TO BEVERAGE PRODUCTION 3.7.1 Determine of added citric acid score The results of regression and ANOVA analysis noticed a close relation between total average sensory point of citric acid and citric acid ratio was detected to the beverage The suitable score of citric acid added to phlorotannin beverage was 0.11% and this score was collected for continous study 30 3.7.2 Determine of added sugar ratio The results announced 17% sugar are adequate to phlorotannin beverage and the score was choiced to study, continously 3.7.3 Determine of the added ratio of citric acid and ascorbic acid The results showed the ratio of citric acid and ascorbic acid was 0.07% and 0.04%, respectively, compared to 200ml beverages, the total sensory point of beverage got the highest value of 18,3 point 3.7.3 Determine of added carrageenan - xanthan gum ratio The results of sensory analysis showed the beverages is the highest sensory point of 18.5 when 0.05% carrageenan and 0.05% xanthan gum was added into the beverages 3.7.5 Determine of added phlorotannin score The results announced Phc content of 30mg is suitable to add to 200ml beverages 3.7.6 Determine of temperature and time of assimilation The results noticed the condition of suitable assimilation was realized at the temperature of 450C in the time of 1-2 minutes 3.7.7 Determine of pasterization time After the pasteurization of beverage at 800C according to different pasterization formulas, the results showed the pasteurization formula of is suitable for the pasteurization of phlorotannin beverage originated from brown algae 3.7.8 Promotion of production process of phlorotannin beverage From all over results, the production process of phlorotannin beverage is suggested and showed in fig 3.15 31 30mg phlorotannin powder for 200ml beverage Saccharose 17% Acid citric 0.07% Water Carrageenan: 0.04% Xanthan gum: 0.05% Acid ascorbic: 0.04% Pasterization Mixing Filter Clean bottle Pasterization Temp: 450C Time: 1-2 minutes Assimilation Pouring Lid closing Pasterization fomular Pasterization Cooling Label sticking Product Fig 3.15 Diagram of production process of phlorotannin beverage 32 * Process explanation + Phlorotannin powder The character of phlorotannin powder originated from brown algae S serratum was as follow: dissolve in the water, dark yellow, the humidity of 5.3%, phlorotannin content got 2.268 ± 0.010g phloroglucinol equivalent, total antioxidant activity was 4.347 ± 0.018g ascorbic acid equivalent, reducing power activity was 9.390 ± 0.024 g FeSO4 equivalent, and DPPH free radical scavenging of 70,020 ± 0,260% for 100g powder + Assimilation The components were used to assimilate the beverage: - Water: water got the standard of Ministry of Publish Health - Sugar: the ratio of 17% sugar was used for 200ml beverages - Carrageenan: Carrageenan ratio of 0.05% was used for 200ml beverage - Xanthan gum: Xanthan gum score of 0.05% was used for 200ml beverage After carrageenan and xanthan gum was dissolved fully in the water, the compound was filtered to remove dregs - Ascorbic acid: Ascorbic acid score of 0.04% was used for 200ml beverage - Citric acid: Citric acid of 0.07% was used for 200ml beverage - Sodium benzoate: Sodium benzoate score of 0.02% was used for 200ml beverage - Postasium sorbate: The ratio of postasium sorbate of 0.02% was used for 200ml beverage - Phlorotannin: 30mg of phlorotanin powder producted from brown algae S serratum was used for 200ml beverage 33 The process of assimilation was realized as follow: the firstly, carrageenan and xanthan gum was fully dissolved into the water After ascorbic acid and citric acid was added into over compound Finally, add phlorotannin and the solution of prepared sugar into final compound and assimilate all added components - Assimilation The solution of beverage was assimilated at temperature of 450C in 1-2 minutes - Pour into bottle The assimilated solution was filtered and poured into a bottle - Lid closing After beverage was poured into the bottle, the semi product was removed the air and closed the lid The semi product was continuously pasteurized - Pasterization The semi product was pasteurized according to the formula of and a part of bacteria was destroyed to extend the storage time of product - Cooling The pasteurized products were cooled - Label sticking The label of product get the standards of decree No 89/2006/NĐ-CP of the goverment, decree No 38/2012/NĐ-CP of the government and joint circular No 34/2014/TTLT-BYT-BNNPTNT-BCT of the Ministry of Publish Health, Agriculture and Rural Development, Industry and Trade - Product The product produced during over process get the quality of safety and 34 hygienic food, the standards of government decree, the standards of ministry joint circular 3.8 TEST PRODUCTION AND EVALUATION OF QUALITY CHANGE OF PHLOROTANNIN BEVERAGE ACCORDING TO THE STORAGE TIME 3.8.1 Testing production and evaluation of product quality Phlorotannin beverage get the quality of good sense and the bacterial standard of Ministry of Publish Health Phlorotannin content and antioxidant activity get the technological standards of built process 3.8.2 Evaluation of product quality according to a storage time The beverages were stored in cold (40C) and room condition After 12 month storage time, the activity of total antioxidant and reducing power, and phlorotannin content of beverages, decreased to 65% compared to the beginning of the shelf-life study After 12 month time of cold storage time, total antioxidant activity and phlorotannin content of beverage was still 75%, compared to the initial shelf-life study In brief, antioxidant activity and phlorotannin content of beverage in the condition of cold storage was also decreased, but the speed of decreasing is slower than that of beverage in the condition of room storage 3.8.3 Counting of materials cost Material cost of 200ml phlorotannin beverage was counted to be 4.257,2 VND 35 CONCLUSION AND PETITION CONCLUSION From all over studies, some conclusions were performed as follow: 1) The optimal condition of the step of phlorotannin extraction from brown algae S serratum was: 96% ethanol, the temperature of 430C, the time of 33 hours, the solvent-to-material ratio of 27:1 (v/w) Phlorotannin content and antioxidant activity (total, reducing power, DPPH) of the received extract got 5.950 ± 0.017 mg phloroglucinol equivalent /g DW, 15.050 ± 0.035 mg ascorbic acid equivalent /g DW, 45.300 ± 0.021 mg FeSO4 equivalent/g DW and 63.98% ± 1.2%, respectively 2) Solvent fractions of n-hexan/ chloroform/ ethyl acetate/ n-butanol have phlorotannin content of 29.8%, 20.6%, 81.8% and 51.94%, respectively, compared to the total phlorotannin content of the initial extract of ethanol Phlorotannin of ethyl acetate fraction purified on Sephadex LH 20 column has peak and their purity of over 92% 3) The suitable condition of spray drying prepare phlorotannin powder such as: aids of maltodextrin, the maltodextrin/ phlorotannin solution ratio of 10%, the pump pressure of 1.0 bar, the speed of pump of 10 rpm, the inlet temperature of 1100C The powder has phlorotannin content of 2.268 ± 0.010g phloroglucinol equivalent/ 100g DW, total antioxidant activity of 4.347 ± 0.018g ascorbic acid equivalent/100g DW and reducing power activity of 9.390 ± 0.024g FeSO4 equivalent /100g DW 4) The phlorotannin powder originated from brown algae S serratum is non-toxic on mice 5) 200ml of antioxidant phlorotannin - rich beverage have the ratio of some ingredients as follow: sugar of 17%, citric acid of 0.07%, carrageenan of 0.05%, xanthan gum of 0.05%, ascorbic acid of 0.04%, phlorotannin of 1.4g, 36 sodium benzoate of 0.04g The ingredients were assimilated at 450C in 1-2 minutes and pasterizated at 800C in minutes PETITION Over studies permit to petited some ideas such as: the design of label, production of the phlorotannin beverage in a larger level to assay the product commerce 37 LIST OF PUBLICATIONS OF THE AUTHOR Dang Xuan Cuong, Bui Minh Ly, Tran Thi Thanh Van, Vu Ngoc Boi, Le Nhu Hau (2015) “Effect of storage time on phlorotannin content and antioxidant activity of six Sargassum species from Nhatrang bay, Vietnam” Journal of Applied Phycology, Springer, Publish online: 23, May, 2015 Dang Xuan Cuong, Vu Ngoc Boi, Tran Thi Thanh Van, Dao Trong Hieu (2015) “Study on production process for antioxidant beverage containing seaweed phlorotannin”, Journal of Agriculture and Rural Development, Ministry of Agriculture of Rural Development, (accepted for publication) Dang Xuan Cuong, Bui Minh Ly, Tran Thi Thanh Van, Vu Ngoc Boi, Le Nhu Hau, Dao Trong Hieu (2014), “Initial study of antioxidant phlorotannin purification from brown algae Sargassum serratum growth in Nhatrang sea”, Journal of Agriculture and Rural Development, Ministry of Agriculture of Rural Development, 2, pp 71-76 Dang Xuan Cuong, Vu Ngoc Boi, Tran Thi Thanh Van, Ngo Dang Nghia (2013), “Screening of antioxidant activity of some brown algae species Sargassum in Khanhhoa, Vietnam”, Journal of Science, CanTho university, B part: Agriculture, Fisheries and Biotechnology, 25, pp 36-42 Dang Xuan Cuong, Vu Ngoc Boi, Tran Thi Thanh Van, Le Nhu Hau (2013), “Evaluation of phlorotannin content and antioxidant activity of brown algae sargassum collected in Nhatrang bay, Vietnam”, Journal of Science and Technology, Thainguyen university, pp 137 Dang Xuan Cuong, Vu Ngoc Boi, Tran Thi Thanh Van (2012), “Accumulation and distribution of antioxidant phlorotannin originated from brown algae Sargassum duplicatum Khanhhoa on the growth time”, Journal of Fisheries Science and Technology, 4, Nhatrang university 38 Dang Xuan Cuong, Tran Thi Thanh Van, Vu Ngoc Boi, Le Xuan Hai (2011), “Multiobjective optimization for phlorotannin extraction and antioxidant activity of brown algae Sargassum serratum collected in Nha Trang bay, Viet Nam using Box-Behnken model”, J Sci Dev., (Eng.Iss 1), pp 155 – 162 Dang Xuan Cuong, Tran Thi Thanh Van, Bui Minh Ly, Vu Ngoc Boi (2011), “Accumulation and distribution of antioxidant phlorotannin of some brown algae species Sargassum Khanhhoa on the growth time”, National conference of Science and Marine Technology, Subcommittee of Biotechnology and Marine resource, 699 - 704 ... algae (Sargassum serratum) in Nhatrang and assay apply to antioxidant beverage? ?? is necessary The target of the thesis: - Research on antioxidant phlorotannin collection from brown algae (Sargassum. .. scope of research: contain parts 1) Extraction and optimization study on phlorotannin collection from brown algae (Sargassum serratum) grown in Nhatrang sea, Khanhhoa 2) Initial fraction and purification... Alternatively, brown algae in Nha Trang sea contains a high content of bioactive substances as fucoidan, phlorotannin, alginate,… Therefore, a study of ? ?Research on phlorotannin collection from brown algae