Nghiên cứu trích ly polyphenol từ củ hà thủ ô đỏ (polygonum multiflorum thunb ) và ứng dụng trong thực phẩm tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL DISSERTATION Major: Food Technology Code: 62.54.01.01 LE PHAM TAN QUOC INVESTIGATION OF THE POLYPHENOLS EXTRACTION FROM POLYGONUM MULTIFLORUM THUNB ROOT AND APPLICATION IN FOOD Can Tho, 2019 THE DISSERTATION WAS COMPLETED AT CAN THO UNIVERSITY Advisor: Assoc Prof Dr Nguyen Van Muoi The doctoral dissertation was evaluated by the Committees at the basic level Meeting at: Meeting room 2, 2nd floor, Campus II – Control Hall, Can Tho University At: 14 p.m., date 17/11/2018 Reviewer 1: Assoc Prof Dr Dai Thi Xuan Trang Reviewer 2: Prof Dr Dong Thi Anh Dao You can find a copy of this dissertation at the library: Learning Resource Center, Can Tho University National Library of Vietnam LIST OF PUBLISHED WORKS Le Pham Tan Quoc and Nguyen Van Muoi 2015 Optimizing the microwave-assisted extraction of phenolic compounds and antioxidant capacity from Polygonum multiflorum Thunb root Journal of Science and Technology, 53(4C): 1-11 (ISSN 0866708X) Le Pham Tan Quoc and Nguyen Van Muoi 2016 Effect of alginate coating and polyphenol extract from Polygonum multiflorum Thunb root on the fresh-cut papaya during storage Science and Technology Journal of Agriculture and Rural Development, Green Agriculture, November, 175-181 (ISSN 1859-4581) Le Pham Tan Quoc and Nguyen Van Muoi 2018 Effect of polyphenol extract from Polygonum multiflorum Thunb root on the storage of minced red tilapia (Oreochromis sp.) Bulgarian Chemical Communications, 50: 26-33 (ISSN 0324-1130) Le Pham Tan Quoc and Nguyen Van Muoi 2018 Phytochemical screening and antimicrobial activity of polyphenols extract from Polygonum multiflorum Thunb root Carpathian Journal of Food Science and Technology, 10(4): 137-148 (ISSN 2066-6845) Le Pham Tan Quoc and Nguyen Van Muoi 2018 Physicochemical properties of Polygonum multiflorum Thunb root powder produced with different carrier agents Chemical Industry & Chemical Engineering Quarterly, 24: 93-100 (ISSN 2217-7434) Nguyen Van Muoi and Le Pham Tan Quoc 2018 The shelf-life of total polyphenol content and the antioxidant capacity of the Polygonum multiflorum (Thunb.) root extract and its spray dried powder according to the Q10 method Bulletin of the Transilvania university of Brasov Series II - Forestry Wood industry Agricultural food engineering, 11: 147-158 (ISSN 2065-2135) CHAPTER INTRODUCTION 1.1 The rationale of the study Nowadays, polluted environment and unhealthy and unhygienic food have caused many diseases related to the formation of large amounts of free radicals in the human body To avoid these effects, foods or pharmaceutical have been fortified with antioxidants to prevent pathogens Currently, 50% of the medicine originated directly or indirectly from natural resources, especially plants Plants are extremely important resources for medicine and health food They could inhibit bacteria, mold and virus In addition, they had the very strong antioxidant capacity, which could prevent and cure many different diseases due to various bioactive compounds, polyphenols in particular Polygonum multiflorum Thunb root is one of the special herbal plants containing high levels of polyphenols and antioxidant capacity It is known as precious medicine to improve the health and extends longevity However, it is quite problematic to use from this material for polyphenols extraction as it is a wild plant that is largely distributed in the mountainous region and is exploited as raw medicine Besides, polyphenols extraction is not simple because we have to maintain total polyphenol content, antioxidant capacity and depend on the extraction methods Hence, finding a suitable extraction method is the priority at present Moreover, applying polyphenols extract from Polygonum multiflorum Thunb root in food technology was limited There are not study that apply polyphenols extract from Polygonum multiflorum Thunb root in food storage and food processing yet For this reason, a study on the application and testing of antioxidant capacity of phenolic compounds from Polygonum multiflorum Thunb root would create the products that have the high nutritional value, better storage time and protect the health of consumers 1.2 Objective of the study Extract the polyphenol compounds from Polygonum multiflorum Thunb root contrasting traditional and modern extraction methods Optimize the extraction process by respond surface methodolody Apply the resulted polyphenols extract in food storage as an antioxidant and anti-microorganism agent Besides, some spray-dried products were made to protect and maintain the total polyphenol content and antioxidant capacity 1.3 Contents Research on storage methods of Polygonum multiflorum Thunb root to maintain total polyphenol content and antioxidant capacity Determining the suitable polyphenols extraction methods and optimizing the factors of extraction process such as solvents, temperature, materials/solvent ratio, solvent concentration, extraction time and extraction power to obtain the highest polyphenols content and antioxidant capacity Determining some physicochemical properties of spray-dried products, anti-microorganism capacity of dryness extract and applying polyphenols extract from Polygonum multiflorum Thunb root to storage the fresh-cut papaya and minced red tilapia Modelling the prediction of changes of total polyphenol content and antioxidant capacity of extract and spray-dried products; and examinig the toxicity of the extract in mice 1.4 Scientific and practical significance The results of this study provided data of total polyphenol content and antioxidant capacity from Polygonum multiflorum Thunb root by some extraction methods The result also showed the suitable polyphenols concentrations for storing the fresh-cut papaya and minced red tilapia At the same time, this study evaluated the anti-microorganism capacity and the quality degradation of polyphenols extract and spray-dried products during storage time The results of this study can improve the economic efficiency of Polygonum multiflorum Thunb root, motivating the cultivation, exploitation and application of this plant in the local area 1.5 New contributions The changes of total polyphenol content and antioxidant capacity in some storage methods were evaluated Total polyphenol content and antioxidant capacity of Polygonum multiflorum Thunb root were optimized by response surface methodology The anti-microorganism capacity and protein-polyphenols interaction were performed The optimum polyphenols concentrations to storing fresh-cut papaya and minced red tilapia were determined The polyphenols extract could be stored in spray-dried products such as maltodextrin and gum arabic The prediction of quality degradation of phenolic compounds based on the the storage temperature was modelled Polyphenols concentration of the extract from Polygonum multiflorum Thunb root was found not toxic in mice 1.6 Outline of the dissertation The dissertation consists of 144 pages with five chapters: Chapter 1: Introduction (pages: 1÷4); Chapter 2: Literature review (pages: 5÷31); Chapter 3: Materials and methods (pages: 32÷58 with 18 experiments); Chapter 4: Results and discussion (pages: 59÷143) and Chapter 5: Conclusions and suggestions (page 144) The primary content has 20 tables and 77 figures The dissertation consists of 434 references (402 English references and 32 Vietnamese references) CHAPTER LITERATURE REVIEW 2.1 Polygonum multiflorum Thunb root Polygonum multiflorum Thunb is a member of the Polygonaceae family, the Fallopia genus Its scientific name is Fallopia multiflora Polygonum multiflorum Thunb It is a wild plant and distributed on the mountainous region in the Northwest and Middle region of Vietnam such as Thanh Hoa, Nghe An, Ha Tinh, Son La, Lai Chau province The compositions of Polygonum multiflorum Thunb consist of many precious compounds that can be used widely in pharmaceutical industry but they degrade easily under the effect of surrounding environment Polygonum multiflorum Thunb root contains starch, protein, lipid, tannins, chrysophanol, rhein, physcion, 2,3,5,4 tetrahydroxytibene-2-O-β-Dglucoside, phospholipid, lecithin, rhaponticin, inorganic compounds and water soluble compounds, especially phenolic compounds, etc These compounds have high antioxidant capacity, anti-aging effect, antihyperlipidemic effect, anti-cancer effect, neuroprotective effect and anti-inflammatory effect 2.2 Extraction methods Currently, there are many extraction methods to separate bioactive compounds from the plants cell such as distillation method, maceration at low pressure, etc which are the traditional extraction methods At the same time, we can use some modern extraction methods such as microwaveassisted extraction, ultrasonic-assisted extraction to improve the extraction yield In addition, extraction method by solvents at high pressure is also performed, including the combination of supercritical carbon dioxide and solvents (Rosa et al., 2009) Besides, some enzymes were used for hydrolyzing and releasing phenolic compounds from interactions of polyphenols with other substances such as carbohydrates, lipids and proteins, etc to improve the extraction yield However, each extraction method also has its advantages and disadvantages, no method is suitable for all materials Choosing extraction methods depends on properties of material, type of solvent, and budget, etc Based on those, the extraction method was chosen to meet some conditions of experiments 2.3 Studies on the polyphenol extraction and its application in food Freezing and drying methods are important to preserve, maintain and reduce the quality degradation of polyphenols in the plant material (Ninfali and Bacchiocca, 2003; Malik and Bradford, 2008), especially the drying method, which is often used to store and maintain the bioactive compounds of medicinal products, especially polyphenols (Cai et al., 2004) Some studies on the extraction of phenolic compounds from plants showed that the extraction process is influenced by many factors, such as the type of material, the extraction method, the sample size, the condition package and preservation time (Cao et al., 1998) Some compounds in the extracts are able to bind and isolate polyphenols such as carbohydrate, lipid, protein (Jakobek, 2015), pectin (Ngo Thi Thanh Tam et al., 2014), cellulose (Kim et al., 2005), etc and thus we can use the enzymes to hydrolyze and release polyphenols In addition, some modern extraction methods widely used in the laboratory and they can be applied to improve and support the extraction process, for instance microwave-assisted extraction (He and Xia, 2011; Simsek et al., 2012), ultrasonic-assisted extraction (Zhao et al., 2013; Sahin et al., 2013), and supercritical carbon dioxide-assisted extraction (Meireles et al., 2013; Fiori et al., 2015) with various types of solvents such as acetone, methanol, ethanol, water, etc Identification and application of phenolic compounds from different plants is a major focus in the medicine industry and healthcare technology In addition, many studies proved that the antimicrobial activity of plants is primarily due to bioactive compounds such as saponins, alkaloids, essential oils and polyphenols (Nascimento et al., 2000) Polyphenols extracts is directly used or we can transform them into spray-dried powders These products can be stored longer and more conveniently to use, for example anthocyanin spray-dried powder, guava leaf extract powder with many carrier agents such as maltodextrin, and gum arabic (Caliskan and Dirim, 2013; Silva et al 2013) Besides, antioxidant compounds in plants extracts can be used in preservation of vegetables such as sliced potatoes (Spanou and Giannouli, 2013), and tomatoes (Boko and Salas, 2015) In the preservation process of meat and fish products, polyphenols extracts from plants can extend the shelf life of mackerel (He and Shahidi, 1997), fillet sturgeon (Haghparast et al, 2011), minced fish (Maqsood et al., 2015) because they have antimicroorganisms capacity and inhibit the rancidity of the product There are some studies on the antioxidant capacity of Polygonum multiflorum Thunb root in the world, but they always used the traditional extraction method and it does not fully evaluate the influencing factors as well as the modern extraction methods In Vietnam, there are also studies on the identification of chemical compounds in Polygonum multiflorum Thunb root (Nguyen Thi Ha Ly et al., 2014) Wine processing from Polygonum multiflorum Thunb root was performed (Le Binh Hoang and Bui Quang Thuat, 2015) The morphology of this plant was also investigated (Pham Thanh Huyen et al., 2015) However, until now there are not research on the utility of polyphenols extracts from Polygonum multiflorum Thunb root in food products and modelling the degradation of total polyphenol content and antioxidant capacity based on storage temperature From the above literature review, the extraction and application of polyphenols extract from Polygonum multiflorum Thunb root in food preservation and food processing is quite feasible and there are many new aspects that can contribute in the food technology CHAPTER MATERIALS AND METHODS 3.1 Materials and methodology 3.1.1 Materials Polygonum multiflorum Thunb roots were harvested from Bao Lam, Bao Lac district, Cao Bang province (Vietnam) Fresh root did not have any physical damage and pest The roots had many different shapes, approximately from 0.5 to kg/root, dark brown skin It was white or light yellow color inside The fresh roots were harvested, sorted, packed and transported to laboratory after or harvesting days 3.1.2 Analysis methods Table 3.1: The analysis methods used in the study No 10 11 Criteria Protein (%) Lipid (%) Starch (%) Reducing sugar (%) Ash (%) Carbohydrate Heavy metal (ppm) Total acidity (%) Hardness (N) Identification of bioactive compounds Total polyphenol content (mg GAE/CK) Methods FAO, 14/7, 1986, p.221 FAO, 14/7, 1986, p.212 AOAC 920.44-2011 TCVN 4594:1988 TCVN: 5253-90 TCVN 4594:1988 TCVN 6971:2001 TCVN 5483:2007 Using Instron 5543 machine to measure the hardness Based on changes of color and precipitation Spectrometric method based on colorimetric reaction of sample with Folin Ciocalteu Spectrometric method based on colorimetric reaction of sample with DPPH, Trolox as standard agents Spectrometric method based on colorimetric reaction of sample with gelatin The paper disc diffusion method for antibiotic susceptibility testing and minimum inhibitory concentration 12 Antioxidant capacity (µmol TE/CK) 13 Interaction of gelatin and polyphenols 14 Anti-microorganism capacity 15 Color parameters Using Minolta (CR-410) machine to measure L*, a*, b* values 16 Amount of microorganism (CFU/g) Counting specific colony on the petrifilm after inoculation time 17 Sensory evaluation 18 PoV (meq/kg) 19 TBARS (µg MDA/kg) 20 21 22 Evaluating physicochemical properties of spraydried products Evaluating shelf-life of product (days) Evaluating toxicity of extract Consumer acceptance test (the 9-point hedonic scale) Iodometric titration method Spectrometric method based on colorimetric reaction of sample with TMP, TBA Determining bulk density, moisture, hygroscopicity, water solubility index, flowability, wettability, particle size, SEM Q10 method Determining the acute and semi-chronic toxicity in mice Some biochemical criterias were measured at 175 hospital 3.1.3 Plan of experiment designs Polygonum multiflorum Thunb root Cleaning, Slicing (thickness: 2-3 mm) Determining the species and composition of material 1st content: Effect of storage methods on TPC and AC of polyphenols of Polygonum multiflorum Thunb root Freezing storage (Exp 1) Exp 2: Research on dried temperature of material Exp 3: Preserving the dried powder at room condition Choosing the suitable storage method nd content: Determining the polyphenols extraction process Reflux maceration method of polyphenols with organic solvents (Exp 4-7) Research on type of solvent; material/solvent ratio, solvent concentration, time and temperature of extraction process Microwave assisted extraction and ultrasonic assisted extraction Effect of ultrasonic on polyphenols (Exp 9) Research on temperature and extraction time Effect of microwave on polyphenols (Exp 8) Research on microwave power and extraction time Choosing the suitable extraction method Optimization of the extraction process by response surface methodology (Exp 10) 3rd content: Research on some properties and application of polyphenols extract (Exp 11-14) - Anti microorganism capacity - Interaction of polyphenols with gelatin - Combination of edible film (alginate) and polyphenols extract to preseve the fresh-cut papaya - Antioxidant capacity of polyphenols extract on minced red tilapia 4th content: Research on the physicochemical properties of spray-dried powder and modelling the degradation of quality of extract and spray-dried powder (Exp 15-16) - Determining the physicochemical properties of spray-dried powder - Modelling the degradation of quality of extract and spray-dried powder 5th content: Research on the toxicity of polyphenols extract in mice (Exp 17-18) - Determining acute toxicity - Determining semi-chronic toxicity Figure 3.1: General plan of the experimental designs Experiment was set up with one factor such as various polyphenols concentration extracts (25x, 50x, 75x and 100x) and the control samples Dead mice ratio (%), urea (mmol/L), creatinine (µmol/L), AST (U/L), ALT (U/L), erythrocytes (M/µL), leukocytes (K/µL), gross morphology of liver and liver histology were analyzed Determining the semi-chronic in mice Objective: Determining the semi-chronic on Swiss mice for weeks at polyphenols concentration extract used for human Experiment was set up with two factors such as polyphenols concentration extract (1x and the control samples) and observed time (0, and weeks) Dead mice ratio (%), body weight (g), urea (mmol/L), creatinine (µmol/L), AST (U/L), ALT (U/L), erythrocytes (M/µL), leukocytes (K/µL), gross morphology of liver/kidney and liver/kidney histology were analyzed CHAPTER RESULTS AND DISCUSSION 4.1 Research on the genealogy and some primary composition of Polygonum multiflorum Thunb root The result showed that the HA sample is Fallopia genus and the same group with Fallopia multiflora taxa (boostrap value of 97%) The distance of genetic between sample and two taxas were zero Hence, the sample was Fallopia multiflora species The result also showed that the composition of material were moisture (78.61±0.54%), protein (2.13±0.12%), lipid (0.42±0.06%), carbohydrate (12.2±0.14%), reducing sugar (2.30±0.28%), starch (8.88±1.58%), and ash (1.03±0.03%) Heavy metal was not detected 4.2 Research on the storage of sliced roots 4.2.1 Effect of the freezing storage methods on TPC and TEAC Frozen storage at -18oC after quick freezing maintained TPC and TEAC better than the slow freezing methods after 180 days TPC and TEAC decreased during the storage time These results were similar with some previous studies for preserving other materials by the freezing methods 4.2.2 Effect of the dried temperature on TPC and TEAC The optimum dried temperature was 60oC for 210 minutes (moisture of sliced root is under 12%) TPC and TEAC were 24.51±1.37 mg GAE/g 12 DW and 175.29±1.09 µmol TE/g DW, respectively This temperature could maintain TPC and TEAC better than other dried temperatures 4.2.3 Research on the changes of TPC and TEAC of the dried root powder at the room condition TPC (24.11±0.5 mg GAE/g DW) and TEAC (174.78±4.91 µmol TE/g DW) of dried root powder could maintain stably for 60 days, then decreased to 20.92±0.93 mg GAE/g DW and 163.94±2.76 µmol TE/g DW after 100 days, respectively 4.3 Research on the polyphenols extraction 4.3.1 The polyphenols extraction by the reflux maceration method Effect of solvents on TPC and TEAC The result showed that acetone concentration of 50% was the optimal solvent TPC (30.43±0.78 mg GAE/g DW) and TEAC (221.56±3.83 µmol TE/g DW) had the best values This result was similar with some studies which believed that the aqueous acetone was the best solvent for extracting polar phenolic compounds Effect of solvent/material ratio on TPC and TEAC An increase of the solvent/material ratio could lead to an increase in extraction yield The result peaked at the solvent/material ratio of 40/1 (mL/g), TPC and TEAC were 33.72±0.44 mg GAE/g DW and 235.52±3.5 µmol TE/g DW, respectively The extraction yield decreased with a further increase solvent/material ratio Effect of acetone concentration on TPC and TEAC The extraction yield increased quickly from 40% to 60% acetone concentration and then decreased strongly with a further increase acetone concentration of 80% The acetone concentration of 60% was the optimal condition for next experiments (TPC and TEAC were 36.5±3.14 mg GAE/g DW and 281.39±3.21 µmol TE/g DW, respectively) Effect of time and temperature on TPC and TEAC TPC and TEAC increased dramatically at the high temperature and long extraction time The yield obtained the best result at 50°C for 90 minutes (TPC and TEAC are 38.60±0.56 mg GAE/g DW and 298.15±2.99 µmol TE/g DW, respectively), then declined quickly with a further increase temperature and extraction time At same time, the cell wall appeared some wrinkles after the extraction process 13 4.3.2 Microwave-assisted extraction of phenolic compounds Effect of microwave power and extraction time on TPC and TEAC Material/solvent ratio of 1/40 (g/mL) and acetone concentration of 60% of the reflux maceration method was used for reseaching on effect of microwave power and extraction time by microwave-assisted extraction The optimum TPC and TEAC values were 44.77±0.25 mg GAE/g DW and 336.83±3.11 µmol TE/g DW at microwave of 127 W/g and extraction time of minutes The yield at this extraction condition was better than others (p