Optimizing the microwave-assisted extraction of phenolic compounds and antioxidant capacity from Polygonum multiflorum Thunb.. For this reason, a study on the application and testing o
Trang 1MINISTRY 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
Trang 2THE 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
Trang 3LIST OF PUBLISHED WORKS
1 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 0866-
708X)
2 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)
3 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)
4 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)
5 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)
6 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)
Trang 4CHAPTER 1 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
Trang 51.3 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
Trang 6The 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 2 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 glucoside, 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
tetrahydroxytibene-2-O-β-D-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 microwave-assisted extraction, ultrasonic-assisted extraction to improve the extraction yield In addition, extraction method by solvents at high pressure is also
Trang 7performed, 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
Trang 8carrier 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
anti-microorganisms 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 3 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 1 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 3 or 4 harvesting days
3.1.2 Analysis methods
Table 3.1: The analysis methods used in the study
Trang 915 Color parameters Using Minolta (CR-410) machine to measure
18 PoV (meq/kg) Iodometric titration method
19 TBARS (µg MDA/kg) Spectrometric method based on colorimetric
reaction of sample with TMP, TBA
Trang 103.1.3 Plan of experiment designs
Figure 3.1: General plan of the experimental designs
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
Freezing storage (Exp 1)
Determining the species and composition of material
Polygonum multiflorum Thunb root
Cleaning, Slicing (thickness: 2-3 mm)
1 st content: Effect of storage methods on TPC and AC of polyphenols
of Polygonum multiflorum Thunb root
Exp 2: Research on dried temperature of material
Exp 3: Preserving the dried powder at room condition
Choosing the suitable storage method
2 nd content: Determining the polyphenols extraction process
Microwave assisted extraction and ultrasonic assisted extraction
Choosing the suitable extraction method
Effect of microwave on polyphenols (Exp 8)
Research on microwave power and extraction time
Effect of ultrasonic on polyphenols (Exp 9)
Research on temperature and extraction time
Optimization of the extraction process by response surface methodology (Exp 10)
3 rd 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
4 th 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
- Determining acute toxicity
- Determining semi-chronic toxicity.
Trang 113.1.4 Data analysis
The experiments were designed randomly with one or two factors, repeated three times The optimum result of the previous experiment was applied for the next one Data were treated by analysis of variance (ANOVA) and LSD post-hoc test to determine the difference between the means of treatments with the support of Statgraphics software (Centurion 15.2 version) Modde 5.0 software was used to calculate the optimum model of some extraction factors by response surface methodology
3.2 Experimental designs
3.2.1 Determining the species and composition of Polygonum multiflorum
Thunb root
Objective: Determination of the phylogenetic tree, the difference of
heredity and primary composition of Polygonum multiflorum Thunb root
Determining DNA, moisture (%), protein (%), lipid (%), carbohydrate (%), starch (%), reducing sugar (%), ash (%) and heavy metal (ppb)
3.2.2 Effect of storage methods on the stability of total polyphenol content
(TPC) and antioxidant capacity (AC) of Polygonum multiflorum Thunb
root
Objective: Research on the changes of TPC and TEAC of materials for the freezing method, drying method and storing dried powder products at room temperature
Determining the changes of TPC and AC for the different storage methods
Thickness of sliced fresh root for freezing method: 2-3 mm
Dried temperatures of sliced fresh root: 50, 60, 70 and 80oC
Dimension of dried powder is <0.5 mm, then packed in vacuum and stored at room condition
The parameters of extraction process:
Solvent: Distilled water Temperature: 50oC
Material/solvent ratio: 1/20 (g/mL) Extraction time: 60 minutes Storage time: 100 days for dried powder and 180 days for fresh sliced root
TPC (mg GAE/g DW) and TEAC (µmol TE/g DW) were determined during the storage time
3.2.3 Determining the optimum polyphenols extraction of Polygonum multiflorum Thunb root
Trang 12Objective: Determining the optimum polyphenols extraction conditions
Solvent concentration: 40, 50, 60, 70 and 80%
Extraction temperature: 30, 40, 50, 60 and 70oC - Extraction time: 30,
Microwave power: 74, 127, 195, 327 and 610 W/g - Extraction time:
Extraction temperature: 30, 40, 50, 60 and 70oC - Extraction time: 5,
TPC (mg GAE/g DW), TEAC (µmol TE/g DW) and some specific polyphenols (mg/g DW) were determined after the optimum extraction process
Trang 133.2.4 Determining the minimum inhibitory concentration (MIC) of dryness extract by the paper disc diffusion
Objective: Evaluating MIC by the paper disc diffusion method for 4
bacteria such as Bacillus subtilis (ATCC 11774), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Salmonella enteritidis (ATCC 13076) and 3 molds such as Fusarium equiseti, Aspergillus niger, Trichoderma asperellum which are isolated and identified in laboratory
Experiment was set up with one factor as dryness extract concentrations: 25, 50, 100, 200, 400, 800 and 1600 mg/mL
The zones of inhibition (mm) of the different dryness extract concentrations were recorded
3.2.5 Evaluating the interaction of polyphenols and gelatin
Objective: Evaluating the polyphenols-protein (gelatin) interaction Experiment was set up with two factors including the extract concentrations (415, 277, 208 and 166 mg GAE/mL) and the gelatin concentrations (30, 60, 90 and 120 mg/mL)
The relative absorbance of supernatant extract (%) was recorded
3.2.6 Research on the combination of edible film (alginate) and extract for fresh-cut papaya storage
Objective: Evaluating the combination of various polyphenols concentrations and alginate for fresh-cut papaya storage at cold temperature
Experiment was set up with one factor such as the concentration extracts, which were diluted by water (415, 277, 208 and 166 mg GAE/mL) with the storage time (0, 2, 4 and 6 days) and the control
Experiment was set up with one factor such as the concentration extracts, which were diluted by water (830, 415, 277, 208 and 166 mg GAE/mL) with the storage time (0, 3, 6 and 9 hours at the room condition;
Trang 140, 1, 2, 3 and 4 days at the mixture of salt and ice; 0, 20, 40, 60, 80 and 100 days at the freezing storage) and the control samples
The quality criteria were analyzed including PoV (meq/kg), pH,
TBARS (µg MDA/kg), color parameters (L * , a * , b *), microbiological analysis (CFU/g), evaluation of sensory acceptability (the 9-point hedonic scale)
3.2.8 Evaluating the physicochemical properties of powder produced with different carrier agents
Objective: Selecting the suitable carrier agent to maintain TPC, AC and evaluating the physicochemical properties of spray-dried powder
Experiment was set up with one factor such as type of the carrier
agents They were maltodextrin (MD, DE 16-19) and gum arabic (GA) The extract (4% Brix, after solvent evaporation) was modified the soluble solid of 25% and 15% by these carrier agents, respectively The solution was well mixed and then fed into a Lab Plant SD-06 spray dryer The feed flow rate, inlet/outlet temperature and air flow speed were approximately set at 500 mL/h, 160/70°C and 5 m/s, respectively
The quality criteria were analyzed including TPC (mg GAE/g DW), TEAC (µmol TE/g DW), yield (%), moisture (%), bulk density (g/mL), hygroscopicity (g/100 g), water solubility index (%), flowability (o),
wettability (s), particle size (mm), color parameters (L * , a * , b *), SEM
3.2.9 Modelling the degradation of quality of extract and spray-dried powder
Objective: Evaluating the degradation of TPC and AC at various
storage temperatures, thus the prediction of quality of phenolic compounds
of extract and spray-dried powder were modelled
Experiment was set up with three factors including materials (extract, gum arabic and maltodextrin), storage temperature (60 and 70oC) and storage time (0, 1, 2, 3, 4, 5 and 6 days)
TPC (mg GAE/g DW), TEAC (µmol TE/g DW) of extract and
spray-dried powder were analyzed
3.2.10 Determining the toxicity of extract
Determining the acute toxicity in mice
Objective: Determining the acute toxicity on Swiss mice for 72 hours
at various polyphenols concentration extracts, which are higher 100 times than concentration extracts used for human