Luận văn thạc sĩ Công nghệ thực phẩm: Study on extract-loaded cashew nut testa using different emulsifiers by double nanoemulsion method

HO CHI MINH NATIONAL UNIVERSITY

HO CHI MINH UNIVERSITY OF TECHNOLOGY

HUYNH LE THANH PHUC

STUDY ON EXTRACT – LOADED CASHEW NUT TESTA

USING DIFFERENT EMULSIFIERS BY DOUBLE

THIS THESIS IS COMPLETED AT

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY – VNU-HCM

Supervisor: Assoc Prof Nguyen Thi Lan Phi

Examiner 1: Assoc Prof Nguyen Vu Hong Ha

Examiner 2: Assoc Prof Mai Huynh Cang

This master’s thesis is defended at HCM City University of Technology, VNU- HCM City on July 14th , 2023

Master’s Thesis Committee:

1 Chairman: Assoc Prof Phan Ngoc Hoa

2 Examiner 1: Assoc Prof Nguyen Vu Hong Ha 3 Examiner 2: Assoc Prof Mai Huynh Cang

4 Council Member: Assoc Prof Nguyen Thi Lan Phi 5 Secretary: Assoc Prof Tran Thi Thu Tra

Approval of the Chair of Master’s Thesis Committee and Dean of Faculty of Chemical Engineering after the thesis being corrected (If any)

CHAIRMAN OF THESIS COMMITTEE DEAN OF FACULTY OF

VIETNAM NATIONAL UNIVERSITY - HO CHI MINH CITY

HO CHI MINH UNIVERSITY OF TECHNOLOGY

SOCIALIST REPUBLIC OF VIET NAM Independent – Liberty - Happiness

THE TASK SHEET OF MASTER’S THESIS

Student’s name: HUYNH LE THANH PHUC Student ID: 2170459 Date of birth: March 3rd, 1998 Place of Birth: HCM City Major: Food Technology Major ID : 8540101

I THESIS TITLE (In Vietnamese):

Nghiên cứu vi bao dịch chiết vỏ lụa hạt điều bằng phương pháp tạo nhũ nano kép sử dụng các chất mang khác nhau

II THESIS TITLE (In English):

Study on extract-loaded cashew nut testa using different emulsifiers by double emulsion method

nano-III TASKS AND CONTENT:

- Analysis of total content of Phenolic, Flavonoid, Catechin compounds and antioxidant capacity of cashew nut testa extract

- Investigation of encapsulation conditions of bioactive compounds in cashew nut testa extract by double emulsion method with different emulsifiers

- Investigation of the stability of the emulsion system of cashew nut testa extract

IV THESIS START DAY: February 2nd, 2023

V THESIS COMPLETION DAY: June 6th , 2023

VI SUPERVISOR : Assoc Prof Nguyen Thi Lan Phi

Ho Chi Minh City, date…

HEAD OF CHEMICAL ENGINEERING DEPARTMENT

ACKNOWLEDGMENT

This thesis is done in Central Experimental Laboratory of Ho Chi Minh City University of Food Industry (HUFI) and Laboratory of Food Processing and Technology B2 of Ho Chi Minh University of Technology (HCMUT) under the instruction of Associate Professor Nguyen Thi Lan Phi

To accomplish this thesis, in addition to my own efforts, I have received a lot of support from my supervisors and friends I would like to express my sincere thanks to everyone who accompanies and helps me

At first, I would like to thank my dear family, I would like to especially express profound love to my parents and brother – who always stand behind and encourage me

Secondly, I would like to express my sincere gratitude to Associate Professor Nguyen Thi Lan Phi and Professor Pham Van Hung Not only has they instructed and given precious advices to me in order to deal with the experimental difficulties and calculations but they also supported favorable conditions as well as chemicals during our thesis

Also, I am very grateful for the helping of MSc Hoang Van Thanh and MSc Truong Thanh An of Central Experimental Laboratory, Msc Mai Nguyen Tram Anh of International University and PhD Tran Thi Tuong An of Laboratory of Food Processing and Technology B2 for supporting the necessary experimental instruments, chemicals and creating comfortable environment as well as giving suitable suggestions during the implement of my thesis

Thank you, all friends who work together in the Central Experimental Laboratory and Laboratory of Food Processing and Technology in the process of implementing the thesis Eventually, I would like to express sincere thanks to the anyone who always motivates and sympathizes with me

ABSTRACT

The bioactive compounds in cashew nut testa has many functional benefits for human health which can be applied in food industry as well as needing to be effectively preserved In the study, the enzyme and ultrasonic - assisted (E-UAE) cashew nut testa extract was emulsified in the 𝑊1/𝑂/𝑊2 system which uses Lecithin as lipophilic and Tween 80, Whey protein Isolate (WPI) as hydrophilic emulsifiers Furthermore, some ratios of components in the system are investigated in order to choose the best ratios as well as investigating the stability of the emulsion in 28 days at room temperature

The cashew nut testa extract has total phenolic content (TPC) is 123.06 ± 0.45 (mg GAE/ g DW sample), total flavonoid content (TFC) is 100.41 ± 0.08 (mg CAT/g DW sample), total catechin content is 22.13 ± 0.41 (mg CAT/ g DW sample) and the antioxidant capacity is 1.08 ± 0.02 (mmol TE/ g DW sample)

The results show the best conditions of the emulsification which include 𝑊1: 𝑂 ratio, Lecithin content, 𝑊2 : 𝑊1/𝑂 ratio, Tween : WPI ratio are 10 : 90 (w/w), 3 % (w/w), 80 : 20 (w/w) and 2 : 3 (w/w), respectively The double nanoemulsion has the encapsulation efficiency (EE) is 91.37 ± 2.48 %, the droplet size is 267.2 ± 2.8 nm, the polydispered index is 0.228 ± 0.007 and the Zeta value is −62.0 ± 0.6

The stability of the double emulsion is also investigated which has encapsulation efficiency is 21.40 ± 1.79 %, droplet size is 257.6 ± 1.4 nm, Polydispered Index is 0.303 ± 0.026 and the Zeta value is −69.4 ± 1.8 after 28 days storage at room temperature

From the results, it can be show the ability of double nanoemulsion systems in the protection of the internal bioactive compounds of cashew nut testa which can be potentially applied in the food industry

TÓM TẮT

Các hợp chất có hoạt tính sinh học trong vỏ hạt điều có nhiều công dụng hữu ích đối với sức khỏe con người, có thể ứng dụng trong công nghiệp thực phẩm cũng như cần được bảo quản hiệu quả Trong nghiên cứu, chiết xuất vỏ lụa hạt điều có sự hỗ trợ của enzyme và sóng siêu âm (E-UAE) được nhũ hóa trong hệ nhũ tương nano kép 𝑊1/𝑂/𝑊2 sử dụng Lecithin làm chất nhũ hóa ưa béo và Tween 80, Whey protein Isolate (WPI) làm chất nhũ hóa ưa nước Ngoài ra, một số tỷ lệ thành phần trong hệ nhũ tương cũng được khảo sát nhằm lựa chọn tỷ lệ tốt nhất cũng như khảo sát độ ổn định của hệ nhũ tương trong 28 ngày ở nhiệt độ phòng

Dịch chiết vỏ lụa hạt điều có hàm lượng phenolic tổng (TPC) là 123,06 ± 0,45 (mg GAE/g chất khô), hàm lượng flavonoid tổng (TFC) là 100,41 ± 0,08 (mg CAT/g chất khô), hàm lượng catechin tổng là 22,13 ± 0,41 (mg CAT/g chất khô) và khả năng chống oxy hóa là 1,08 ± 0,02 (mmol TE/g chất khô)

Kết quả cho thấy tỷ lệ thành phần tốt nhất của quá trình nhũ hóa bao gồm tỷ lệ 𝑊1: 𝑂, hàm lượng Lecithin, tỷ lệ 𝑊2 : 𝑊1/𝑂, tỷ lệ Tween : WPI lần lượt là 10 : 90 (w/w), 3 % (w/w), 80 : 20 (w/w) và 2 : 3 (w/w), tương ứng Nhũ tương nano kép tốt nhất có hiệu suất bao bọc (EE) là 91,37 ± 2,48 %, kích thước giọt là 267,2 ± 2,8 nm, chỉ số polydispered là 0,228 ± 0,007 và giá trị Zeta là -62,0 ± 0,6

Độ ổn định của nhũ tương kép tốt nhất cũng được nghiên cứu với hiệu suất bao bọc là 21,40 ± 1,79 %, kích thước giọt là 257,6 ± 1,4 nm, Chỉ số đa phân tán là 0,303 ± 0,026 và giá trị Zeta là -69,4 ± 1,8 sau 28 ngày bảo quản ở nhiệt độ phòng

Từ kết quả thu được, có thể cho thấy khả năng của hệ nhũ tương nano kép trong việc bảo vệ các hợp chất có hoạt tính sinh học bên trong vỏ hạt điều và cho thấy tiềm năng trong việc ứng dụng trong công nghiệp thực phẩm

COMMITMENT

I hereby declare that this is my own independent scientific work The results in this study do not reproduce or infringe the copyright of any other source The reference to the document has been cited and the source of the reference is recorded in accordance with regulations

Student

2.1.1 Classification and distribution 2

2.1.2 Production of cashew nut in the world and Vietnam 3

2.3.4 Bioactivity potentials of cashew nut testa 11

2.4 Double nanoemulsions and other types 13

2.4.1 Definition 13

2.4.2 Preparation 15

2.4.3 High – energy methods 18

2.4.4 Emulsifiers 19

2.4.5 Droplet size, Polydispersity index and Zeta – potential 22

2.4.6 Practical Studies 23

CHAPTER 3: MATERIAL AND METHODS 25

3.1 Materials and chemicals 25

3.2 Intrustments 26

3.3 Material Preparation 26

3.4 Extraction of Cashew nut Testa 27

3.5 Preparation of Double Nanoemulsion 𝑾𝟏/𝑶/𝑾𝟐 28

3.6 Investigating the components of Double Nanoemulsion 𝑾𝟏/𝑶/𝑾𝟐 30

3.6.1 Investigation of 𝑾𝟏: 𝑶 ratio 30

3.6.2 Investigation of Lecithin content 31

3.6.3 Investigation of 𝑾𝟐 : 𝑾𝟏/𝑶 ratio 31

3.6.4 Investigation of Tween 80 : WPI ratio 31

3.7 Determination of Total Phenolic content (TPC) 31

3.8 Determination of Total Flavonoid content (TFC) 32

3.9 Determination of Total Catechin content (TCC) 32

3.10 Determination of DPPH radical scavenging assay 33

3.11 Determination of Encapsulation Efficiency 33

3.12 Determination of Droplet Size, Polydisperity Index and Zeta Potential 34

3.13 Determination of Stablity of double nanoemulsion 34

3.14 Statistical Analysis 34

CHAPTER 4: RESULTS AND DISCUSSION 35

4.1 Polyphenol compounds content in cashew nut testa 35

4.2 Investigating the components of Double Nanoemulsion 𝑾𝟏/𝑶/𝑾𝟐 36

4.2.1 Investigation of 𝑾𝟏: 𝑶 ratio 36

4.2.2 Investigation of Lecithin content 38

4.2.3 Investigation of 𝑾𝟐 : 𝑾𝟏/𝑶 ratio 40

4.2.4 Investigation of Tween 80 : WPI ratio 41

4.3 Determination of Stablity of double nanoemulsion 42

CHAPTER 5: CONCLUSION AND RECOMMENDATION 44

5.1 Conclusion 44

5.2 Recommendation 45

REFERENCES 46

APPENDIX A: ANALYTICAL METHODS 60

APPENDIX B: ANALYTICAL RESULTS 63

APPENDIX C: SAMPLE PICTURES 69

LIST OF FIGURES

Figure 2.1 Cashew tree and their fruit 2

Figure 2.2 The column chart of global cashewnut yield from 2010 – 2020 3

Figure 2.3 Cashew nut cross – section 4

Figure 2.4 Cashew nut testa 6

Figure 2.5.Comparison between 3 types of emulsions 15

Figure 2.6 Schematic illustration of high and low – energy emulsification methods 17

Figure 2.7 Schematic illustration of cross – flow (left) and pre – mix (right) membrane emulsification methods 17

Figure 3.1 Sample preparation process 26

Figure 3.2 Cashew nut testa enzymatic extraction process 27

Figure 3.3 Cashew nut testa double emulsion preparation process 29

LIST OF TABLES

Table 2.1 Major components in cashew nut 5

Table 2.2 Major chemical components in cashew nut testa 7

Table 3.1 List of experimental intrustments 25

Table 4.1 Chemical parameters of Cashew nut testa 35

Table 4.2 Encapsulation efficiency and physical parameters of double nanoemulsions at different 𝑊1: 𝑂 Ratios 37

Table 4.3 Encapsulation efficiency and physical parameters of double nanoemulsions at different Lecithin concentrations 38

Table 4.4 Encapsulation efficiency and physical parameters of double nanoemulsions at different 𝑊2 : 𝑊1/𝑂 ratios 40

Table 4.5 Encapsulation efficiency and physical parameters of double nanoemulsions at different Tween 80 : WPI ratios 41

Table 4.6 Effect of storage time on the EE and physical parameters of double nanoemulsion at room temperature 43

DW Dry weight

TPC Total phenolic content TFC Total flavonoid content TCC Total catechin content EE Encapsulation efficiency PDI Polydispersity Index

PGPR Polyglycerol polyricinoleate GAE Gallic acid equivalents CAT Catechin equivalents TE Trolox equivalents

DPPH 2,2-diphenyl-1-picrylhydrazyl SET Single electron transfer

Z – value Zeta potential

HR Conventional heat flux method DLS Dynamic light scattering UV–Vis Ultra Violet-Visible

CHAPTER 1: INTRODUCTION

The cashew nut (Anacardium occidentale) is one the most popular exporting agricultural commodities in Vietnam There are many by-products from the cashew nut processing such as outer hard shell, cashew apple or the cashew nut testa

The cashew nut testa is a rich source of bioactive compounds such as catechin and epicatechin which is the primary flavonoids [1] These bioactive compounds have potential applications in food or pharmaceutical industry because of their antioxidant, antibacterial or anticancer activities However, the testa is only usually used for livestock feeding, leather production or biomass So that, the potential of bioactive compounds in the testa are not fully ultilized

Emulsification is the method of combining two liquids to create a colloidal suspension, in which the components of one liquid are dispersed but not dissolved in the other The method has many potential in encapsulation of hydrophilic compounds and protected, including volatile compounds, vitamins, minerals, amino acids and polyphenols to enhance the sensory qualities of specific goods and cover the odor of particular molecules [2] This method is widely applied in many product in food industry such as including milk, cream, coffee creamer, soft drinks, nutritional beverages, sauces, dips, deserts, dressings, mayonnaise, ice cream, margarine, and butter [3]

The bioactive compounds are easily impact by the external environment such as ultraviolet radiation, temperature, climate which have many influences in the amount of bioactive compounds [4] So that the emulsification can be a proper method for protection of these compounds from external environment

In this study, the cashew nut testa was extracted by the enzyme and ultrasonic – assisted extraction (E – UAE) then encapsulated the extract by the emulsification The study aims are investigated the best ratios of components for the highest encapsulation of bioactive compounds in the extract as well as investigating the stability of the double nanoemulsion

CHAPTER 2: LITERATURE REVIEW

2.1 Anacardium genus

2.1.1 Classification and distribution

Anacardium is a genus which comes from Anacardiaceae family as well as cashew

family or sunac family There are about 83 gerena and 860 species have been found around

the world [5] in this family The most well – known species used in food industry is

Anacardium occidentale or cashew nut

Anacardium genus has origin from tropical regions in South America like Southern Honduras to Panama, Brazil, and Eastern Paraguay while Anacardium occidentale may come

from the tropical savanna area in Central Brazil [6] Nowaday, cashew nut tree is mainly planted in India, Vietnam, Côte d'Ivoire, Guinea-Bissau, Tanzania, Benin, Brazil and other countries in East and West Central Africa and South East Asia

In Vietnam, the cashew tree is mainly cultivated in Binh Phuoc, Dong Nai, Daklak, Gia Lai province In Binh Phuoc, because of the suitably tropical climate as well as red basaltic and acrisols soil on ancient alluvium so this province is the most cultivated in cashew tree which has planted area is about 170.000 ha in 2020 In details, Bu Dang, Bu Gia Map, Phu Rieng and Dong Phu districts is 4 area where the tree mainly plant in Binh Phuoc province Dong Nai province is the second most fertilized area for planting cashew tree which area is 36.000 ha

Figure 2.1 Cashew tree and their fruit

2.1.2 Production of cashew nut in the world and Vietnam

The global yield of cashew nut product has been fluctuated through the time As in Figure 2, From 2010 – 2020, the yield is range from 3.18 – 4.41 million tons which highest yield is in 2015 and the lowest is in the next year In 2020, the production value is 4.18 million tons which is increased in comparison of the last year

Figure 2.2 The column chart of global cashewnut yield from 2010 – 2020

The main exporters of cashew nut is Ivory Coast, India, Vietnam, Tanzania,Nigeria Ghana, Burundi, Benin, Mozambique, Madagascar,… According to FAO, in 2020, the production yield of cashew nut in Viet Nam is 348.504 tons, India is 772.779 tons and the largest exporter is Ivory Coast which yield is 848.700 ton

In Vietnam, the cashew is mainly cultivated in Southeast and Central Highlands region which consist of Binh Phuoc, Dong Nai, Daklak, Gia Lai According to FAO, in the period 2010 – 2020, the cashew yield is fluctuated from 215.765 – 352.029 tons which reaches the highest in 2015 and drops significantly in 2017 The export turnover of cashew nut contributes to Vietnam economy over 3 billion USD in 2020 which main imported market is America, Netherland, Australia, England, Canada

2.2 Cashew nut

2.2.1 Description

Anacardium occidentale tree has height from 10 – 15 m with short, unbalanced shape

grown on tropical or subtropical regions The leaves of the cashew tree has leather color, ellipse or obovate shape and the flower is small, greenish color at first then becomes crimson when mature

The cashew fruit has yellow, red or yellowish - red colour when maturity which length is about 5 – 11 cm It is also juicy, spongy, fibrous and has astringent taste At the end of the fruit has a kidney – shaped nut which kernel covered by a double shell consisted of inner thin layer (testa or husk) and outside thick layer (nut shell)

Figure 2.3 Cashew nut cross – section [7]

The cashew fruit and nut has independent growth which the former become maximize the weight after 50 days and the latter is 30 days Moever, the fruit growth faster after the nut reaching maximum size and the nut shell also become dehiscent, harden in this time which make the nut weight reduce about 10 – 40% [8]

The harvesting season of cashew nut depends on geographic location of cultivated countries Generally, the harvesting period of cashew nut ranges from February to June or July The riped fruits drop from the trees are collected by farmers Then, the nuts separated from the fruits and dried by sun heating in 2 – 3 days in order to reduce the moisture content Finally, they are stored in suitable conditions for selling to merchants The remain fruits are usually ultilized for fertilizer and cattle feeding but sometimes the Indian used for fermentation to make cashew alcohol (Feni)

2.2.2 Chemical composition

The cashew nut is a rich source of fat which includes mono - and polyunsaturated fatty acids occupy about 75% total lipids Moreover, the main fatty acid in the nut is oleic acid which is dominant in the former and linoleic acid in the latter [9] The carbohydrate content in the nut is also high which mainly composed of starch, sugar and fiber Furthermore, the main sugar in the nut is sucrose which occupied over 98% in total sugar content, the remains are glucose and fructose according to USDA The other pretty high amount composition is protein which the most abundant amino acid is glutamic acid (about 4.6 g/100 g), the following are Arginine (2.2 g/100 g) and Aspartic acid (1.9 g/100 g) [10]

Table 2.1 Major components in cashew nut [9]

Furthermore, the cashew nut shell can be extracted to obtain the oil or liquid which main compounds are anacardic acid which has many bioactive activities such as such as antitumor, antioxidant, gastro-protective, and antibiotic Moreover, the heating product of anarcadic acid create cardanol (60 – 65%) and cardol (15 – 20%) which are main components in the oil [12] However, these compounds are also allergic factor which cause mouth – burning, pruritic, reddish - brownish rash on skin prolong about 4 – 5 weeks to the consumers [13]

2.3 Cashew nut testa

2.3.1 Chemical compositions

The cashew nut testa or husk is the reddish - brown inner thin layer of the kernel which occupies 1 – 3% total weight of the whole nut [1] Usually, they are used for animal feed, tanning industry, animal feed, fuel burning, fertilizer, compost making,… because of the remarkable protein and fat content

In the dry matter of cashew nut testa composed of 190 g/kg crude protein, 103 g/kg crube fiber, 20.1 g fat and 20.2 g/kg ash [14] which are the high energy source for farming animal feeding Moreover, the testa has the acid detergent fiber and neutral detergent fiber contents which are 14.91 % and 40.23 %, respectively as well as the remarkable hydrolysable tannins content is about 22.03 % [15]

Figure 2.4 Cashew nut testa

Table 2.2 Major chemical components in cashew nut testa [14]

2.3.2 Phenolic compounds in cashew nut testa

There are many kind of phenolic compounds in the cashew testa but the most notable group is Flavan-3-ols (the subclass flavanols) which is found in many foodstuff under many

structural types such as monomer (catechin and epicatechin), oligomers, polymers (proanthocyanidins), and other derivative compounds (such as theaflavins and thearubigins) [16] The structure of the group is C15 (C6-C3-C6) structure which consist of benzopyran sub-group (A and C rings) combined with aromatic ring (B ring) linked to carbon C-2 of C ring

In the study of (Chandrasekara and Shahidi, 2011) [11], the raw defatted cashew nut testa samples were investigated for analysing total phenolic compounds (TPC) which resulted in soluble phenolics and bound phenolics are 269.05 ± 9.77 GAE mg/g and 1.36 ± 0.1 GAE mg/g respectively Moreover, the phenolic profile of the testa consisted of catechin, epicatechin,

epigallocatechin, syringic acid, p – coumaric acid, gallic acid which catechin was the most dominant compound

According to the study of (Trox et al.,2011) [17], the dry matter of cashew nut testa sample were analysed by using RP – HPLC, LC – MS methods which results revealed the significant

amount of catechin and epicatechin in the testa are 5.7 and 4.46 g/kg dry matter, respectively

The other study of (Matthew and Parpia, 1970) [1] showed that catechin and epicatechin content in the testa were 6 % and 7.5 % in dry samples correspondingly and contributed over 40 % total polyphenols Furthermore, the polymer proanthocyadinidins occupied less than 40 % and the monomeric proanthocyadinins structure consists of 2 leucocyanidins and 2 leucopelargonidin Leucocyadinin was the majority leucoanthocyadinin

In the cashew nut testa, the tannin content ranges from about 24 % to 26 % [18] In the other view, the tannin material content is 25 %, non-tannin material content is 11 % which has some similar effects in compare of the wattle bark using in leather industry [19]

The tannins content determined by (Ukoha et al.,2010) [20] used three methods: Shakes, Tannic acid and Folin – Ciocalteau which results were 19.87 %, 22.1 % and 21.01 %, respectively Additionally, the tannins in the testa were condensed tannins which components predominantly was catechin, the following was epicatechin and the others are Quercetin, Myricetin, Azaleatin, Cyadinin and Delphinidin

2.3.3 Extraction methods

The phenolic compounds in the cashew nut testa are extracted by many methods which are investigated by many studies such as solvent extraction, enzyme extraction, ultrasonic – assist extraction (UAE) or the combination of them

There are some disadvantages in catechin extraction Firstly, the extraction is taken place in the tissue of the plant bound with sugars, proteins or lead to polymerization between inside components Thus, these newly formed derivative have many degree of dissolubility, different chemical structures as well as their interaction with other components which is inadequately investigated Secondly, catechin is easily sensitive compound under oxidation, light, high temperature and alkaline environment and it is challenging in finding out suitable extraction

methods [21] For details, the suitable pH for catechin is lower than 4 but it is become creasively unstable when pH is increased to alkaline environment The interaction between catechin with protein compounds is also causing the precipitation inside the solution which is called “cream’’ formation [22]

• Ultrasonic – assist extraction (UAE)

The ultrasonic – assist extraction method is used in order to reduce the impact of heating effect to the samples instead of using thermal extraction as well as higher yield than conventional extraction The method takes advantage of the acoustic energy which is not absorb by the molecules and is transmitted through the solvent to the target compound in low temperature The cycles of compression and rarefaction affect the molecule of solvents which finally creates the bubbles or cavities when the ultrasound intensity is in high level The cycle of bubble which are forming, growing and imploding which release high temperature and pressure creates the shear force to break the cell wall for extraction the target compounds

In the study of (Kumar and Lokeswari, 2014) [23], by using UAE method with cashew nut testa in the frequency 20 kHz and analysis the tannin content by spectrophotometric at 650

nm wavelength which result was about 6.66 mg/g sample

By using the combination of Solvent and UAE method in Pistachio nuts skin and nut samples at 25 ℃ and the frequency 40 kHz, (Tomaino et al, 2010) [24] determined the total flavonoid content which were 70.27 ± 5.42 mg Catechin/g fresh weight (f.w) for the former and 0.46 ± 0.03 mg Catechin/g f.w for the latter In detail, the Catechin content in the skin was remarkablely higher than nut which were 377.45 ± 24.36 µg/g f.w and 2.41 ± 0.18 µg/g f.w while Epicatechin was only found in skin with the content was 104.8 ± 10.56 µg/g f.w

The walnut, almond, and pine nut shells was extracted by the combination of Water – Ethanol and Ultrasonic at 50 ℃ and 60 minutes in the study of (Queirós et al, 2019) [25] which results in the flavonoid content were 197.6, 99.4, and 114.6 mg Catechin/g extract , respectively Morever, the condensed tannin content in walnut, almond and pine nut samples are 60 mg, 34.6 and 30.4 mg Catechin/g extract, respectively

Another 11 Spanish almond samples were extracted by sonication in 10 minutes at 4 ℃

conducted by (Gracia et al, 2021) [26] shows that the catechin content ranged from 8.21 ± 1.39 mg/ 100 g fresh weight (f.w) to 16.58 ± 3.22 mg/ 100 g f.w and the epicatechin content was 5.25 ± 0.51 to 13.81 ± 1.04 mg/ 100 g f.w

A comparative study conduct by (Das and Eun, 2018) [27] illustrated that the ultrasonication extraction in the condition 35 kHz was more effective than the conventional extraction in green tea samples In detail, the Catechin content in the UAE samples were averagely higher than the conventional extraction samples 2 folds in all investigated conditions which temperatures were 60, 70, 80 °C and the time range from 5 to 30 minutes

• Enzyme – assisted extraction (EAE)

Enzyme-assisted extraction is a method of extraction which using the enzyme to disrupt the cell wall for the solvent go into the cell for target compounds extraction It is an environmental - friendly alternatives for the conventional methods which can cause many environmental concerns For example, in solvent extraction, hexane is usually used but it is harmful for environment The most commonly used enzymes for the extraction are cellulases, hemicellulases and pectinases which can be achieved from animal or fruit sources

The EAE (Pepsin) showed the recovery of catechin from milk tea samples more effective than solvent extraction (MeOH, HCl) in the study of (Ferruzzi and Green, 2006) [28] which the enzymatic treatment recovery rate were 89–102% while the other two are 78–87% and 20–74% total catechin, respectively

In the study of (Maier et al, 2008) [29], the extraction of grape pomance skin samples by using pectinolytic and cellulolytic enzyme at ratio 2:1, T = 40 ℃ and pH = 4.0 which were optimal environment The results illustrated the phenolic, non-anthocyanin flavonoids and anthocyanins yield were remarkable increased which were 91.9, 92.4, and 63.6%, sequentially

The using of enzyme tannase in green tea extraction also help remarkably increased the total phenolic content which was conducted in the study of (Shao et al, 2020) [30] More detail,

in the environment at T = 70 ℃ in 40 minutes enzymatic extraction, the phenolic content in samples were improved from 137 g/kg to 291 g/kg while the Gallic acid, Epicatechin Gallate and Epicatechin content were also significantly risen

A comparative study of many types of enzymes such as Celluclast, Cytolase, Econase, Pectinex, Rapidase, Ultraflo, Viscozyme, Tannase used in phenolic extraction of green tea (Hong et al, 2013) [31] show the phenolic content and flavonoid content were higher than the non – enzymatic samples 4 – 15% and 12 – 33 % respectively Moreover, the Visozyme samples had the highest content among investigated enzymes in both tests

In the (Olawuyi et al, 2021) [32] study, the plum juice samples are treated by ultrasonic, enzymatic and combined extraction The results showed that the enzymatic treatment had higher phenolic content and flavonoid content than conventional and ultrasonic methods Furthermore, the combined methods in 15 minutes also showed the highest phenolic content and flavonoid content than others investigated methods

(Balasubramaniam et al, 2019) [33] implemented a comparative study in the extraction of finger millet by ultrasonic (UAE), enzyme – assisted ultrasonic (UEA) and conventional heat flux methods (HR) The results showed the using of xylanase enzyme in UEA significantly increased total phenolic content 2.3 fold than HR method Morever, the total flavonoid content and tannin content was also improved which was 1.3 and 1.2 fold higher than HR method, sequentially

In our study, the combination of enzyme and ultrasonic was applied in the extraction of cashew nut testa in order to maximally exploit the phenolic compounds in the sample

2.3.4 Bioactivity potentials of cashew nut testa

The cashew nut testa has many functional properties because of the remarkable phenolic

profile which mainly contains catechin flavonoid The flavonoid is considered having

antioxidant, antibacterial activity as well as anti – inflammation effect

In antioxidant activity, the raw cashew nut testa has the remarkably higher activity in

comparison with the interior kernel or the whole nut which illustrates in all 3 antioxidant methods (DPPH, ORAC and OH radical scavenging) In detail, the insoluble and soluble

antioxidant activity of defatted raw cashew nut testa by DPPH, ORAC and OH radical

scavenging methods ranged from about 33.07 ± 1.65 – 708.49 ± 6.32 GAE mg/g of defatted

meal, 0.046 ± 0.002 – 74088 ± 2956 TE µg/g of defatted meal, 684.24 ± 13.65 – 1091.52 ± 71.7 CE µg/g of defatted meal, respectively [11] Another study illustrated the ability of antioxidant of ethanolic – extracted cashew nut testa by using 5 different antioxidant assay which were ABTS radical scavenging, superoxide scavenging assay, deoxyribose oxidation assay, lipid peroxidation, iron chalation assay The results showed that the extract had antioxidant activity in all methods which 𝐸𝐶50 value are 1.30 ± 0.02 µg/ml in ABTS assay, 10.69 ± 1.13 µg/ml in superoxide scavenging assay, 17.70 ± 0.05 µg/ml in deoxyribose oxidation assay, 24.66 ± 0.32 µg/ml in lipid peroxidation assay and 6.00 mg/ml in iron chelation assay [34] In the analysis of three cashew product samples, the skin – on sample had significantly higher antioxidant activity than raw and dry – roasted samples which about 25 times higher in DPPH assay or about 6 – 7 folds higher in ORAC assay These results demonstrated the presence of skin on the samples has great impact on the antioxidant activity [18] From aforementioned studies, the cashew nut testa has been proved to be a promising

natural antioxidant source which can be applied in food industry

The study about antibacterial activity of the cashew nut testa aqueous extract was

conducted by disc diffusion assay which results show that it could effectively inhitbit

Escherichia coli and Staphylcoccus aureus in the zone of 6 mm and 12 mm diameters,

respectively [35] Another study showed that the free phenolic fraction extracted from cashew nut testa contained the highest total phenolic and flavonoid content in comparison with esterified and bound fractions So that, the antibacterial ability of the free fraction was also

highest which can inhitbit pathogen such as Staphylcoccus aureus, Escherichia coli, Micrococcus luteus, Bacillus cereus at the concentration below 3 mg/ml which reach the

maximum zone of inhibition in disc diffusion assay [36] Therefore, the testa is a promising source of natural antibacterial agent which can improve the consumers health as well as applied in some industries such as pharmaceutical, therapeutic as well as food manufacture

The anticancer ability of the cashew nut testa is also investigated in order to fully exploit

the potential of the by – product of cashew The aqueous extract of the testa was demonstrated

can inhitbit some cancerous cells such as HepG2, HEK293 which viability were reduced to 52 % and 47 %, sequentially [35] Although these studies are still limited, the catechin flavonoid was proved to be a anticancer source in many studies The flavonoid was investigated the inhibition of many cancerous cells such as lung, breast, ovarian, gastric, colon,… and has been shown many positive effect in the viability reduction as well as inhibition of metastasis [37] For example, the catechin flavonoid at the concentration of 600 µmol/L had positive effect against the profileration of non – small cell lung cancer A549 which reached 19.76% inhition rate by increasing the expression of p21, p27 as well as inhibiting cyclin E1, phosphorylation of protein kinase [38] Hence, the cashew nut testa which is the rich source of catechin flavonoid is hopefully the potential agent in the functionally treatment and effectively inhibition of the cancers

From these functional properties mentioned above, the cashew nut testa is promised to be the excellent natural bioactive source which hopefully has effective impact on human health as well as applying in functional food production

2.4 Double nanoemulsions and other types

2.4.1 Definition

When mixing two non – miscible liquids with the assistance of mechanical shearing and

emulsifiers, the homogeneous mixture obtained after the process is called Emulsion (Macroemulsion) which the droplet sizes range from 1 – 100 μm However, because of the effect of the force of gravity, the particle size of emulsion mixture is increased through time which lead to the separation, so the normal emulsions are not thermodynamically unstable system, easily affect by creaming, cracking (breaking), flocculation and phase inversion phenomenon [39] Generally, there are two main emulsion systems: water – in – oil (w/o) and oil – in – water (o/w)

The double emulsion is the more complex system than the normal emulsion which formation is by homogenizating or ultrasonificating the primary emulsion w/o or o/w with

primary emulsifiers then blending with secondary emulsifiers The final products are the water-in-oil-in-water (w/o/w) or oil-in-water-in-oil (o/w/o) double emulsion which is the

former is more common type Beside many similar thermodynamic destabilization mechanisms of single emulsion like gravitational separation, flocculation, coalescence and Ostwald ripening process, double emulsion has other factors which result in instability which occur by interior water phase For detail, the inner and outer water phase diffuse together or the former is ejected through the oil phase which lead to the unstable state of double emulsion [3]

Double emulsion increase the protection of bioactive compounds from exterior environment such as temperature, pH changing, chemical and biochemical intereaction by isolation in the inner water phase as well as biocompatible, biodegradable characteristics [3, 40] Moreover, the using of both hydrophilic and lipophilic phases can provide good encapsulation efficiency of hydrophilic and lipophilic compounds [41] Because of good potential bioactive compounds protection and delivery, it has many applications in many fields especially food industry and pharmaceutical [42, 43]

Microemulsion is other type of developed conventional emulsion which is isotropic,

transparently homogeneous, thermodynamic stable system and has the droplet size range from 10 – 100 nm [44, 45] Microemulsion has been applied especially pharmaceutical industry due to higher absorption rate, enhancing bioavailability, improving stability of drug compounds, solubilization of both hydrophobic and hydrophilic drugs as well as cost – effective method [46] The method also faces some limitation such as requiring higher emulsifiers concentration, easily impact by temperature and salinity [46]

By the development of technology, the conventional emulsion size is become smaller which has positive impact in the bioactiave compounds protection and transportation One of new-

developed emulsions is Nanoemulsion which spherical size range from 20 to 500 nm [47] in

compare with 0.1 to 100 um of normal emulsion [48] Moreover, the color of the nanoemulsion can be changed from transparent (50 – 200 nm) to milky (up to 500 nm) which depend on the size of the droplets [49] The nanoemulsion has been shown many potential industrial applications because of many advantages such as external environmental protection, kintetically stable, decreasing particle size, moderately using surfactant and simple manufacture [50] Additionally, the small spherical size is useful in the retention and delivery of drug compounds

through membrane, endothelium or circulatory system inside body as well as prevent the rapid excretion of these compounds [51] Besides these benefits, the nanoemulsion still faces many unstable factors such as gravitational separation, flocculation, coalescence but less serious than the normal emulsion because of the smaller droplet size and the nonionic surfactants [52] so that nanoemulsion is seemingly thermodynamically stable than normal emulsion [53] However, the main factor lead the unstability of nanoemulsion is the Ostwald ripening phenomenon caused by the diffusion of the small solubilized oil droplets over time which can be solved by using very low water – solubility oil such as soy, sunflower, fish oil or non – polar inhibitors [54]

Double nanoemulsion is a remarkable novelty which has the characteristics of both double

emulsion and nanoemulsion The w/o/w double nanoemulsion is the covering of the inner aquaeous phase by large - sized oil droplets then dispersed in the external water phase with the support of surfactants or vice – versa in the o/w/o emulsion The nano-sized in double nanoemulsion can ameliorate the thermodynamically instability which are frequently met in normal double systems [55] The double nanoemulsion has many potential applications in many industrial fields such as pharmaceutials, foods and cosmetics [56]

Figure 2.5 Comparison between 3 types of emulsions [47]

2.4.2 Preparation

The preparation of double emulsion such as W/O/W system is the combination of the

intensively mixing primary W/O phase contains hydrophobic surfactant with the external phase mixed with hydrophilic surfactant by moderate shear force in order to avoid the broken of internal droplets [57] Generally, double emulsion is prepared by using conventional

apparatus to create shear stress, turbulence, cativation effect such as rotor – stator system and high – pressure homogenizer [58] The main advantages of traditional methods are flexibility, suitable for both lab or high – volume production as well as pretty low price [59] However, these methods face some drawbacks such as uncontrollable droplet size, high – energy consumption [58]

In order to decrease the emulsion to nano – scale particles, the low – energy methods and high – energy methods are applied The former group are consisted of emulsion inversion

point (EIP), phase inversion temperature (PIT), spontaneous emulsification and the latter group are rotor – stator mixing, high – pressure homogenization (HPH), microfluidizer as well as ultrasonication [47, 54, 60]

Although the low – energy methods has some advantages such as energy – efficiency,

suitable for encapsulation thermosensitive compounds, simple implement, the methods still face many drawbacks like easy coalescence while storing, using high amount of surfactant which maybe has negative effect in the sensory value or cost of production as well as limitation

using in some types of oils and surfactants [61, 62, 63, 64] The high – energy methods are

also less complex preparation processes as well as formation of fine emulsion from wide range of materials as well as capable of large – scale production [63, 65]

Beside these aforemetioned methods, the membrane emulsification is pretty newly

developed methods which the coarse pre – mix permeate through the membrane (pre – mix method) or the dispersed phase passed through and the continuous cross – flow the membrane (cross – flow method) [66] These methods use lower energy – consumption in comparision with high – energy methods but require higher energy than low – energy methods such as phase inversion temperature [60] The membrane techniques has shown many potentials such as easily controllable droplet size through the pore size of the membrane , lower shear stress and less complex operation conditions than high – energy methods and ability of large – scale manufacture [67, 68] Nevertheless, the methods also have some drawbacks such as low - rate emulsification, low flow rate when the viscosity of dispersed phase is high which make the process become time – consuming as well as the fouling phenomenon [69, 70] Additionally,

Shirasu-porous-glass (SPG) membrane widely used in the emulsification is pretty costly, easily broken and lengthy purification [71].

Figure 2.6 Schematic illustration of high and low – energy emulsification methods [64]

Figure 2.7 Schematic illustration of cross – flow (left) and pre – mix (right) membrane

emulsification methods [66]

2.4.3 High – energy methods

The commonly applied high – energy methods are rotor – stator systems, high pressure homogenization (HPH), microfluidization and ultrasonication

Rotor – stator system is the basic high – energy approach in the formation of emulsion

The system create the hydrodynamic stress in the form of velocity gradients and intensive turbulence to break up the coarse emulsion droplets [60] The method is oftenly combined with other high – energy methods in order to create the complete nanoemulsion because the single method often create the large particles size as well as wide range distribution of particle size [72] Regarding dispersed phase volume fractions and intermediate-to-high viscosities, it is often regarded as a standard approach for emulsion production [73]

High pressure homogenization is the method uses high pressure to form the emulsion

particles at nano – size which applied for low to intermediate viscosity fluids [74] In the method, coarse emulsion is pushed through the tiny aperture with high pressure plus repeatedly homogenization cycles which create the vigorous turbulence and highly localized hydraulic shear to maximally minimize the droplet size [39] In order to have smaller droplet size for the industrial demands, the emulsion is sometime homogenized by extremely high pressure as well as repeatedly homogenizing multiple times because of the linear relationship between size and magnitude of pressure [63] The drawback of the method is low thermal efficiency which lead to high energy losses during the process as well as increasing the temperature of product [74]

Microfluidization, like the HPH method, when a coarse emulsion is forced through a

small hole by high pressure to disrupt the droplet However, in the microfluidizer, the coarse emulsion is split in two and the two streams are then forced to collide in the interaction chamber The two swiftly moving streams of the input emulsion clash inside the interaction chamber, creating powerful disruptive forces that effectively break the droplets in order to form fine emulsion [63] The droplet size of emulsion is flexibly control in this method which can improve the stability of the product as well as the wide range of materials can be processed [75]

Ultrasonication uses the ultrasonic sound waves from the probe which create the

cativation effect to break up the droplets of coarse emulsion In the process, the sound waves from the probe create the sinusoidal pressure in fluid which destroy the bubbles formed from the cavitation by the cycles of contraction and expansion Finally, the explosion of these bubbles releases high – shear stress which disrupts the coarse droplets to become fine droplets [60] The method has shown many advantages such as requiring low surfactant concentration, stable dispersion, prevention of microbial contanmination Additionally, the method is easy to clean and industrial scale – up as well as low energy consumption in comparison with microfluidization [76] However, there is a risk of metallic pollution during processing because of the cativation which cause the ionic metal in the probe releases into the emulsion mixture as well as less suitable for thermo – sensitive compounds [73, 76]

Because each method has their own advantages and drawbacks In order to, improve the

former as well as ameliorating the latter, several studies about combination of these methods

has been conducted recently For example, the combination of HPH and ultrasonication shows that the energy – consumption and required homogenization pressure are decreased which can lead to reduce the industrial expenses [76] Another study illustrated that ultrasonication combines with rotor – stator system create the smaller droplet size, lower surface tension, higher encapsulation efficiency than exclusive implementation of latter which result in better stability [77] Additionally, the combination also reduced the processing time which over 3 times shorter than only applies rotor – stator system, increases kinetic stability as well as more homogenous droplet size distribution [78] These improvements are potential in industrial fabrication which can effectively cut down the production cost and processing time as well as increasing the shelf – life of the final products

In this study, the integration of rotor – stator and ultrasonication will be applied in order to create the double nanoemulsion which can effectively encapsulate interior bioactive compounds from cashew nut testa extract

2.4.4 Emulsifiers

Emulsifiers are surface-active molecules that are added to emulsions to increase the

stability of emulsion by adhering on the surface of the droplets which help reduce the surface

tension as well as facilitate the breakage of droplets during homogenization Therefore, emulsifiers can prevent the coalescence of emulsion droplets with others which improve the stability of the products [63] There are many types of emulsifiers use in fabricating nanoemulsion droplets which consisted of small molecule surfactants, proteins and hydrocolloids [72]

Small molecule surfactants are considered highly effective in fabrication of

nanoemulsion by both low – energy and high – energy methods Commonly, small molecule surfactants are the most popular emulsifiers used in industrial manufacturing which divided into 3 smaller categories based on electrical characteristics: ionic, nonionic and zwitterionic [72] The molecular weight of this kind of emulsifiers varies about from 250 g/mol to 1200 g/mol and usually simultaneously contains hydrophilic head and hydrophobic tail in the main structure [73]

• Ionic surfactant has positive charge or negative charge on their hydrophilic groups

The surfactant can form nanoemulsion by both low and high – energy approaches However, because of irritative effect when consumes high concentration, they are limited in large – scale processing which requires high level of surfactant [54] Some examples of these surfactants are lauric alginate, sodium lauryl sulfate, benzalkonium

chloride, centrimonium bromide,…

• Non – ionic surfactant not have electrical charge on the hydrophilic groups The

surfactant is considered to be less harmful for consumers, fast decomposition in natural and eco – friendly production such as sucrose fatty esters emulsifiers [63] Another popularly used non – ionic emulsifiers in manufacture are Tween groups which can prevent nanoemulsion from aggregation in various pH value and ionic strength value but less stable from coalescence at the temperature close to PIT point [63] Some examples include Tween groups, Span groups, lactic acid esters of mono-and

diglycerides groups, Mono and diglycerides groups,…

• Zwitterionic surfactant has both positive and negative electric charge group on the

same molecule which the final charge is depended on the pH level of medium [54] Phospholipids group is the most common representative example for this surfactant

which are naturally producted from animals, plants, microorganisms Zwitterionic surfactant has shown many potentials in the nanoemulsion formation such as small droplets, more stable than other surfactant such as Tween 20, sucrose monopalmitate [73] Examples are Lecithin, Lauryldimethylamine oxide, Myristamine oxide,…

Protein surfactants are amphiphilic molecules which orginated from animals, plants or

synthesized by micrioorganisms These surfactants have ability to adsorp on the nanoparticles surface and create viscoelastic layer in order to protect the droplets from destabilization such as coalescence phenomenon [73] The protein surfactants like plant – based protein has some advantages such as environmentally friendly properties, wide range of diversity in nature, cost – efficiency Additionally, the nanoemulsion fabricated by these surfactants has high encapsulation efficiency, excellent protection from external environmental factors such as centrifugal force, pH value, storage as well as oxidant factors [79] Beside these benefits, protein surfactants is unsuitable in the formation of nanoemulsion by low – energy methods, the aggregation of droplets at the pH near isoelectric point as well as flocculation when increasing temperature because of the denaturation of proteins structure [54, 73, 80] Some notable examples include Whey protein isolate (WPI), Whey protein concentrate (WPC), Soy protein isolate, Casein,…

Hydrocolloid surfactants are the group of polysaccharide compounds such as Gum

arabic, modified starch, modified cellulose, pectin which have both polar and non – polar on the structure While surface activity at pectin and gum arabic is related to proteins that are coupled to hydrophilic polysaccharides via covalent bonds, surface activity at modified starch and modified cellulose is associated with the nonpolarity of the chemical groups attached to hydrophilic polysaccharides [80] These polysaccharide surfactants stabilize the nanoemulsion droplets by the formation of thick hydrophilic interfacial films which have steric repulsion effect Additionally, the layers are shown to be more sustainable at wide range of pH level as well as thicker than the layers create by protein surfactants which tend to aggregate at pH level near isoelectric point However, they are often ineffective in formation of small droplets as well as requiring high level in using because of low surface activity [63] Other notable examples are corn fiber gum and soy soluble polysaccharide

Each type of emulsifier have their own pros and cons so that many studies try to

investigate the combination of these emulsifier in order to fabricate optimal nanoparticles

For example, the beta - carotene nanoemulsion used mixture of WPI and Tween 20 at ratio 1:1 as emulsifier had higher stability than used only Tween 20 Additionally, the mixture also helped the particle size became smaller than only using WPI [81] Another study showed that the combination of WPI and Tween 80 at different ratios had positive impact on the stability of Phosphatidylserine Emulsion after 28 days by the formation of viscoelastic interface by WPI and enhancing steric stabilization by the presence of Tween 80 [82] In the formation of Mexican oregano essential oil double emulsion by using the combination of Lecithin in the primary emulsion as well as mixture of WPC and Tween 80 in the secondary emulsion, the emulsion was stable after 25 days storage Moreover, the combination could fabricate the smaller droplet size than only using WPC in the secondary emulsion [83]

In this study, Lecithin was used as stabilizer in the primary emulsion and the combination of WPI and Tween 80 was applied in the second emulsion in order to fabricate stable double nanoemulsion which could effectively encapsulate the bioactive compounds in the cashew nut testa extract

2.4.5 Droplet size, Polydispersity index and Zeta – potential

Droplet size is the one of the most important factor which contribute to the stability of

the emulsion as well as evaluating the impact of the emulsifying factors such as the method or the emulsifier concentration The smaller size of the droplets, the more effective they can resist the destabilized factors which usually lead to separation such as gravitational separation, coalescence, flocculation, and Ostwald ripening Additionally, due to the small droplets size, Brownian motion effects may counteract gravity forces, which can also prevent droplet motion [61]

Polydisperity index demonstrates the distribution of particle size in the emulsion mixture

The parameter is the ratio of standard deviation and mean droplet size The low PDI indicates that the droplet size in the mixture is highly uniform [48] The value of PDI range from 0 (shows the droplet size in the emulsion sample is completely uniformly distributed which called monodispered) to 1 (indicates that there are many different droplet size in emulsion

sample which called polydispered) [84] Generally, the PDI value which can be determined by dynamic light scattering method (DLS) below 0.05 is deemed to be monodispered distribution and above 0.7 is polydispersed [85] When the PDI value is remained unchanged through storage time and external conditions, the emulsion is considered to be stable

Another important value used to evaluate the stability of emulsion sample is Zeta potential which illustrates the electrical charge of the droplets at the shear plane and

electrostatic repulsion between particles The value is considered to be the more representative parameter in compare with surface charge density or surface potential because of easier in measuring as well as inherently explanation of the adsorption of any charged counter – ion [3] The nanoparticles is considered to be stable when the Zeta value is higher than + 25 mV or lower than – 25 mV which can be varied depend on droplet size, concentration, pH of the medium or temperature [85]

2.4.6 Practical Studies

There are many studies about the encapsulation of phenolic compounds by the emulsion in order to effectively protect them from external factors as well as applying in the food industry or pharmaceutical

In the study of (Aditya et al, 2014) [86], catechin and curcumin were simultaneously encapsulated into the W/O/W double emulsion which was used polyglycerol polyricinoleate (PGPR) as lypophilic and gelatin as hydrophilic surfactants Additionally, the emulsion was prepared by probe sonication method and the ratio between W/O phase and outer aqueous phase was 25:75 The results show single – nutraceutical and co - loaded emulsions had high encapsulation efficiency which ranged from 88 – 97 % Moreover, the emulsion was significantly stable in the digestive environment which bioaccessibility of the co – loaded emulsions was 4 times higher than freely mixing these compounds as solution

Another study of (Snoussi et al, 2020) [2] about the encapsulation of catechin in double emulsion by the combination of rotor – stator and ultrasonication methods The emulsion used PGPR emulsifier in primary emulsion and sodium casinate in outer water phase The emulsion had excellent encapsulation efficiency which was 99.35 % and the average droplet size was

63 nm Furthermore, the impact of external environment such as flash pasteurization was not significantly impact the encapsulation efficiency and the antioxidant activity 𝐼𝐶50 of the emulsion was higher than pure catechin which proves the good protection of the inside compounds from outside factors

By the nano - sized encapsulation of catechin from green tea ethanolic – extract with the support of Lecithin and Tween 80, the nanoemulsion of (Tsai and Chen, 2016) [87] was highly stable system which encapsulation efficiency was about 88 %, droplet size was 11.45 nm and zeta potential was – 66.3 mV The droplet size, PDI, catechin content, encapsulation efficiency of the nanoemulsion were not significantly changed after 120 days storing at 4 ℃ which illustrated excellent stability Furthermore, the nanoemulsion showed remarkably better 𝐼𝐶50of PC – 3 cancerous cells than ethanolic catechin extract which was 8.5 µg/ml in comparison with 15.4 µg/ml, respectively

In the study of (Bhushani et al, 2016) [88], the nanoencapsulation of catechin was prepared by high pressure homogenizer method which used soy protein isolate as emulsifier The nanoemulsions were show highly stable after 15 days storing at 4 ℃ which encapsulation efficiency of catechin was all over 80 % Furthermore, the E4 emulsion which contained 10% wt oil and 0.5% wt catechin has 2.78 times higher bioaccessible than unencapsulated sample as well as good protection against instable factors such as creaming, phase separation and sedimentation

The cashew nut testa is an rich source of catechin so that it can be potentially ultilized in many field of industries In our study, the catechin from testa extract was double nanoencapsulated at many ratios of ingredients and phases in order to find the best emulsion which could effectively prevent interior catechin from degradation

CHAPTER 3: MATERIAL AND METHODS

3.1 Materials and chemicals

The main material used in the experiments was cashew nut testa which origin from Binh Phuoc Province The raw cashew nut testa was red – brownish color, small pieces, dry and contained in the plastic bag

The chemicals used for the making W/O/W nano emulsion process consisted of testa powder extract for the water phase and vegetable oil for the oil phase Furthermore, Lecithin was used for lipophilic emulsifiers and Whey protein isolate (WPI) as well as Tween 80 are used as hydrophilic emulsifiers

There were also other chemicals used for sample analysis like methanol, ethanol, Ciocalteu reagent, gallic acid, catechin, sodium carbonate (𝑁𝑎2𝐶𝑂3), aluminium chloride (𝐴𝑙𝐶𝑙3), sodium nitrite (𝑁𝑎𝑁𝑂2), DPPH, deionized water, double – distilled water, vanillin, acid sulfuric (𝐻2𝑆𝑂4) solutions

Folin-3.2 Intrustments

Table 3.1 List of experimental intrustments

pH/mV – meter

WTW, Germany

3 Heating circulation bath CORIO™ CD-B19 Julabo, Germany

5 Heating magnetic stirrer ARE Velp, Italy

9

Spectrophotometer PhotoLab® 7600

10 Nano–Particle size analyzer SZ-100 Horiba, Japan

3.3 Material Preparation

The obtained material was prepared following the procedure as shown in Figure 8 After drying in oven (Memmert, Germany) at 45 ℃ in 24 h the dried testa was fine grinded by grinder (SEKA SK200, Japan) then screened by 35 mesh sieve

Figure 3.1 Sample preparation process Cashew nut testa

3.4 Extraction of Cashew nut Testa

Figure 3.2 Cashew nut testa enzymatic extraction process 90 ℃ and 10 minutes Enzyme