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
LITERATURE REVIEW
Anacardium genus
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
Cashew nut
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)
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]
Cashew nut testa contains many phenolic compounds such as catechin, epicatechin, epigallocatechin which are dominant, syringic, gallic acid and p-coumaric acid [11] which has anti-hyperlipidaemic, anti-inflammatory, antioxidative, anticarcinogenic, and cytoprotective effect for human health
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]
Cashew nut testa
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]
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,
8 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
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
9 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]
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 àg /g f.w and 2.41 ± 0.18 àg /g f.w while Epicatechin was only found in skin with the content was 104.8 ± 10.56 àg /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
Double nanoemulsions and other types
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
14 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
15 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]
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
16 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]
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]
MATERIAL AND METHODS
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, Folin-Ciocalteu reagent, gallic acid, catechin, sodium carbonate (𝑁𝑎 2 𝐶𝑂 3 ), aluminium chloride (𝐴𝑙𝐶𝑙 3 ), sodium nitrite (𝑁𝑎𝑁𝑂 2 ), DPPH, deionized water, double – distilled water, vanillin, acid sulfuric (𝐻 2 𝑆𝑂 4 ) solutions.
Intrustments
Table 3.1 List of experimental intrustments
No Intrustment Product Type Origin
1 pH Meter Ino Lab pH 7110 SET 4 pH/mV – meter
2 Shaking water bath WNB 22 Memmert, Germany
3 Heating circulation bath CORIO™ CD-B19 Julabo, Germany
4 Ultrasonic Bath WUC-A03 Daihan, India
5 Heating magnetic stirrer ARE Velp, Italy
7 Sonicator Sonics VCX 500 Newtown, CT, USA
10 Nano–Particle size analyzer SZ-100 Horiba, Japan
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
Extraction of Cashew nut Testa
Figure 3.2 Cashew nut testa enzymatic extraction process
The cashew nut testa powder was extracted by enzymatic combined with ultrasonic methods which are based on the studies of (Santana and Macedo, 2019) [89] and (Giahi et al, 2021) [90] Firstly, the powder was mixed with double distilled water at ration 1 : 30 (w/w) Then, the enzyme Pectinex – SPL was added into the mixture with enzyme : water ratio 2 % (v/w) and incubating at 50 ℃ in shaking water bath (Memmert, Germany) After 60 minutes incubation, the enzyme was inhibited by heating at 90 ℃ in 10 minutes in heating circulation bath (Julabo, Germany) Next, the mixture was ultrasonicated at 40 kHz; 40 ℃ ; 40 minutes in ultrasonic bath (Daihan, India) and centrifugated at 5500 rpm in 10 minutes after that Finally, the extract was filtered by the filter papers in order to remove the remain residue The mixture was preserved in the refrigerator for making nanoemulsion and analysis.
Preparation of Double Nanoemulsion 𝑾𝟏/𝑶/𝑾𝟐
The preparation of the double nanoemulsion was based on the method of (Wang et al, 2017) [91] with some modifications The primary 𝑾 𝟏 /𝑶 phase was formed by mixing a mixture of Cashew nut Testa Extract with the oil phase which consists of lipophilic emulsifier Lecithin and soy bean oil Finally, the primary phase was mixed with the outer water phase
𝑾 𝟐 which composed of double distilled water, Tween 80 and Whey protein isolate (WPI) in order to form complete Double nanoemulsion 𝑾 𝟏 /𝑶/𝑾 𝟐 Many paramters such as the 𝑶 :
𝑾 𝟏 ratios, Lecithin content in primary phase and 𝑾 𝟐 : 𝑾 𝟏 /O ratios, Tween 80 : WPI ratios in the double emulsion were investigated at different ratios for finding the best parameters
Firstly, the cashew testa extract was mixed with Soy bean oil and Lecithin by using the homogenizer (IKA, Germany) at 20,000 rpm in 5 minutes Then, the mixture was ultrasonicated at 20 kHz; 40% amplitude in 10 minutes by sonicator (Newtown, CT, USA) in order to obtain the 𝑾 𝟏 /𝑶 emulsion The primary emulsion was mixed with external water phase with are consisted of distilled water, Tween 80 and WPI by homogenizer (IKA, Germany) at the same parameters in the first metioned Finally, the mixture was ultrasonicated in 6 minutes to complete the process The 𝑾 𝟏 /𝑶/𝑾 𝟐 double nanoemulsion was preserved in the refrigerator for further analyzing
29 Figure 3.3 Cashew nut testa double emulsion preparation process
Cashew nut Testa extract Soy bean oil
Investigating the components of Double Nanoemulsion 𝑾𝟏/𝑶/𝑾𝟐
There are four parameters which were investigated in order to have the best protective emulsion system The investigation begins from the internal to the external sequence of the emulsion Firstly, in the 𝑾 𝟏 /𝑶 emulsion , the ratio of 𝑾 𝟏 : 𝑶 (w/w) as well as the Lecithin content (% w/w) are investigated which parameters were 10 : 90, 20 : 80, 30 : 70 (w/w) and
3 %, 5 %, 7 % (w/w), sequentially Then, the parameters of external 𝑾 𝟐 phase consisted of the ratio of 𝑾 𝟐 : 𝑾 𝟏 /𝑶 together with the ratio of Tween 80 : WPI were investigated at 90
The total flavonoid content (TFC) was analyzed then calculated the encapsulation efficiency (EE) and compared between the investigated samples The parameters had the most highest EE were chosen and used to make the best nanoemulsion system
In the experiment, the primary nanoemulsion were preapared with three different 𝑾 𝟏 : 𝑶 ratios which were 10 : 90, 20 : 80 and 30 : 70 (w/w) and the Lecithin content was fixed at 5% (w/w) In the secondary emulsification, the ratio of 𝑊 2 : 𝑊 1 /𝑂 and Tween 80 : WPI were constant at 80 : 20 (w/w) and 2 : 3 (w/w), respectively The best 𝑾 𝟏 : 𝑶 ratio was constantly applied for subsequent experiments The experimental procedure was implemented following the section 2.5
In the experiment, the primary nanoemulsion was prepared with the best 𝑾 𝟏 : 𝑶 ratio found out in previous section and the Lecithin content were investigated at 3%, 5% and 7%
(w/w) In the secondary emulsification, the ratio of 𝑊 2 : 𝑊 1 /𝑂 and Tween 80 : WPI were constant at 80 : 20 (w/w) and 2 : 3 (w/w), respectively The best Lecithin content was constantly applied for subsequent experiments The experimental procedure was implemented following the section 2.5
In the experiment, the primary nanoemulsion was prepared with the best 𝑾 𝟏 : 𝑶 ratio and
Lecithin content found out in previous sections In the secondary emulsification, the ratios of 𝑾 𝟐 : 𝑾 𝟏 /𝑶 were investigated at 90 : 10, 80 : 20, 70 : 30 (w/w) and the ratio of Tween 80 : WPI was constant at 2 : 3 (w/w) The best 𝑾 𝟐 : 𝑾 𝟏 /𝑶 ratio was constantly applied for subsequent experiments The experimental procedure was implemented following the section 2.5
3.6.4 Investigation of Tween 80 : WPI ratio
In the experiment, the primary nanoemulsion was prepared with the best 𝑾 𝟏 : 𝑶 ratio and
Lecithin content found out in previous sections In the secondary emulsification, the best ratio of 𝑾 𝟐 : 𝑾 𝟏 /𝑶 found out from previous section was fixedly applied and different Tween
80 : WPI ratios are investigated at 5:0, 4:1, 3:2, 2:3, 1:4, 0:5 (w/w) The best Tween 80 :
WPI ratio was constantly applied for the formation the final double nanoemulsion which was storage in refrigerator at 4 ℃ for further analysis as well as next experiments.
Determination of Total Phenolic content (TPC)
The total phenolic content in the samples was analyzed by using the Folin - Ciocalteu reagent assay which based on the method of (Hung and Morita, 2008) [92] with modifications 0.5 ml sample was mixed with 0.5 ml Folin - Ciocalteu reagent, 1 ml saturated sodium carbonate solution and added distilled water until the final volume was 10 ml The mixture samples were measured by the UV – VIS spectrophotometer (WTW, Germany) at 725 nm The standard curve was made by dilution of gallic acid at different concentrations and the TPC of samples were expressed by milligrams gallic equivalent per gram dry weight sample (mg GAE/ g DW sample)
Determination of Total Flavonoid content (TFC)
The total flavonoid content was determined according to the method of (Dias et al, 2019) [93] with modifications 0.15 ml sodium nitrite (5%) was added into 0.5 ml sample then incubated in 5 minutes After that, 0.15 ml aluminium chloride (10%) was added and the incubation was continued in 6 minutes Then, 1 ml sodium hydroxide and 1.2 ml distilled water were added into the mixture for the complete reaction Finally, the mixture was centrifuged at 13.000 rpm in 5 minutes and measured by UV – VIS spectrophotometer (WTW, Germany) at 510 nm The standard curve was made by dilution of catechin at different concentrations and the TFC of samples were expressed by milligrams catechin equivalent per gram sample (mg CAT/ g sample).
Determination of Total Catechin content (TCC)
The total catechin content was analyzed base on the modified method of (Sun et al, 1998) [94] by using Vanillin as reagent For detail, 1 ml of sample was mixed with 2.5 ml Vanillin (1% w/w in MeOH) and 2.5 ml 𝐻 2 𝑆𝑂 4 9 N After vortexing, the mixture was incubated in the dark in 15 minutes at room temperature The Catechin content of the samples was measured by using the UV – VIS spectrophotometer (WTW, Germany) at 500 nm The standard curve was made by dilution of catechin at different concentrations and the TCC of samples were expressed by milligrams catechin equivalent per gram dry weight sample (mg CAT/ g DW sample).
Determination of DPPH radical scavenging assay
The DPPH assay was based on the method of (Hung and Morita, 2008) [92] with some modifications for the determination of antioxidant capacity Briefly, 0.1 ml of sample was mixed with 3.9 DPPH solution which concentration was 0.075 mM and incubate in the dark in 30 minutes For the blank sample, 0.1 ml MeOH was used as alternative sample mixed in 3.9 ml 0.075 mM DPPH solution then analysis immediately The sample mixture was measured by UV – VIS spectrophotometer (WTW, Germany) at 515 nm The calculation was elicited from the following formula:
Which 𝐴𝑏𝑠 𝑡=0 is the absorbance of the blank sample at t = 0; 𝐴𝑏𝑠 𝑡0 is the absorbance of the sample at t = 30
The standard curve was made by dilution of Trolox at different concentrations and the antioxidant capacity of samples were expressed by milimol trolox equivalent per gram dry weight sample (mmol TE/ g DW sample).
Determination of Encapsulation Efficiency
The encapsulation efficiency of the sample was analyzed according to the methods of (Aditya et al, 2014) [86] and (Cruickshank-Quinn et al, 2014) [95] Firstly, the nanoemulsion samples were cool centrifuged at 13.000 rpm, 4 ℃ and 30 minutes The lower layer was taken and dilute 10 times by absolute MeOH solution for protein precipitation After centrifuging at 13.000 rpm, 4 ℃ and 30 minutes, the supernatant was taken and filtered by 0.22 àmsyringe filters and analyzed the TFC by UV – VIS spectrophotometer (WTW, Germany) at 515 nm The encapsulation efficiency was calculated by the equation based on the study of (Evageliou et al, 2019) [96]
Determination of Droplet Size, Polydisperity Index and Zeta Potential
The Physical Properties of the nanoparticles are measured by the method of (Aditya et al, 2014) [86] which uses the Dynamic Light Scattering (DLS) Technique Briefly, the samples were diluted by deionized water at the ratio 1 : 4000 then analyzed Droplet size, PDI and Zeta Potential by the Nanoparticle Analyzer (Horiba, Japan) Each measurement was done by triplicate
Determination of Stablity of double nanoemulsion
The stability study was implemented by investigating total flavonoid content as well as physical properties such as droplet size, PDI and zeta potential of optimized nanoemulsion samples in 0, 7, 14, 21 and 28 days in the storage condition at room temperature (25 ℃).
Statistical Analysis
All the experiments were done by triplicate (n = 3) and the results were shown as mean ±
SD Morever, the latter were analyzed by one – way analysis of variance (ANOVA) method which implemented by Statgraphic Centurion version 19 software and the significance was calculated with the level of confident interval is 95 %
RESULTS AND DISCUSSION
Polyphenol compounds content in cashew nut testa
The determination of TPC, TFC, TCC of the cashew nut testa powder and DPPH of the extract were carried out by using UV–Vis Spectrophotometry which results were illustrated in the Table 4.1
Table 4.1 Chemical parameters of Cashew nut testa
Total phenolic content (mg GAE/ g DW sample) 123.06 ± 0.45
Total flavonoid content (mg CAT/g DW sample) 100.41 ± 0.08
Total catechin content (mg CAT/ g DW sample) 22.13 ± 0.41
DPPH capacity (mmol TE/ g DW sample) 1.08 ± 0.02
In cashew nut testa powder, the TPC, TFC and TCC were 123.06 ± 0.45 (mg GAE/ g DW sample), 100.41 ± 0.08 (mg CAT/ g DW sample) and 22.13 ± 0.41 (mg CAT/ g DW sample), respectively The DPPH capacity of the extract was 1.08 ± 0.02 (mmol TE/ g DW sample)
The TPC, TCC of the dry testa powder in the study was lower in comparison of the study of (Chandrasekara and Shahidi, 2011) [11] which TPC is 269.05 ± 9.77 (mg GAE/ g deffated sample) and TCC is 47.289 ± 3.76 (mg/ g deffated sample) The reason of the differences mainly in the extraction method of the compared study which was using ethanol as solvent and samples treatment The TPC, TCC in ethanolic extraction was shown to be more effective than using aqueous extraction In the study of (Yu et al, 2005) [97], the TPC of the peanut skin extracted by 80 % ethanol was significantly higher than using only water which was 1.58 fold higher in direct peeling preparation In the case of TCC, (Gramza et al, 2005) [98] reported that the 95% ethanolic extraction of green tea and black tea remarkable increased the total catechin content which was about 1.7 times and 2.2 times higher, respectively in comparison with aqueous extracts
The TFC of the dried cashew testa skin was shown to be higher than some other types of nut skins such as Pistachio and Halzenut In the study (A Tomaino et al, 2010) [24], the TFC of Pistachio skin was 70.27 ± 5.42 mg Catechin/ g f.w which was about 30% lower the TFC
36 of the cashew testa in the current study Another study of (Tas and Gokmen, 2015) [99] in 14 varieties of halzenut skins showed that except the Uzun Musa halzelnut, the 13 remaining samples had the lower TFC than the cashew nut testa which varied from about 30.9 ± 0.39 - 93.6 ± 2.41 mg Catechin/ g sample
In the study of (Pekal et al, 2012) [100], the antioxidant capacity of 6 dry tea samples determined by DPPH assay in 20 minutes range from 0.77 ± 0.05 - 1.14 ± 0.06 (mmol TE/ g dry tea) which showed that the antioxidant ability of cashew testa in current study was higher than the majority of tea samples in the comparative study Another study of (Pycia, Kapusta and Jaworska, 2019) [101] in 3 different types of walnuts in 3 months illustrated that their DPPH capacity vary from 19.49 ± 0.76 – 73.54 ± 0.34 (mmol TE/ 100 g dry mass) which were lower than cashew testa in the present study.
Investigating the components of Double Nanoemulsion 𝑾𝟏/𝑶/𝑾𝟐
After the analysis, the encapsulation efficiency (EE) as well as physical parameters of the investigated nanoemulsion were illustrated in the Table 4.2 The EE in the double nanoemulsion which 𝑾 𝟏 : 𝑶 Ratio was 10 : 90 shows the highest value among three investigated ratio and the EE of this nanoemulsion was about 77% In the study of (Li et al, 2012) [102], EGCG was entrapped in β-lactoglobulin treated at 85 ℃ and pH = 7 to obtain the final nanoparticle had EE value reach about 78 % which was pretty similar to the current study
By using the hydrophobic medium, encapsulation efficiency of the water – soluble compounds was stable at high value because the medium can prevent these compounds migrate out the emulsion effectively [103] So that, the 10 : 90 nanoemulsion, which had the highest oil phase content had the remarkable higher EE value than the other samples because of the protective ability of the inner flavonoids from leaking out by the high outer oil phase
Table 4.2 Encapsulation efficiency and physical parameters of double nanoemulsions at different 𝑊 1 : 𝑂 Ratios
𝑊 1 : 𝑂 Ratio EE (%) Size (nm) PDI Z - Value (mV)
About the physical properties, the droplet sizes of the nanoemulsion varied from 219.2 nm to 258.7 nm which all were in the range of the nano – size (20 – 500 nm) as well as having milky appearance [49] The results was pretty similar to the results in the study of (Bhushani, Karthik and Anandharamakrishnan, 2016) [104], which the droplet size of 4 catechin nanoemulsion samples varied from about 244.67 – 268.67 nm
The PDI of the 10 : 90, 20 : 80, 30 : 70 nanoemulsion samples were 0.289, 0.276, 0.201, respectively The results showed the downward trend which could be explained by the higher probability of coalescence phenomenon when increased the oil phase content [105] Moreover, the droplet size of the 10 : 90 and 20 : 80 samples were larger than the 30 : 70 nanoemulsion which indicated that the droplet of formers fusing more easily than the latter as well as having higher PDI value All the samples has the PDI below 0.7 which illustrates that they were not polydispered distribution [85]
The Zeta – potential of the samples vary from – 62.8 to – 63.8 mV which were considered to be negatively stable nanoemulsion which can excellently prevent them from unstable factors such as coalescence [85] The results also show that the lower oil phase content resulted in decreasing the Zeta value which may be attributed to the insufficient oil coverage the protein molecules [105] In the study of (Lu, Li and Jiang, 2011) [106], the tea polyphenol was loaded in the lecithin/cholesterol mixture and the final liposome products had the Zeta potential was
- 67.2 mV which was nearly consistent with the Zeta potential of the samples in the current study
According to the results in the experiment, the 10 : 90 sample had the significantly higher
EE and Zeta – Value than 20 : 80 and 30 : 70 nanoemulsions Although the droplet size and PDI was not the best among the samples, these parameter of the 10 : 90 sample was still in the
38 acceptable range which can be considered to be stable emulsion Hence, the chosen 𝑾 𝟏 : 𝑶
Ratio as the best parameter for the next experiments was 10 : 90
In the study, three different Lecithin content were investigated which were 3%, 5% and 7% (w/w) The final results which included EE, droplet size, PDI and Zeta – potential were demonstrate in the Table 4.3 According the results, the EE had the downward trend when increased the Lecithin content and the 3% sample had the highest EE which was nearly 91% These results was similar in the study of (Goncalves et al, 2015) [107] which the EE of quercetin emulsions is reduced from 66.7 % to 37.2 % when increased the Lecithin concentration from 25 g/L to 60 g/L Additionally, the rise of organic and water ratio do not change the reduction of EE when increased the Lecithin concentration The phenomenon could be attributed to the aggregation of droplet due to the increasing of viscosity when added more lecithin into the emulsion [108, 109]
Table 4.3 Encapsulation efficiency and physical parameters of double nanoemulsions at different Lecithin contents
Lecithin content EE (%) Size (nm) PDI Z - Value (mV)
The increasing of lecithin content also led to the decreasing of droplet size which from 268.1 nm to 242.3 nm In the study of (Li et al, 2023) [110], when increased the zein : lecithin mass ratio from 1 : 1 to 2 : 3 and 1 : 2, the droplet size was reduced from 278 nm to 132 nm and 139 nm, respectively, which similar to the current study The reason behind the reduction was explained due to the combination between lecithin and zein to form the compact structure which lead to the smaller size of nanoparticles Zein is prolamine protein which is has high content of proline amino acid mainly found in corn (maize) In the WPI used in current study also contains proline so that the combination between the lecithin and proline could be occurred in the emulsion which results in decreasing in particle size All the samples had droplet size in the nano – range which from 20 nm to – 500 nm
The PDI of the samples were also have downward trend like the particle size which range from 0.310 to 0.196 The results showed that the higher lecithin content had the positive effect in the particle uniformity because the temperature increased during the ultrasonication may be improve the mobility and flexibility of the lecithin which lead to enhancing the fluidity of the bilayer and forming smaller as well as more uniform particles [111] All the samples had the PDI below 0.7 which illustrates that they were not polydispered distribution [85]
The Zeta – potential is fluctuated when increased the lecithin content which ranged from – 62.4 mV to – 54.7 mV For detail, when the lecithin content was rised from 3% to 5%, the Zeta value was reduced from – 59.2 mV to – 62.4 mV which may be caused by the exposure of negative charged groups because of the binding of lecithin led to the protein unfolding Moreover, the electrostatic interaction between the positively charged amine groups in protein and the negatively charged phosphate groups in lecithin also helped to reduce the zeta- potential [112] The Zeta value was increased from – 62.4 mV to – 54.7 mV when further increased lecithin content from 5% to 7% which could be explained by the adsorption of excessive lecithin on the interface resulted in the cover of charged groups [112] All the samples had Zeta value lower than – 25 mV which meaning the emulsions were stable systems [85]
According to the results in the experiment, the 3% sample had the significantly higher EE value than 5% and 7% nanoemulsions Although the droplet size, PDI and Zeta value were not the best among the samples, these parameter of the 3% sample was still in the acceptable range which could be considered to be stable emulsion Therefore, the best Lecithin content was 3% (w/w) because have the highest superior EE among 3 samples which promisingly had the best protective of internal flavonoids from external environmental factors
In the experiment, the double nanoemulsion was investigated with three different 𝑊 2 :
𝑊 1 /𝑂 ratios which were 90 : 10, 80 : 20, 70 : 30 (w/w) According to the results, the EE had fluctuated trend when increased the primary emulsion content In detail, when the 𝑊 2 : 𝑊 1 /𝑂 ratios changed from 90 : 10 to 80 : 20 made the EE increased which from about 71 % to 93
% The EE of the 80 : 20 sample was pretty consistent with the double emulsion prepared by
(Wang et al, 2017) [91], which used Tween 80 as surfactant in the external water phase and the final EE was nearly 95 % When further decreased the 𝑊 2 content, the EE was reduced to about 85% The reason behind the reduction was assumed to be the increasing of extract volume and the loss of WPI/Tween 80 which led to insufficiently covering the inside compounds
Table 4.4 Encapsulation efficiency and physical parameters of double nanoemulsions at different 𝑊 2 : 𝑊 1 /𝑂 ratios
𝑊 2 : 𝑊 1 /𝑂 ratio EE (%) Size (nm) PDI Z - Value (mV)
About the physical properties, the particle size, PDI as well as Zeta potential were all have upward trend when changed 𝑊 2 : 𝑊 1 /𝑂 ratio from 90 : 10 to 70 : 30 For detail, the size was growth from 200 nm to 300 nm The phenomenon could be explained because of the increasing content of high viscosity primary emulsion as well as decreasing content of low viscosity of external water phase which make the shear force more difficult in breaking the droplets [113] The similar situation was occred in PDI value which increased from 0.206 to 0.399 when decreased external water phase content All the PDI value are below 0.5 which could be deemed to be narrow distribution There was also the decreasing in the Zeta – value from about – 60.1 mV to – 63.6 mV which could be explained by the intereaction between Leicithin and WPI due to the increasing of 𝑊 1 /𝑂 content There was no significant difference between the Zeta value of the 80 : 20 and 70 : 30 samples The low Zeta value also showed that all 3 samples were stable nanoemulsion
From the results, the 80 : 20 sample had the highest EE among three samples Although the droplet size and PDI were not the best but they were still in acceptable range Thus, the best 𝑾 𝟐 ∶ 𝑾 𝟏 /𝑶 ratio was 80 : 20 which used for next experiments
4.2.4 Investigation of Tween 80 : WPI ratio
The experiment was conducted by investigating the impact of different Tween 80 : WPI ratio in the double nanoemulsion EE and physical properties in order to choose the best one
The investigated ratio were 0 : 5, 1 : 4, 2 : 3, 3 : 2, 4 : 1 and 0 : 5 (w/w), which results were demonstrated in the below table When added more WPI into the external water phase, the EE was gradually have upward trend which reached the maximum value 92 % at 1 : 4 sample then decreased to about 86 % when the outer phase contained only WPI The increasing of WPI had positive impact on the EE which could be explained by the adsorption on the oil surface results in the formation of stable and resistant layer over the oil droplets [114]
Table 4.5 Encapsulation efficiency and physical parameters of double nanoemulsions at different Tween 80 : WPI ratios
EE (%) Size (nm) PDI Z - Value (mV)
About the physical parameters, the droplet size was generally increased from the 5 : 0 to
Determination of Stablity of double nanoemulsion
In the experiment, the effect of storage time was investigated on the best nanoemulsion which was conducted in 4 weeks at room temperature (25 ℃) The EE and physical properties were analyzed which was illustrated in the Table 9 According the results, after 28 days, the
EE was decreased from about 91 % to 21 % and the most dramatically reduction was occurred from day 14 to day 28 These results indicated that the Tween 80 could keep the nanoemulsion stable for a short time In the study of (Li et al, 2021) [117], the Tween 20 was also shown to be able to effectively stabilize vitamin 𝐵 12 – emulsion in about 14 days, after this time, the EE was shown to be dynamically decreased In detail, the EE of vitamin 𝐵 12 – emulsion was reduced from about 93 % to about 47 % after 4 weeks storage in the room temperature Additionally, Tween 20 was shown to have better ability in lowering surface tension as well as interfacial tension than Tween 80, which indicated that the former may create more stable emulsion than the latter [118] This was may be the reason of the lower EE of the nanoemulsion in the current study than the emulsion in the comparative study after 28 days investigation
Table 4.6 Effect of storage time on the EE and physical parameters of double nanoemulsion at room temperature
Storage time EE (%) Size (nm) PDI Z - Value (mV)
From the physical perspective, the nanoemulsion was generally keep their good parameters after 28 days storage at room temperature The droplet size was reduced in the first week investigation then continously increased from about 242 nm to nearly 258 nm The droplet size was shown to have better stability than the E1 emulsion prepared in the study of (Bhushani, Karthik and Anandharamakrishnan, 2016) [104], which consisted of 5 % oil, 0.3 % green tea catechin, 4 % soy protein isolate and 90.7 % aqueous phase The droplet size of the E1 emulsion was increased from about 244 nm to 275 nm after 15 days storage in room temperature
The PDI and Zeta Value had the same fluctuated trend which range from 0.225 to 0.332 and – 59.1 mV to – 69.4 mV, respectively The change in the Zeta value may be attributed to the change of pH during storage which could affect the electric charged groups in WPI For details, the change of pH could make the electric charge of carboxyl groups in protein become more negative which resulted in the decreasing of Zeta – value [119]
CONCLUSION AND RECOMMENDATION
Conclusion
The double nanoemulsion has many potential applications in many important industrials such as pharmaceutical, food and beverage or cosmetic because of highly protective properties of internal target compounds from external factors such as light, temperature, pH or chemical reactions as well as improvement in sensory value of the products In this study, the cashew nut testa extract is encapsulated in W/O/W nanoemulsion by high – energy methods with the support of lipophilic surfactant lecithin and hydrophilic surfactants WPI as well as Tween 80 After analysing the chemical properties of cashew extract, investigation of components as well as investigating the stability of the final double nanoemulsions, we have some overall conclusions about the study
The chemical properties of the cashew testa extract included TPC, TFC, TCC and DPPH capacity which were 123.06 ± 0.45 (mg GAE/ g DW sample), 100.41 ± 0.08 (mg CAT/ g DW sample), 22.13 ± 0.41 (mg CAT/ g DW sample) and 1.08 ± 0.02 (mmol TE/ g DW sample), respectively
The best formulation of the double nanoemulsion were prepared with 𝑊 1 : 𝑂 ratio Lecithin content, 𝑊 2 : 𝑊 1 /𝑂 ratio, Tween 80 : WPI ratio are 10 : 90 (w/w), 3 % (w/w), 80 : 20 (w/w) and 2 : 3 (w/w), sequentially The flavonoids in the cashew testa extract were effectively encapsulated by the best nanoemulsion which EE was over 91 % Moreover, the best nanoemulsion also had good physical properties which demonstrated through droplet size, PDI and Zeta value In detail, the droplet size was about 267 nm, the PDI was 0.228 and the Zeta value was – 62 mV
In the stability study, the best double nanoemulsion was show effective EE in about 14 days then dramatically decreased to about 21 % at the end of investigation The physical parameters were also changing which droplets size, PDI and Zeta value were recorded at about
257 nm, 0.303 and – 69.4 mV, respectively at day 28
In conclusion, the cashew nut testa is promising bioactive sources and the double nanoemulsion is potential method in protection of these bioactive compounds in order to prolong storage time which can be applied in many field of industry.
Recommendation
After the study, we have some recommendations for expand studies:
• Investigation of flavonoid profile in the cashew nut testa extract
• Using another enzymes or the combination of them in order to more effectively improve the extraction of cashew nut testa
• Investigation more parameters in the 𝑊 1 : 𝑂 ratio, Lecithin content, 𝑊 2 : 𝑊 1 /𝑂 ratio, Tween 80 : WPI ratio
• Determination of the pH of the double nanoemulsion during storage
• Investigating the stability of nanoemulsion at other temperatures
• Taking the scanning electron microscope image of the double nanoemulsion for more physical descriptions
• Investigation antimicrobial and anticancer abilities of the double nanoemulsion
• Applying and investigating the double nanoemulsion in the spray drying system
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The Folin – Ciocalteau assay is one of the most popular method in the determination of total phenolic compound because of simplicity and less time - consumption The principle of the method is the reduction of tungsten and molybdate in the Folin reagent which causes by the phenolic compounds in the sample in the alkaline condition Hence, the yellow color of the reagent is became blue color which can be determined by UV – Vis spectroscopy
Figure A.1 Reduction mechanism of Folin when react with phenolic compounds
Figure A.2 Reaction mechanism of 𝐴𝑙𝐶𝑙 3 method with the presence of 𝑁𝑎𝑁𝑂 2
One of most common methods in the determination of flavonoid content is the aluminium chloride (𝐴𝑙𝐶𝑙 3 ) assay which results in the formation of stable chelates of Al – flavonoid complex by the binding between aluminum and the high affinity of oxo and hydroxyl groups in the flavonoids However, the method is unsuitable for catechin because of the lack of carbonyl groups so that the chromophores are not created This problem can be solved by the adding of sodium nitrite (𝑁𝑎𝑁𝑂 2 ) which can conjugate with 𝐴𝑙𝐶𝑙 3 to significantly increase the maximum wavelength as well as reacting with catechin to form the chromophores
The mechanism of the method is the reaction between vanillin and catechin in the acidic environment which form the chromophore The method show more reliable in the result than another catechin analytical method such as Polyvinylpolypyrrolidone – Folin method which is 99% accuracy in compare with 83 % of the latter method in the analysis of purified quebracho tannin However, the catechin is easily vunerable flavonoid so that the temperature in the catechin determination should be carefully controlled as well as the temperature Furthermore, the using of sulfuric acid (𝐻 2 𝑆𝑂 4 ) instead of hydrochloric acid (𝐻𝐶𝑙) can improve the sensitivity of the method
Figure A.3 The chemical reaction between Catechin and Vanillin
The DPPH assay is one of the most popular approaches to measure antioxant capacity which mechanism is SET In the method, the DPPH reagent which has purple color when dissolve into methanol or ethanol solution reacts with the antioxidant factors in the sample After a period of time, the purple color is changed to pale yellow which depends on the nature of the testing samples The DPPH test has the benefit of being a simple, affordable, and quick way to assess the radical-scavenging capacity of non-enzymatic antioxidants However, it is necessary to carefully control some external environment during analysing the sample such as reaction time and temperature which can affect the final results
Figure A.4 Reaction Mechanism of DPPH assay