EFFECT OF WHEY PROTEIN ISOLATE-MENIRAN EXTRACT (Phyllantus niruri L.,) EENCAPSULATION USING CASEIN HYDROLYSATE ON FOAM, OPTICAL MICROSCOPY, PARTICLE
SIZE, AND VISCOSITY
Lukman Hakim1), Nicolays Jambang1)
1) National Research and Innovation Agency of the Republic of Indonesia (Research Center for Food Technology and Processing), Jl. Jogja-Wonosari Km. 31.5, RT 4/ RW 5, Gading IV, Gading, Kec. Playen, Kab.
Gunung Kidul, Prov. D.I. Yogyakarta, 55861
*Corresponding Email: [email protected] Submitted 31 June 2023; Accepted 31 July 2023
ABSTRACT
The purpose of this study was to determine the effect of whey protein isolate (WPI)-extract of meniran (Phyllanthus niruri L.) encapsulation using casein hydrolyzate on foam overrun, optical microscopy, particle size and viscosity. The materials used was whey protein isolate, an extract of meniran (Phyllanthus niruri L.) encapsulated using casein hydrolyzate. The method used was a completely randomized design (CRD) with 5 treatments of casein hydrolyzed as encapsulation material, 0%, 2%, 4%, 6% and 8%, namely P0, P1, P2, P3 and P4 repectively, with 3 replications.
The variables observed were foam overrun, particle size, and viscosity which were analyzed using analysis of variances (ANOVA). If there is a significant or very significant difference, analysis will be continued with the DMRT (Duncan Multiple Range Test). Variable of optical microscopy was analyzed descriptively. The results showed that whey protein isolate-meniran extract encapsulation using casein hydrolyzate gave a very significant difference (P<0.01) on in viscosity and foam overrun but did not give significant difference (P>0.05) on particle size. The average foam overrun value was 325% - 400%, the viscosity was 0.985 cP - 1.015 cP, the particle size was 0.8944 nm - 1.5031 nm, and optical microscopy showed that the most abundant distribution and amount of foam was observed in P4, namely whey protein isolate-meniran extract encapsulated with 8% casein hydrolyzate. Along with the addition of casein hydrolyzate with different concentrations (2%, 4%, 6%, 8%), the distribution and shape of the foam became more even and uniform. The conclusion of this study was that the addition of 6% casein hydrolyzate on the encapsulation effect of whey protein isolate-meniran extract was able to maintain the physical properties of WPI-meniran extract on viscosity, optimal particle size, foam overrun, and optical microscopy with a shape, uniform foam distribution and lamella tend to be thick.
Keyword : Casein hydrolyzate; foam; meniran; optical microscopy
INTRODUCTION
Casein and whey protein are the two main protein components in milk. Casein counts for 80% of the total protein in milk, while whey protein contributes 20%. Whey protein is a group of globular proteins isolated from whey, a cheese byproduct made from cow's milk. Whey contains less than 1%
protein which mainly consists of β- lactoglobulin (β-LG), α-lactalbumin (α-LA), bovine serum albumin (BSA), immunoglobulin and proteose peptones, as well as several proteins including lactoferrin, lactolin, glycoprotein, lactoperoxidase and transferrin (Gangurde et al., 2011).
Whey protein are micron-sized aggregates or gel particles with important surface characteristics to adsorb at the air/water interface and produce very stable froth (Lazidis et al., 2014). Rehydrated whey protein gel particles will produce foam with increased stability than protein at the same concentration (Lazidis et al., 2016). Having hydrophilic and hydrophobic groups in its structure, milk protein is an excellent surfactant. Milk proteins help create froth by dispersing and concentrating at the air-liquid interface, which leads to a reduction in surface tension. The protein then stretches with hydrophilic and hydrophobic groups towards the liquid and air phases, respectively, and generates a highly visco-elastic interfacial layer to stabilize the air bubbles (Xiong, et al., 2020).
Meniran (Phyllanthus niruri L.) belongs to the Spermatophyta division, Angiospermae subdivision, class Dicotyledonae, order Geraniles, family Euphorbiaceae, genus Phyllanthus (Webster 1986; Padua et al., 1999 in Oktavidiati, et al., 2011). The active
ingredients of the meniran plant (Phyllanthus niruri L.) are filantin, hypophyllanthin, potassium, resin, tannins, saponins and flavonoids. This makes the plant efficacious as a diuretic, antidiarrheal, mouth ulcer remedy, fever reliever (antipyretic), medicine for gonorrhea, kidney evaluation, blood purifier, and treatment of hepatitis. The active constituents of tannins can protect liver cells from viral and bacterial infections, while phyllanthin and hypophyllanthin protect against toxins. The meniran plant (Phyllanthus niruri L.) used was extracted using the Microwave-Assisted Extraction (MAE) method. Meniran extract is not only beneficial for health, but its use in food ingredients, especially whey protein, can improve functional quality, namely facilitating the absorption of the human body, and used as a functional food. The addition of meniran extract to whey protein will produce a covalent bond and a non-covalent bond in the form of hydrophobic, van der Walls and hydrogen bonds interactions.
Encapsulation is a process of coating a core material (Wu, et al., 2000 in Julkarnain, et al., 2016). This process is also a technique of entrapment of the core material in certain encapsulating materials. The advantage of the encapsulation technique is to protect and control the release of the active ingredients (Palupi, et al., 2014). Microencapsulation is a process in which small particles or droplets are surrounded by a coating to give a small capsule. Microcapsules are small spheres with a relatively simple shape. The material inside the microcapsule is called the core, internal phase, or filler, while the wall is called the shell, coating, or membrane (Jyothi, et al., 2012). The wall material determines the stability of the microparticles, process
*Corresponding author:
Lukman Hakim
Email: [email protected]
National Research and Innovation Agency of the Republic of Indonesia (Research Center for Food Technology and Processing), Jl. Jogja-Wonosari Km. 31.5, RT 4/ RW 5, Gading IV, Gading, Kec.
Playen, Kab. Gunung Kidul, Prov. D.I. Yogyakarta, 55861
How to cite:
Hakim, L., & Jambang, N. (2023). Effect of Whey Protein Isolate-Meniran Extract (Phyllantus niruri L.,) Eencapsulation Using Casein Hydrolysate on Foam, Optical Microscopy, Particle Size, and Viscosity. Jurnal Ilmu dan Teknologi Hasil Ternak (JITEK), 18 (2), 139-148
efficiency and degree of protection for the core. Wall materials commonly used for microencapsulation include synthetic polymers and natural biomaterials (usually a lot of materials derived from carbohydrates and proteins) (Bakry, et al., 2016). Protective materials can be divided into 3 namely carbohydrates, proteins, and fats.
Encapsulations derived from carbohydrates are classified based on their origin, such as from plants, maltodextrin, starch, cellulose, gum arabic, mesquite gum, guar gum, galactomannan, cyclodextrin and pectin, from animals or microbes such as xanthan, gellan, dextran and chitosan and from marine ingredients. such as carrageenan. and alginate.
Encapsulations derived from proteins are classified based on their origin, such as from plants, there are soy proteins and pea proteins, and from animals, such as casein, whey protein and gelatin. Encapsulations derived from fat include milk fat, phospholipids, beeswax and carnauba wax (Calvin, 2020).
The main protein contained in milk is casein. Easy to obtain, non-toxic and very stable. Casein is often used as a supplement and emulsifier in the food industry (Diak, et al., 2007). Casein is used as an emulsifier in the encapsulation process (Lestari, et al., 2019). Casein has good water retention and binding ability, which can help increase the viscosity of the emulsion. High viscosity can lower the emulsion droplet sedimentation coefficient, thereby increasing stability. (Li, et al., 2015).
Hydrolyzed casein or casein which has been processed by enzymes has an emulsification function, binds water and forms foam (Pratama, et al., 2019). Casein hydrolyzate has two functions as an encapsulant that protects particles during the manufacturing process from heat and other factors and as an addition to the protein source. This addition will produce hydrophobic interactions that reduce surface tension and widen the surface elasticity of the film (Staszweski, et al., 2012). Whey protein isolate-meniran extract is expected to have the effect of changing viscosity, foam overrun, increased particle size and optical microscopy
values and the form of thick foam and uniform distribution.
MATERIALS AND METHODS
Materials
The research material for whey protein isolate-extract meniran (Phyllanthus niruri L.) encapsulated, the ingredients were whey protein isolate (WPI) 90 plain obtained from the online shopping application at Shopee, Tryptone Type-1 casein hydrolyzate (Casitose Type-I) (Himedia), meniran powder (Phyllantus niruri L.,) obtained from Balai Materia Medica Batu.
Methods
The research method used a completely randomized design (CRD) with 5 treatments and 3 replications. The treatment was the encapsulation of wpi-meniran extract using casein hydrolyzate (0, 2, 4, 6 and 8% (w/v)), P0, P1, P2, P3 and P4 respectively.
Meniran extraction
3 g of meniran powder dissolving in 100 ml of distilled water and homogenizing, maceration process is carried out during 24 hours. Meniran extraction using with Microwave Assisted Extraction (MAE) at 70ºC for 10 minutes, every one minute turned on MAE then turned off for 2 minutes (performed until the 10th minute). This aims to maintain the temperature not exceeding 80ºC and prevent the degradation of meniran bioactive compounds.
Meniran extraction results have thick characteristics (coarse extract) and brownish green in color. The extract was allowed to cool at room temperature, filtered using filter paper and stored in the refrigerator at 4ºC. Liquid meniran extract was taken as much as 10 ml then dried using an evaporator at 50ºC (medium) for 6 minutes to reduce the water content of meniran. Whey Protein Isolate (WPI) was prepared, weighed 30 g WPI 90 and then dissolved with 564 ml of distilled water. Meniran extract was added as much as 36 ml to become a combination of meniran extract and WPI 90 into a liquid sample.
Spray Drying
The combination of wpi-meniran extract was then added to the encapsulant, namely casein hydrolyzate in the amount of the percentage of each treatment (g). The sample was then homogenized using a magnetic stirrer for 40 minutes. The sample used for the spraying process was 600 ml at a temperature of 162, 163 or 165ºC and the spraying process was divided into two sessions using a sample of 300 ml/session.
Data analysis used the experimental method with a completely randomized design (CRD), the average data obtained was
calculated, followed by an Analysis of Variance (ANOVA). If there is a significant or very significant difference, the DMRT (Duncan Multiple Range Test) will be continued.
Foam overrun
A sample of 3 g of powder and 20 ml of aquadest was taken and then placed in a tube.
Stir with a hand mixer for 3 minutes, waited for 30 seconds. The sample was measured using a 100 ml measuring cup. Foam Overrun calculation is performed with the formula:
OV (%)=Vt/V0 x 100%
Description : Vt : the final volume of the solution after stirring (ml) V0 : initial volume of solution (ml).
Viscosity
Take a sample of 15 g of powder and 100 ml of distilled water. Homogenized, Viscosity test was carried out using a Brookfield Viscometer with spindle No. 40 at 37ºC and 20 rpm for 20 seconds. Record the results of the viscosity test.
Particle size
Take a sample of 5 g of powder. Added 15 ml of aquadest mixed until homogeneous.
The particle size test was carried out using Horiba LA 500 (Horiba Instrument, Irvine CA) laser diffraction particle size distribution.
Record the results of the particle size test.
Optical Microscope
Take a sample of 3 g and 20 ml of distilled water. Drop 1 drop of foam formed on the object glass, then cover with a cover glass 3. Observed using an optical microscope with a magnification of 40x and 100x.
Photographed the results of observations using a camera.
RESULT AND DISCUSSION
Foam Overrun
The effect of encapsulating whey protein isolate-meniran extract with different
concentrations of 0%, 2%, 4%, 6%, 8% in spray drying, gives a very significant difference in effect (P<0.01) for encapsulated casein hydrolyzate foam overrun, can be seen in Table 1.
Based on the results of analysis of variance in Table 1, the effect of using whey protein isolate-extract meniran encapsulation with different concentrations of casein hydrolyzate showed a very significant (P<0.01) difference in effect on foam overrun.
This is because whey protein isolate-meniran extract contains flavonoids that interact and bind to each other so that they change the structure of the protein and affect its ability as a good foaming agent. Data on the ability to form foam encapsulation of whey protein isolate-meniran extract with different concentrations in treatments of casein hydrolyzate at P0, P1, P2, P3 and P4 yielded an average value of 325%, 350%, 375%, 400% and 370.83% respectively. The highest foam overrun was found in whey protein isolate-meniran extract encapsulated casein hydrolyzate (P3), which was 400% and the lowest was found in whey protein isolate- meniran extract which was not encapsulated casein hydrolyzate (P0), namely 325%.
Rahayu, et al., (2015) reported the results of foam overrun values of whey protein with the
addition of green tea leaf extract with different concentrations resulting in foam overrun values between 44.79 – 46.52%.
Meniran extraction generates heat energy, which damages cell walls and tissues while extracting the compounds found in meniran, such as flavonoids, phenolics, and polyphenols. This compound will interact with amino acids in whey protein, causing reduced foaming power. This is in accordance with the results of a previous study that showed the more concentration of meniran extract added to whey protein, the lower the percentage of foaming power. Nagy, et al., (2012) stated that β-lg in whey protein has many binding sites on polyphenols. The bond
between whey protein and polyphenols is a non-covalent bond interaction, which consists of hydrophobic interactions, van der Waals-, hydrogen bridges and ionic interactions.
According to Jiang et al., (2018) protein- polyphenol mixtures can explain their increased foaming ability, enabling more efficient transport of protein molecules to the air-water interface. According to Hakim, et al., (2013) foam strength, foam stability, and a decrease in surface tension are all related to foam power. Foam overrun refers to the foaming ability and stability of the foam associated with a decrease in surface tension in a water-air system due to adsorption of protein molecules.
Table 1. Foam Overrun
Treatment Average ± SD (%)
P0 325.00 ± 0.00a
P1 350.00 ± 0.00b
P2 375.00 ± 0.00d
P3 400.00 ± 0.00e
P4 370.83 ± 7.22c
This also causes the foam overrun to experience an increase in foam along with a decrease in viscosity. Martinez, et al., (2011) stated that an increase in froth in general as a result of pressure can be attributed to a decrease in the viscosity of the solution which allows incorporation of air, as it is less constrained by lower viscosity. This significant difference in effect is thought to be due to differences in the percentage of casein hydrolyzate used resulting in different foam overrun values in each treatment. The results of this analysis are based on the value of P0 (a combination of 0% casein hydrolyzed meniran extract), P1 (2%), P2 (4%), P3 (6%), P4 (8%), namely 325%, 350%, 375 %, 400%, 383.33%. This is in accordance with the opinion of Xiong, et al., (2020) which states
that the foaming ability of milk protein is influenced by a number of parameters, including the amount of protein, the ratio of casein hydrolyzate to whey protein, heat treatment, and ionic environment. Martinez, et al., (2011) stated that the increase in foam overrun is related to the interaction between surface pressure and particle size which is directly related, thus encouraging an increase in the ability of foam overrun.
Viscosity
The use of whey protein isolate-meniran extract encapsulation yielded highly significant (P<0.01) differences in effect on the viscosity of the combination of meniran extract encapsulated by casein hydrolysate, which can be seen in Table 2.
Tabel 2. Viscosity
Treatment Average ± SD (cP)
P0 0.993 ± 0,003b
P1 1.015 ± 0,002 cd
P2 1.001 ± 0,003c
P3 1.008 ± 0,003d
P4 0.985 ± 0,002a
Data on the viscosity analysis of whey protein isolate-meniran extract with different concentrations in treatments P0, P1, P2, P3 and P4 yielded an average value of 0.993 cP, 1.015 cP, 1.001 cP, 1.008 cP and 0.985 cP.
The highest viscosity was found in whey protein isolate-meniran extract encapsulated casein hydrolyzate (P1), namely 1.015 ± 0.002 cP and the lowest was found in whey protein isolate-meniran extract encapsulated casein hydrolyzate (P4), namely 0.985 ± 0.002 cP. Saragih, et al., (2022) reported the results of the viscosity of whey protein added to meniran extract with different concentrations resulting in a viscosity value between 4.21 – 10.42 cP. The initial protein content of whey protein raw material determines the viscosity because protein can bind to water molecules. According to Saragih, et al., (2022) stated that high viscosity has a high protein content because protein which dissolves in water forms globules which are hydrophobic on the inside and hydrophilic on the outside with a higher water holding capacity. The addition of casein hydrolyzate which is hygroscopic causes the ability to bind water to WPI-meniran extract to increase thereby reducing the viscosity value.
The addition of casein hydrolyzate wpi-meniran extract contains amino acids which react and result in a decrease in peptide
size. This is in line with the opinion of Susanty and Indrati (2021) which states that enzymatic hydrolysis of proteins can reduce the size of peptides so that they can change the functional properties of proteins and improve their quality.
The hydrolyzed protein obtained after hydrolysis produces a protein composed of free amino acids and short chain peptides which provide advantages as a functional food because of its amino acid profile. The difference in viscosity values is also affected by the distribution of protein molecules in solution and the molecular weight of the protein. According to Anggraini and Yunianta (2015) the value of viscosity is affected by the distribution of protein molecules, as well as the molecular weight of the protein in solution, while the molecular weight of a protein is directly related to the length of its peptide chain. The shorter the peptide size of a protein, the lower the molecular weight and the easier the distribution of molecules in solution, resulting in a lower viscosity.
Particle Size.
The use of whey protein isolate- meniran extract encapsulation gave no significant effect (P>0.05) on the particle size combination of meniran extract encapsulated by casein hydrolyzate, which can be seen in Table 3.
Tabel 3. Particle Size
Treatment Average ± SD (nm)
P0 1.1776 ± 0.15007
P1 0.8944 ± 0.23212
P2 1.0677 ± 0.26563
P3 1.1603 ± 0.40894
P4 1.5031 ± 0.45824
Particle size data of whey protein isolate-extract meniran with different concentrations in the P0, P1, P2, P3 and P4 treatments yielded an average value of 1.1776 nm, 0.8944 nm, 1.0677 nm, 1.1603 nm and 1 .5031nm. The highest particle size was found in whey protein isolate-meniran extract encapsulated casein hydrolyzate (P4) which was 1.5031 nm and the lowest was found in
whey protein isolate-meniran extract encapsulated casein hydrolyzate (P1) which was 0.8944 nm. This is affected by the high temperature during spray drying and makes the results stick to the wall a lot so that it affects the particle size. The higher the spray drying temperature used, the higher the yield of chlorophyll extract powder produced.
Higher spray drying temperatures result in
drier powders. Fewer products stick to the tube, and less product loss (Annisa et al., 2017 in Mardaningsih et al., 2012). Differences in the concentration of the addition of casein hydrolyzate in each treatment consisted of control (P0); 0% (P1); 2% (P2); 4% (P3); 6%
and (P4); 8% gave a difference which showed no significant difference in each treatment (P>
0.05) resulting in values of 0.15007 nm, 0.23212 nm, 0.26563 nm, 0.40894 nm and 0.45824 nm. Rahayu, et al., (2021) reported the results of particle size values with different catechin concentration treatments resulting in particle sizes between 159.43- 213.6 nm.
Particle size can be affected by the composition of the casein hydrolyzate, namely proteins, lipids and carbohydrates (Dupont, et al., 2015). The difference in particle size values is because each treatment has a different ability to trap air so that the value of foam overrun increases. According to
Martinez et al., (2011) said that the influence between particle size has a rapid decrease in stress (or increase in surface pressure) for the treated solution which may be associated with an increase in surface activity (ie surface hydrophobicity) as a result of an increase in protein surface contact. This can be explained by a decrease in the size of whey protein aggregates leading to increased mobility in the bulk phase and release of hydrophobic groups.
Optical Microscopy
Microscopic examination of the foam formed was carried out using a microscope with a magnification of 40x and 100x with one drop of sample preparation on an object glass.
This microscopic test of foam aims to determine the shape of the foam and the distribution of foam that is formed evenly on each side or not. The optical microscopy image of the wpi-meniran extract froth that is formed is presented in Figure 1.
a1 a2 b1 b2
c1 c2 d1 d2
e1 e2
Figure 1. Optical Microscopy of wpi-meniran extract encapsulation using casein hydrolyzate at 40x (a1) P0 : 0%; (b1) P1 : 2%; (c1) P2 : 4%; (d1) P3 : 6%; (e1) P4 : 8% and optical microscopy at 100x (a2) P0 : 0%; (b2) P1 : 2%; (c2) P2 : 4%; (d2) P3 : 6%;
(e2) P4 : 8%.
The results of microscopic observations of wpi-meniran extract foam with casein hydrolyzate encapsulated looked different in each treatment sample. The results of the distribution and amount of froth that were abundant in P4 were whey protein isolate- meniran extract encapsulated casein hydrolyzate as much as 8%. Along with the addition of casein hydrolyzate with different concentrations (2%, 4%, 6%, 8%), the distribution and shape of the foam became more even and uniform. This can be seen using a 100x magnification microscope in Figure 1.a3, b3, c3, d3, e3.
Whey protein isolate-meniran extract which is not encapsulated by casein hydrolyzate (P0) produces froth that tends to be large but uneven and non-uniform. During the preparation stage with object glass that has been dripped with foam and then covered using a cover glass, the foam at P0 tends to break easily compared to other treatments.
This is in accordance with the data from the analysis of foam overrun in Table 1. It shows that the value of foam overrun in whey protein isolate-meniran extract that is not encapsulated by casein hydrolyzate (P0) is lower, namely 325%.
During the heating process at a temperature of 65-70ºC for 40 minutes, whey protein does not form aggregates so that a film layer is formed on the bubbles so that it spreads on the surface. This is in accordance with the statement of Norwood et al., (2016) who stated that non-aggregated protein contributes to foam formation because it diffuses to the interface more quickly, then unfolds and is adsorbed to form an aqueous film surrounding the air bubbles. Proteins that unfold at this interface can also serve as anchors for aggregated proteins.
Optical microscopy analysis which can be seen in Figure 1. The addition of casein hydrolyzate tends to become thicker, and the shape of the lamella changes from spherical to polyhedral. There are non-uniform foam shapes and sizes, but when casein hydrolyzate is added, the air pressure in the small bubbles (Figure a2. marked with red circles) diffuses to the larger bubbles (Figure c2. marked with
yellow ovals), resulting in almost uniform air bubbles (Fig. e2. marked with blue circles).
CONCLUSIONS
The results of the study adding 6%
casein hydrolyzate to the encapsulation effect of Whey Protein Isolate-meniran extract were able to maintain the physical properties of WPI-meniran extract on viscosity, optimal particle size, foam overrun and optical microscopy with a shape, uniform foam distribution and lamella tend to be thick.
ACKNOWLEDGMENT
This research was supported by Universitas Brawijaya through Hibah Doktor Lektor Kepala programmed 2021.
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