Microencapsulation of gamma oryzanol using inulin as wall material by spray drying: optimization of formulation and characterization of microcapsules

Abstract

Gamma oryzanol (GO) is the rice bioactive compound which presents various therapeutic effects. However, GO is relatively unstable to environmental factors during processing and storage. The objective of this work was to produce GO microparticles encapsulated with inulin and Tween80 (GOINs) by spray-drying. Response surface analysis was used for the optimization of the encapsulation to get maximum % encapsulation efficiency (%EE) of GO. Three process variables for the concentration of 10–20% inulin (w/v), 3–5% Tween 80 (w/v), and 3–5% GO (w/v) were investigated. Quadratic polynomial regression model for the optimization with R² at 0.92 was obtained from the study The optimum condition was 20% inulin (w/v), 3% Tween 80 (w/v), and 3% GO (w/v) which yielded a high % EE of 82.63% and particles size at 1,154.60 ± 28.85 nm Fourier transform infrared spectroscopy demonstrated that GO was encapsulated inside the inulin matrix. Our study provided potential and improved hygroscopicity ranged from 6.51 to 10.22 g H₂O/100 g dry weight of GO in spray-dried microcapsules.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13197-024-05988-0.

Introduction

Rice is widely cultivated and consumed as a staple food in many Asian countries including Thailand. Gamma oryzanol (GO) is a rice specific bioactive substance consisting of polyphenols such as ferulic acid (4-hydroxy-3-methoxy cinnamic acid) esters with phytosterols and triterpene alcohols (Lerma-García et al. 2009). GO components have shown the potential of many therapeutic properties such as reduction of cholesterol levels, modulation of the pituitary secretion, inhibition of the gastric acid secretion and platelet aggregation (Ferrannini et al. 2007; Rodsuwan et al. 2021a, b, c). GO also has potential applications as a natural antioxidant claimed to prevent lipid oxidation on thermal stability and act as a preservative in cosmetics (Sunil et al. 2015).

The recent trend of consuming nutritional food products demonstrates the incorporation of inulin into food products, which contributes to enhancing the product and has a beneficial effect on humans (Tsokolar-Tsikopoulos et al. 2015). Inulin is one of plant fructans that naturally occur as a plant storage carbohydrate in the Asteraceae family. It is a polymer of β-(2,1)-linked D-fructose units with a terminal glucose residue linked by different lengths of fructose unit chains (Barclay et al. 2016) The molecular structure of inulin has only one atom of the polymer backbone attached to the fructose ring, making inulin very flexible and resulting in a variety of different polysaccharide structures (Barclay et al. 2016). Among the natural sources of inulin, chicory (Cichorium intybus L., var. sativum) is most used due to its high content of inulin with a high degree of polymerization. Inulin and fructo-oligosaccharides are essential natural ingredients used in the food industry because of their desirable and diversified functional properties. These compounds also behave as prebiotics, meaning they are non-digestible by human but only by certain colon bacteria (probiotics) such as Bifidobacteria (Roberfroid, 2000). Thus, this selective stimulation of gut bacteria can provide several benefits to the host health. Inulin, presented a low glycemic, has been utilized as a wall material for encapsulation of essential oils, garlic extract, pineapple peel extract, and non-dewaxed propolis using spray-drying (Fernandes et al. 2014).

The microencapsulated bioactive compounds have been widely used to overcome a number of challenges, including preserving the chemical stability of bioactive compounds throughout processing and storage, masking the unpleasant taste, and avoiding undesired interactions with the matrix for a variety of food applications. Encapsulation can be achieved by processes such as spray-drying (Suwannasang et al. 2021; Rodsuwan et al., 2024c), freeze-drying (Ahmed et al. 2022), extrusion (Bamidele and Emmambux 2021), liquid–liquid dispersion (Rodsuwan et al. 2021a, b, c; Pithanthanakul et al. 2021), and coacervation (Oliveira et al. 2022). Spray-drying is a popular process for microencapsulation and for drying antioxidant foods, natural food colors, flavors, and oils (Jimenez-Gonzalez et al. 2022; Suwannasang et al. 2022; Lourenço et al. 2020).

Rodsuwan and coworkers (2021b) successfully used the liquid–liquid dispersion technology to encapsulate GO with zein and dried the nanocapsules obtained by freeze-drying, which led to an encapsulation efficiency (EE) of 67.77–84.97%. To save the cost and drying time, it is worth of studying conditions of spray-drying the GO-loaded nanocapsules with an additional wall material. In a study, GO, low methoxyl pectin, and Tween 80 were blended to prepare emulsions that were then sprayed into a calcium chloride solution to cross-link pectin, and the EE after spray-drying emulsions was between 82.15 and 96.78% (Lee et al. 2009). The need of additional calcium cross-linking step increases the complexity and cost of encapsulation. We hypothesize that an EE of 80% and higher can be achieved by substituting pectin with flexible inulin.

The first objective of this study was to optimize emulsion formulations using a three-factor, three-level central composite design (CCD) to prepare microcapslues using spray-drying. The three factors were inulin, Tween 80, and GO concentrations used in emulsification. The conditions were optimized for the maximal EE using response surface methodology (RSM) to prepare microcapsules that were characterized for particle size, structure, and interactions.

Materials

GO was supplied by Tsuno Rice Fine Chemicals Co., Ltd. (Wakayama, Japan). Distilled water was obtained from a Milli-Q Plus purification system. All other chemicals and solvents were analytical grade except inulin from Fuji Nihon Thai Inulin Co., Ltd. (Bangkok, Thailand) and Tween 80 from Union Chemical 1986 Co., Ltd. (Bangkok, Thailand), which were a food grade ingredient.

Methods

Preparation of GO and inulin suspension and spray-drying

The optimization of inulin (wall materials), Tween 80 (surfactant), and GO (core) concentrations was carried out. The GO emulsions were prepared following the method of Lee et al. (2009) with inulin substituting for pectin and changing the component concentrations. Briefly, inulin was dissolved in distilled water, followed by adding GO and Tween 80 to form mixtures containing 10–20% (w/v) inulin, 3–5% (w/v) Tween 80, and 3–5% (w/v) GO. The suspensions were thoroughly mixed on a stirrer (IKA^® C-MAG HS 7, Staufen, Germany) at 500 rpm for 30 min. The prepared suspensions were then spray-dried at 160 °C using a spray-dryer (B-191 Mini Spray-dryer, Büchi Labortechnik AG, Flawil, Switzerland) with a 0.7 mm diameter nozzle. The mixture was fed into the drying chamber at a feed rate of 3–4 mL/min. A three-factor, three-level CCD was used to optimize the conditions of SDMs to obtain maximal %EE. The experimental design consisted of 15 experimental points (Table 1) that included replicated center points, and the midpoints of each edge in the multidimensional cube were defined as the region of interest. The three factors, inulin, Tween 80, and GO concentrations, were examined as independent variables. The actual values of each variable were coded at three levels (low, mid, and high) for statistical analysis (supplementary Table 1). The ranges of the independent variables were initially determined from preliminary experiments, at which the effects of optimized conditions on the EE were examined. The quadratic model was expressed for the prediction of the optimal point using the following equation:

$$Y={\beta }_0+\sum _{i=1}^3{\beta }_i{X}_i+\sum _{i=1}^3{\beta }_{\mathit{ii}}{X}_i^2+\sum _{i=1}^2\sum _{j>i}^3{\beta }_{\mathit{ij}}{X}_i{X}_j$$ 1

where Y is the predicted response (%EE), and β₀, β_i, β_ii, and β_ij are constant, linear, quadratic, and cross-product regression coefficients, respectively. The statistical analysis of experimental data was performed using Minitab version 18 (Minitab Inc., State college, PA, USA).

Table 1.

Yield, experimental and predicted values of GO encapsulation efficiency, a_w, and hygroscopicity of spray-dried microcapsules obtained at different runs

Runs	Coded factor levels			Real factor levels			Yield	Encapsulation efficiency (%)		aw	Hygroscopicity(g H2O/100 g dry weight)
	A	B	C	A	B	C		Experimental	Predicted
1	− 1	− 1	− 1	10	3	3	68.96	73.36	73.34	0.15 ± 0.004	10.02 ± 0.18
2	1	− 1	− 1	20	3	3	52.67	82.63	81.76	0.20 ± 0.003	6.51 ± 1.08
3	− 1	1	− 1	10	5	3	64.13	75.35	74.9	0.18 ± 0.003	9.57 ± 0.09
4	1	1	− 1	20	5	3	55.07	78.36	80.11	0.19 ± 0.004	9.83 ± 0.22
5	− 1	− 1	1	10	3	5	34.83	70.89	69.66	0.14 ± 0.001	10.22 ± 0.65
6	1	− 1	1	20	3	5	47.65	77.07	78.04	0.16 ± 0.002	10.18 ± 0.27
7	− 1	1	1	10	5	5	65.34	72.16	73.55	0.15 ± 0.004	7.00 ± 1.31
8	1	1	1	20	5	5	51.85	78.17	78.71	0.20 ± 0.001	9.32 ± 0.93
9	− 2	0	0	7	4	4	41.11	68.23	68.66	0.20 ± 0.002	7.64 ± 0.24
10	2	0	0	23	4	4	53.06	81.25	80.08	0.29 ± 0.004	6.94 ± 0.19
11	0	− 2	0	15	2	4	52.13	75.65	76.58	0.16 ± 0.001	8.15 ± 0.36
12	0	2	0	15	6	4	61.87	80.12	78.46	0.29 ± 0.003	10.11 ± 0.35
13	0	0	− 2	15	4	2	54.69	79.14	79.15	0.15 ± 0.003	9.46 ± 0.26
14	0	0	2	15	4	6	60.72	75.62	74.88	0.17 ± 0.002	7.72 ± 0.18
15	0	0	0	15	4	4	48.83	78.32	77.03	0.15 ± 0.001	8.57 ± 0.35

Determination of yield and encapsulation efficiency (EE)

The EE of SDMs was determined following the method of Rodsuwan et al. (2021a, b, c). Briefly, approximately 5 mg of the microcapsule powder was suspended in 4 mL of methanol and dichloromethane (1:1, v/v). The suspension was sonicated for 10 min and filtered with a nylon syringe filter (0.45 µm). The GO content in the permeate was measured with high performance liquid chromatography (HPLC) using an Agilent 1200 HPLC system (Agilent Technology, Germany). The HPLC system was equipped with a UV–vis detector, a Zorbax Eclips XDB-C 18 analytical column (Agilent, 250 mm × 4.6 mm, 5 µm), and a Zorbax XDB-C18 guard column (Agilent, 12.5 mm × 4.6 mm, 5 µm). The detector was set at 330 nm. The mobile phase was a mixture of methanol, acetonitrile, dichloromethane, and acetic acid at a ratio of 55:35:9.5:0.5. The mobile phase was run at the isocratic mode with a flow rate of 1.4 mL/min. The sample injection volume was 20 µL, and the run time was 25 min. The EE and yield of spray-drying was calculated as follows:

$$\text{EE (\% )}=\frac{\text{GO}\text{amount}\text{in}\text{SDMs}}{\text{Total}\text{GO}\text{amount}\text{added}\text{to}\text{the}\text{preparation}}\times 100$$ 2

$$\text{Yield (\% )}=\frac{\text{Mass}\text{of}\text{SDMs}}{\text{Total}\text{mass}\text{of}\text{Tween},\text{inulin},\text{and}\text{GO}\text{used}\text{to}\text{prepare}\text{SDMs}}\times 100$$ 3

Water activity (a_w) and hygroscopicity

The A_w was measured using an Aqua Lab Lite water activity meter (Decagon Devices Inc., Pullman, USA). The hygroscopicity of the SDM powders was determined following the method described by Cai and Corke (2000) with some modification. Approximately 0.5 g of each sample powder was placed in a desiccator containing a saturated solution of NaCl (75.3% relative humidity). The results were determined after 7 days of ambient storage as the mass of moisture absorbed per 100 g dry weight of sample.

Morphology of the particles

Size distribution of SDMs were analyzed by laser diffraction particle size analyzer 131 (Partica mini LA-350, Horiba, Japan) using distilled water as the dispersant before a measurement. The mean diameters over volume (D 43) of dried powder were reported.

In addition, the morphology of SDMs was studied using scanning electron microscopy (JSM-6610LV, Jeol Ltd, Tokyo, Japan). The SDMs were placed on strips of double-faced carbon tape (Ted Pella, Inc., Redding, CA) before fixing on aluminum stubs. SDMs were fractured for cross-sectional SEM by slicing a razor blade perpendicularly through a layer of microcapsules prior to sprinkling on the tape. Images of the microparticles were captured with an acceleration voltage of 15 kV and a current of 1,750 mA.

Fourier-transform infrared (FTIR) spectroscopy

FTIR spectra of GO, inulin, and SDMs were determined following the method of Luo et al. (2011). In brief, the samples were mixed with potassium bromide (KBr) at 99% (w/w) to form a pellet and analyzed with an FTIR 4100 instrument (Jasco International Co., Ltd, Tokyo, Japan). The measurement was recorded at a wavenumber range of 4000–400 cm^{−1 (}each an average of 14 scans).

Results and discussion

Yield and EE of SDMs

The yield of SDMs in 15 experimental runs ranged from 34.83 to 68.96%, as shown in Table 1. The minimum EE, 68.23%, and the maximum EE, 82.63%, was observed in experiments No. 9 and 2, respectively. The regression analysis on experimental data of EE was obtained as follows.

$$\begin{array}{cc}\hfill EE(\%)& =61.60+2.51A-0.36B-3.14C-0.03AA+0.12BB\hfill \\ \hfill & -0.05CC-0.16AB-0.002AC+0.58BC\hfill \\ \hfill \end{array}$$ 4

where, A, B, and C represent inulin, Tween 80, and GO concentrations (% w/v), respectively.

The effectiveness of the regression equation and the significance level of the model were assessed by an Analysis of Variance (ANOVA) test. According to the result of ANOVA in Table 2, the p value for the model was 0.001, indicating the high significance of the model. The lack of fit was also statistically significant (p ≤ 0.05). Bashir et al. (2010) suggested that there may be some systematic variations unaccounted for in the hypothesized model or the exact replicate values of the independent variable in the model that provide an estimate of pure error. The predicted values of EE obtained using Eq. (4), and the experimental values of EE are presented in Table 1. Data from the verification experiments indicated small differences between the experimental and predicted results. The maximum EE of SDMs, 82.63%, was observed for the formulation with 20% inulin, 3% Tween 80, and 3% GO. Meanwhile, the minimum predicted EE of SDMs (68.66%) was obtained at 6.59% inulin, 4% Tween 80 and 4% GO. Our study showed that A, C, and A² were less than 0.05, indicating significant effects on the variable counts. The order of variables affecting viable counts was as follows: inulin (A) > GO (C) > Tween 80 (B). The correlation coefficient for a good-fit model should be at least 0.80. The value of correlation coefficient (R²) obtained in the present study for EE was 0.9238, indicating that the response model did not explain only 7.62% of the total variations. The SDMs were then produced at conditions resulting in the maximum EE for characterizing the morphology, particle size, and functional groups with FTIR.

Table 2.

The ANOVA of spray-dried microcapsules in the central composite design with encapsulation efficiency as the response value

Source	Degree of freedom	Sum of squares	Mean square	F-value	p-value
Model	9	205.319	22.813	13.46	0.001
A	1	157.422	157.422	92.89	0.001
B	1	4.238	4.238	2.5	0.145
C	1	21.991	21.991	12.98	0.005
A2	1	12.705	12.705	7.5	0.021
B2	1	0.431	0.431	0.25	0.625
C2	1	0	0	0	0.987
AB	1	5.168	5.168	3.05	0.111
AC	1	0.001	0.001	0	0.981
BC	1	2.703	2.703	1.59	0.235
Error	10	16.947	1.695	–	–
Lack-of-Fit	5	14.398	2.88	5.65	0.04
Pure error	5	2.548	0.51	–	–
Total	19	222.265	–	–	–

The pareto chart indicates significant factors on EE of SDMs. The linear and the quadratic models for inulin (A) followed by GO (C) were positively correlated and highly significant (supplementary Fig. 1). Among linear and quadratic terms, AA was found to be positively correlated to EE, but BB and CC were found to be insignificant. The presence of inulin improved drying properties of the wall matrix, most likely by enhancing the formation of a dry crust around the atomized droplets (Fernandes et al. 2014).

The results of this study showed that EE depends mainly on the ratio between the GO (core) and inulin (wall material). The higher EE at higher wall material and core concentrations is probably related to the higher amounts of solids facilitating drying and entrapment of the core. The trend agrees with the results of other researchers (Tupuna et al. 2018). Inulin is a low-molecular weight polysaccharide, and its function as a plasticizer may prevent the drying-induced surface shrinkage of microparticles to enhance the EE (da Silva Carvalho et al. 2016). Finally, the results suggest that inulin is advantageous in relation to GO loading since Tween 80 mainly acts as an emulsifying agent and matrix forming material while inulin serves as a coating agent.

The two-dimensional contour plots can describe the effects of mutual interactions between the independent variables inulin, Tween 80, and GO concentrations on EE. As shown in Fig. 1a–c, the influence of inulin and GO concentrations on EE were more significant than that of Tween 80 concentration, which had limited effects on EE. The contour plot in Fig. 1a–c shows weak mutual interactions between parameters AB, AC and BC, indicating that the mutual interactions between inulin, Tween 80, and GO concentrations were not significant. However, the increased amounts of inulin increased the extent of GO diffusion into the gelling medium during the microparticle formation in the spray-drying process. This suggests that Tween 80 at 2.3% concentration was sufficient to produce SDMs with a high EE. Daza et al. (2016) reported the increased yield with the increased carrier agent during spray-drying, which might be due to improved water removal at higher temperatures. Moreover, the protective effect of a carrier at higher concentrations can prevent the adhesion of particles to the chamber wall.

Fig. 1.

Contour plots showing interactions of variables on encapsulation efficiency% of spray-dried microcapsules: inulin and Tween 80 concentrations A; inulin and GO concentrations B; Tween 80 and GO concentrations C

a_w and hygroscopicity

Moisture content and a_w are important properties to be considered for the stability of food products during processing and storage. SDMs prepared from all conditions exhibited a_w lower than 0.3 (Table 1), suggesting a microbiological stability of the products since limited or no microorganisms can grow at a_w lower than 0.3 (Syamaladevi et al. 2016). Hygroscopicity is a characteristic of wall materials that could cause the absorption of moisture from surrounding atmosphere (Barbosa-Cánovas et al. 2020). Inulin in the amorphous powder form has hygroscopicity and acts as a humectant (Wong et al. 2022). In this study, the hygroscopicity of SDM powders ranged from 6.51 to 10.22 g H₂O/100 g dry weight (Table 1). These results are lower than 15.7–18.8 and 13.8–19.4 g H₂O/100 g dry weight, respectively, for cagaita microencapsulated with gum arabic and inulin prepared by spray-drying (Daza et al. 2016). Lisiecka et al. (2023) examined the effects of pectin replaced inulin as the wall material for black hollyhock flowers extracts obtained through spray-drying. The study found that increasing the proportion of pectin, while replacing inulin, led to a higher hygroscopicity and reduced solubility in water. The observed low hygroscopicity in SDMs powders in this study offers distinct advantages over comparable microcapsules. These characteristics may contribute to enhanced stability, improved flowability, and an extended shelf life, providing practical benefits for applications. The variable that most affects the hygroscopicity of SDM powders is inulin, and the highest concentration of inulin, 20%, resulted in the lowest hygroscopicity of 6.51 ± 1.08 g H₂O/100 g dry weight (Table 1). The results agree with those observed for cagaita powders obtained by spray-drying, which showed a decrease in hygroscopicity with an increase in carrier concentration (Daza et al. 2016). The results in the present study confirm the ability of inulin as a wall material to improve the hygroscopicity of SDM powders important to the stability during storage and application.

Morphology and particle size distribution of the optimized SDMs

Physical characteristics of SDMs prepared at the optimized conditions were evaluated for morphology and particle size. The powder showed agglomeration (Fig. 2), and individual particles revealed sphericity with surface dents. Microcapsules aggregate due to excessive wetting during wet quenching, which was facilitated by the co-current direction of the coating solution spray. Because of this, some of the particles formed bridges among themselves and joined together to make huge, and wet clumps (Anandharamakrishnan 2015). The agglomeration made the product to be more flowable in the process, caused more aggregation and resulted in an increased particle size. Similar findings were reported by Morelo et al. (2019). A collision among particles during drying, single droplets from a solubilized feed may combine and form agglomerates particles.

Fig. 2.

Scanning electron microscopy images of the microparticles A and cross-section B of spray-dried microcapsules prepared with 20% (w/v) inulin, 3% (w/v) Tween 80, and 3% (w/v) GO. Scale bars represent 5 and 10 µm in (A) and (B), respectively

The distribution and PDI (0.21) of the SDMs produced at the optimized conditions are shown in Fig. 3. The sample had a mean diameter of 1,154.60 ± 28.85 nm. Kim et al. (2010) reported that the mean particle size of microparticles with GO loaded in calcium pectinate was approximately 29.78 µm, which was larger than the size of SDMs in this study. The small particles of SDMs in the present study may be significant to properties such as appearance, flowability, and dispensability.

Fig. 3.

The particle size distribution and PDI of spray-dried microcapsules prepared with 20% (w/v) inulin, 3% (w/v) Tween 80, and 3% (w/v) GO

FTIR

The structures of GO, inulin and SDMs prepared from the optimized formulation were characterized with FTIR to investigate functional groups and possible interactions between GO and carrier materials after spray-drying, shown in Fig. 4. The bands from 3000 to 3700 cm⁻¹ in all samples are related to hydrogen bonds and O–H stretching in carbohydrates, polyphenol, and carboxylic acids (Santos et al. 2018). The characteristic absorption peaks of inulin were located at 2942 (CH₂ stretching), and 1037 cm⁻¹ (C–O–C bending) (Akram and Garud 2020). The FTIR spectra of inulin and GO in the present study have similar peaks to those reported by Akram and Garud (2020) and Rodsuwan et al. (2021a, b, c), respectively. The FTIR spectrum of SDMs showed main peaks at the same wavenumbers as those of GO and inulin but the peaks were broadened, possibly because the formulation contained around 20% (w/w) dry basis inulin. Some peaks from 1256 to 1690 cm⁻¹ of SDMs shifted and disappeared. The peaks of SDMs at 1265 and 1521 cm⁻¹ are due to C–O and C = C stretching of GO. Major changes in the spectrum of SDMs were slight shifts at 1513–1521 cm⁻¹, which might correspond to the possible interaction of GO with carbonyl ester groups at the interfacial part of the SDMs The FTIR results confirm the absence of new bonds formed from production of SDMs.

Fig. 4.

FTIR spectra of GO, inulin, and spray-dried microcapsules prepared with 20% (w/v) inulin, 3% (w/v) Tween 80, and 3% (w/v) GO

Conclusion

We have demonstrated the optimization of encapsulation conditions for EE of SDMs based on central composite design (CCD) with response surface methodology (RSM). The EE was significantly affected by the concentrations of inulin and GO and was the highest at inulin concentration of 20% (w/v). The hygroscopicity of SDMs decreased with an increase of inulin concentration. For the SDMs produced at the optimized formulation with 20% (w/v) inulin, 3% (w/v) Tween 80, and 3% (w/v) GO, SEM examination revealed the sphericity of individual particles that were agglomerated, the particle size analysis showed a narrow peak with a mean diameter of 1154.60 nm and a PDI of 0.21, and FTIR confirmed the physical interaction between components. Findings from the present study showed the possibility of using Tween 80 to emulsify GO and inulin as a wall material in spray-drying. Further research will explore various topics, including free GO before and after spray-drying, response effectiveness such as yield, hygroscopicity, and solubility as affected by relevant parameters, nutrient release characteristics during simulated gastrointestinal digestion, in vitro and in vivo bioavailability, assessing physical and chemical stability under varying environmental conditions, and ultimately developing SDMs with desired properties as food ingredients and enhanced health benefits.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

UR conceived the study, conducted experiments and data curation, and wrote the manuscript draft. VR carried out conceptualization, funding acquisition, writing—reviewing, and editing of this work. BT and SV carried out the visualization and data analysis. KT carried out the conception and supervision. QZ carried out the supervision, review, and editing. SP carried out data support and funding acquisition.

Funding

Thailand Institute of Scientific and Technological Research (886109) Thailand Research Fund, under the Research and Researchers for Industries Program (PHD59I0053).

Code availability

Not applicable.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

All the authors mutually agree for submitting this manuscript Journal of Food Science and Technology (original research article).