Optimization of orange juice formulation through using lactose‐hydrolyzed permeate by RSM methodology

Abstract Permeate is the by‐product of the process of ultrafiltration in a kind of cheese making process in which a semipermeable membrane filters the liquid. It mainly contains 4.5%–4.8% lactose and 0.44%–0.47% mineral salts which make it a safe disposal issue. This study was conducted to use permeate and its lactose as an alternative to sugar, and to use these useful permeate compounds in an optimized orange juice formulation. Milk permeate, as a waste disposal of dairy companies, was applied in lactose hydrolyzed form as the cost effective sugar and water substitution in production of orange juice. The RSM optimization method was applied for formulating beverage mixture. The heated and nonheated permeate samples were incubated with β‐glycosidase enzyme in three thermal ranges (35, 40, and 45°C), 3 time intervals (60,150, and 240 min), and 3 enzyme levels (0%, 0.1%, and 0.2%). The degree of hydrolysis was determined by MilkoScan analyzer. In the next step, optimization of orange juice was accomplished with a mixture of sugar (10%–40%) and hydrolyzed permeate (10%–40%) with specific Brix through RSM statistical design. The physicochemical properties and sensory evaluation were measured during 8 weeks of storage. At the first stage of the study, the heated sample with 0.1% enzyme density, which was incubated for 150 min at 40°C, was yielded the best result. At the second stage, which was the juice production and evaluation, the statistical analysis showed increasing trend of pH and sugar content, but density and vitamin C showed a decreasing trend during storage time (p < .05). The optimal condition was obtained in taking 35% permeate and 41 days of storage in which the values of formalin, vitamin C, and sensory tests were in the highest levels.

This study intended to use lactose-hydrolyzed permeate to replace a large part of water and also the sugar in an optimized orange drink formulation to produce a marketable, natural, and healthy product with high nutritional qualities in addition to decrease the wasted disposal and the environment pollution. The freezing point was considered as an indicator of lactose hydrolysis. Introducing lactose-hydrolyzed permeate in orange juice formulation was studied through RSM optimization method. The produced orange juices were evaluated through physicochemical analysis including formalin index, reducing sugar amount, ascorbic acid measurement, and sensory evaluation.

| Materials
The permeate was obtained from the ultrafiltration of whole milk in the Pegah Dairy Co. The orange concentrate was prepared from Noosh Co. and stored in refrigerated condition (−20°C) before its utilization. The β-galactosidase of the Christian Hansen was used to hydrolyze permeate samples.

| Permeate enzymatic hydrolysis
The heated and nonheated permeate samples were incubated and hydrolyzed by β-galactosidase in different thermal range,

| Beverage preparation
The beverage samples were prepared by using orange concentrate and sugar mixture (10, 14.5, 25, 35, and 40) and hydrolyzed permeate (10, 14.5, 25, 35, and 40), which provided 50% of the total Brix of the beverage. The samples were placed in the beaker on a magnetic stirrer and they were mixed up together and then were pasteurized at 90°C for 5 min (Singh et al., 2014). In the next step, the cold juice was poured into sterile capped bottles and stored at a refrigerated temperature. The physicochemical properties of the samples including pH, reducing sugar, vitamin C, and sensory properties were measured during 60 days storage.

| ME A SUREMENT OF PHYS ICOCHEMIC AL INDICE S
The acidity was measured in terms of lactic acid based on titration and the pH was measured by a digital laboratory pH meter (WTW537) with glass electrode. The protein was measured by the Kjeldahl analysis method, the density was measured by pycnometer, and the Gerber method was used to measure the fat content in permeate. The reducing sugar was investigated by using Lane and Eynon volumetric method (AOAC, 923.09), and formalin indices were assessed using standard methods (Institute of standards and industrial research of Iran; fruit juices, test methods; (No. 2685).

| Formalin index
Briefly, for formalin index measurement, 25 ml of orange juice was titrated in a beaker with 0.25 N NaOH to pH 8.1 on the pH meter; then, 10 ml of neutralized formaldehyde solution was added. The solution was titrated potentiometrically to pH 8.1 with 0.25 N NaOH after about 1 min. The formalin index was calculated as follows: where V is the volume of the utilized NaOH, N is the normality of NaOH, and V 0 is the volume of the sample.

| Sensory evaluation
The sensory evaluation was performed by 10 trained panelists.
Briefly, 60-70 ml of cool beverage was given to the panelists. Their location was constant during the sensory test, and the time of the sensory tests was selected around 11 a.m. The sensory testing was performed according to the linear method and the general acceptance of the beverage samples by the reviewers. Each time, the panelists were asked to mark the appropriate form after testing the drink, while considering the sensory properties such as color, taste, appearance, and sensation of the mouth. Between each assessment, the panelists consumed water to eliminate the taste of the previous sample. For linear evaluation, a linear length of 20 cm was drawn, and its two ends were defined as very desirable and very undesirable and the optimal term was placed in the middle. Each of the evaluators marked a linear line on this horizontal line after testing the samples that they were responsible for their evaluation. Finally, the sensory evaluation score was determined based on a related scale in which each 1 cm was considered a score (Sharma, Choudhary, Thakur, & Thakur, 2019).

| Ascorbic acid measurement
The iodine titration test (iodometry) was used to measure vitamin C (Aghajanzadeh, Kashaninejad, & Ziaiifar, 2016). This method was used to determine the endpoint of titration from starch as a reagent.
First, 20 ml of the orange drink was transferred to a 250 ml flask and mixed with 150 ml of distilled water. After adding 1 ml of 1% starch indicator solution, the solution was titrated with iodine solution until a blue color appeared.

| Experimental design
The response surface methodology (RSM) was employed in two stages: (a) to study the effects of three qualitative factors (enzyme content, incubation temperature, and incubation time) and one nominal factor (heat treatment) in permeate lactose hydrolysis and (b) to evaluate the effect of the storage time (8 weeks) and the permeate amount (10%-40%) in the orange beverage prepared through using selected treatment from the first stage. After implementation, the variance and the regression analysis were performed on the data and the Fisher distribution was used to determine the significant effects (p < .05).

| Sensory evaluation
The sensory properties of the orange beverage during 8 weeks storage at refrigerator temperature are shown in (Figure 2). As reflected by the coefficients in Table 3, the interaction effect of the storage time and the permeate amount on sensory evaluation of the beverages were significant (p < .05). With the increase of the permeate's percentage and by the predominance of its taste in the beverages, its acceptance by the evaluators was expected to decrease. According to the chart, samples with a higher percentage of permeate had a lower score, and samples with a lower percentage of permeate, even in a longer period of time, were able to receive a higher score in sensory evaluation. Findings of Sakhale, Pawar, and Ranveer (2012) on a whey-based mango beverage showed that the reduction of existing water in mango juice beverage declines overall desirability score.
As shown in Figure 2, the less overall desirability was reached by the increment of the amount of the permeate and the storage time, which is probably due to the production of the aromatic compounds caused by the chemical reactions in the beverage. Therefore, the highest overall desirability was found for the samples with 35% of permeate at day 41.

| Reducing sugar analysis
The effect of the permeate amount on reducing sugar was significant (p < .05). As shown in Figure

| Total solid changes
We found that the permeate amount had a significant effect on the total solid (p < .05). By increasing the proportion of the beverage's permeate, a decreasing trend was seen in TS amount ( Figure 5). By increasing the permeate percentage, the amounts of sugar were decreased, and therefore, reducing the amount of TS was expected.
Finally, it is expected that the slight increase of TS at higher amounts of the permeate is because of the increase of the permeate related salts and compounds in the beverage.

| Density measurement and formalin index
The permeate percentage significantly declined the product density and the formalin index (p < .05). Indeed, by increasing the permeate

| Vitamin C analysis
Our analysis indicated that the effect of the permeate percentage on the trend change of vitamin C was significant (p < .05). In addition, the high adjusted R 2 value (.99) was obtained for the corresponding predictive model (Table 3). As shown in Figure  The increase of the storage temperature is also another degrading factor, and it has been reported that pasteurization reduced the amount of ascorbic acid up to 2%-6% (Das, Bhattacharjee, & Bhattacharjee, 2013;Torregrosa, Esteve, Frigola, & Cortes, 2006).

| CON CLUS ION
The orange beverage prepared from the permeate not only has the higher nutritional value compared to other available fruit beverages

ACK N OWLED G M ENTS
The authors thank the research fellows at the food science laboratories in Sari Agricultural Science and Natural Resource University, Iran, for their technical assistance.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
The human and animal testing was unnecessary in the current study.