Development and Storage Stability of Whey Sugarcane Based Functional Beverage

ABSTRACT Whey and sugarcane juice contain many functional components like immunoglobulins and antioxidants, which are very beneficial for human health. Pakistan is the world’s 4th and 5th largest producer of milk and sugarcane, but we are still importing dairy products and sugar from foreign countries. Using whey and sugarcane juice to make value-added functional drinks can enhance its consumption and ultimately be exported to foreign markets. In this research, sugarcane juice was incorporated in whey in five different Blends; 10, 20, 30, 40, and 50%. The product was subjected to different physicochemical analyses at 0-day, 7-day, 14 days, 21 days and 28 days of storage. Results were analyzed through two-way factorial under CRD, which indicated that there was a highly significant effect (p < .01) of blends on all the proximate analyses (fat, protein, total solids, and ash) of the product, while the effect of storage (days) was non-significant (p > .05). Moreover, the interaction effect (Blends*storage days) was non-significant (p > .05) on all the test parameters. However, the individual effect of days and Blends on other physiochemical tests; pH, acidity (%), sedimentation (%), viscosity (c.p), and antioxidants (µg/mL) was highly significant (p < .01). While density (g/ml) and total phenolic contents were significantly affected (p < .01) by Blends with a non-significant (p > .05) effect of storage (days). Apart from that, the sensory analysis indicated that all the parameters were affected by both Blends and storage (days) with a highly significant effect (p < .01). Conclusively this whey-based sugar cane beverage is an innovative and newly emerged functional drink with a lot of nutrition and health-promoting effects. However, it needs further research to evaluate the proper storage conditions and the use of different preservatives to enhance its keeping quality.


Introduction
Consumers are now demanding functional drinks full of these sorts of phenomenal nutrients; therefore, the beverage industries intend to research and develop novel compositions and enhanced functionality. [1]Consequently, products like whey-based functional drinks, naturally flavored drinks, whey-based fruit drinks, functional energy drinks, whey-based tea and so on, are transferring the future of the beverage market from traditional liquids foods like 100% juices, colas, milk, teas and others. [2]A significant proportion of world milk production is utilized for the manufacturing of cheese, out of which 95% comes out as whey, a liquid obtained after the coagulation and curdling of milk. [3]The production process classifies whey into two main categories: sweet and acidic.Whey is a rich source of lactose.
Moreover, whey contains other functional components, including immunoglobulins, α-lactalbumins, β-lactoglobulins, lactoferrins, and serum proteins.The digestibility and biological value of whey proteins are more than the egg protein. [3]The products of whey in the world market are, i.e. concentrates, isolates, hydrolyzates, whey powders and other fractions like β-lactoglobulin, α-lactalbumin, casein glycomacropeptide (GMP), lactoperoxidases and lactoferrins. [4]These components of whey are associated with improved immunity, augmenting anticancer attributes, boosting antimicrobial and anti-adhesive effects in opposition to pathogenic properties and antiviral properties. [5]Approximately 85 million tons of whey is produced in the Indo-Pak region, out of which more than 40% is wasted, which is putting the environment on the verge of pollution. [6]Various whey-based functional drinks consisting of plain, alcoholic, carbonated, and fruit flavored have been successfully developed and marketed worldwide. [7]ugarcane juice is extracted from the stem of the sugarcane plant it contains about 20% total solids, high in polyphenolic compounds up to 160 mg/L.Additionally, it also possesses flavonoids and phenolic acids, which promote its antioxidant activity. [8]About 35 types of phenolic compounds have been reported so far, belonging to the flavonoid group capable of fighting against free radicals.Sugarcane juice is a relatively cheaper source of antioxidants, vitamins (A, C and D), minerals (Fe, Ca, P, K and Mg) and energy due to its higher sucrose contents. [9]Conclusively, whey-based drinks are helpful for muscle rehabilitation, muscle cramping, and nucleotide depletion due to the presence of functional components.Developing whey-based functional drinks is also beneficial in managing whey-based waste to reduce environmental pollution.Thus, in this study, sugarcane juice is utilized in whey with different proportions as whey and sugarcane are surplus in Pakistan and the incorporation of whey into sugar cane to develop a functional drink is an innovative idea.

Procurement and raw material analysis
The raw materials, milk and sugar cane, were purchased from university farms and then whey was obtained by acidification of milk.The sugar cane juice was squeezed from the sugar cane stems.Sigma-Aldrich Ltd. (Burling-ton, MA, USA) supplied all chemicals required for the test.The whey and sugar cane juice were subjected to different physiochemical analyses.In addition, a functional beverage was made by making a blend plan given in Table 1.

Determination of pH:
The pH of the functional drink was noted by using a digital pH meter as described in standard methods of AOAC. [10]The pH meter (InoLab 720, Germany) was calibrated with 4.00 and 7.00 buffer solution.The sample was taken in a beaker, and pH electrode was dipped in the sample until a stable reading was noted.

Determination of acidity:
Acidity is estimated by titrating milk samples against NaOH solution (0.1 N) using phenolphthalein as an indicator.The method (920.124)AOAC [10] was used and acidity (%) calculated by following the formula: Determination of fat: Crude fat content in beverages was assessed by following method no.932.06 described in AOAC, [11] using the Gerber method.Fat content was noted as the reading of the butyrometer (used for Gerber fat estimation).
Crude protein: Protein content was determined using the Kjeldahl apparatus as described in AOAC [10] method (991-20).Thus, the proportion of nitrogen in the samples was estimated as a percentage by using the equation given below.While the protein (%) was calculated by multiplying the nitrogen percentage by 6.25: Percent Protein = Percent Nitrogen × 6.25 Total solids (TS): Moisture content was assessed by following method no.926.05 directed in AOAC, [10] using a hot air oven.The moisture content was calculated in percentage using the following formula: Whereas, the total solids were estimated by: Total solids (TS) = 100 -% moisture Total ash: Ash content estimation in each oven-dried sample was done by following the practice described in method no.942-05 of AOAC [10] using a muffle furnace.Samples (pre-dried in the oven) were taken in crucibles, charred on the burner, and finally put in a muffle furnace (550-600°C temperature).
Viscosity: Apparent viscosity was measured by following the method described by Sodini et al. [12] using a viscometer at 60 rpm for 15 sec.against 100 ml of sample.
Density: The density of the product was determined by using the methods described by AOAC . [10]00-ml pycnometers were used to determine density.Each pycnometer was calibrated with distillate water at 20, 50, and 80 C to measure thermal expansion.Temperatures were kept consistent using a Colora Ultra thermostatized bath with heating resistors and centrifugal circulation.The samples and heating water temperatures were controlled using previously calibrated mercurial and digital thermometers.Pycnometers were filled with samples and heated in a bath without their tops.The tops were replaced, eliminating air occlusion.Pycnometers were immediately weighed in a Sartorius AG balance.
Sedimentation: The samples were placed in 25 mL graduated cylinders, then kept at 25°C for 24 hours to evaluate the sedimentation.The sedimentation index (SI), as defined by Silva et al., [13] was acquired using Eq.
According to this protocol, just 5 mL methanol (0.1 M) solution of 2, 2-diphenyl-1-picrylhydracyl radicals were taken in test tubes having 0.1 mL of methanol excreted samples.After resting at temperature 27°C just for precisely twenty minutes.After this resting time, samples were analyzed on a spectrophotometer at 517 nm wavelength.The antioxidant level was calculated by this equation: Total phenolic contents: Total phenolic contents were analyzed using folin-ciocalteu colorimetric reagent, a method copied by Arslan and Ozcan, [14] with minor modification to get excellent results.0.1 mL (100 µL) of the extracted sample was taken with the help of a micro pipette in a test tube.Also, heaving 0.2 mL (200 µL) and distilled water 2 mL was also added, giving rest time 30 minutes for incubation at room temperature.After these 30 minutes of incubation, just 1 mL (1000 µL) and 20% solution of sodium carbonate after 1 hour of incubation mixture were analyzed on a spectrophotometer at a standard wavelength of gallic acid 765 nm.Sensory analysis: Sensory evaluation was carried out for different attributes such as smell, taste, appearance, mouth feel, and overall acceptability by a panel of trained judges using a 9-point hedonic scale described by Meilgaard et al. [15]

Statistical analysis
Analysis of variance was done on all the data for each parameter (ANOVA).The data was run in three repetitions, and results were conferred as mean ± SD.The differences were indicated significance p < .05. [16]

Ash contents
Minerals are a vital component of food as they perform plenty of functions in the body.The results of this research indicated that there was a highly significant effect (p < .01) of blends on the ash contents of wheybased sugar cane beverages.That is just because sugar cane contains less minerals than whey.As the percentage of sugar cane increased in the respective blends, the ash content was reduced.Blend zero WFDC; 0% sugar cane juice) had the highest percentage of ash, about 0.44%, while blend 5 WFD (50%) containing 50% sugar can juice had minimum ash; (0.33%,) while other Blends; WFD (10%), WFD (20%), WFD (30%), and WFD (40%) had varying amounts of ash, i.e. 0.41 ± 0.01%, 0.38 ± 0.01%, 0.36 ± 0.00% and 0.35 ± 0.01% respectively.On the other hand, effect of storage on ash of whey-based sugar cane beverage shows that it remained constant with time from 0 to 28 days and there was non-significant (p > .05)effect of storage on ash% of the beverage; the percentages of ash of whey based functional drink were found constant (0.36 ± 0.00%) during the entire storage period.The factors, which affect the ash percentage, are mainly the presence of inorganic minerals like Ca, Na, K, Fe, Mg, and many others.

Protein value
Proteins are building blocks of the body and essential components of food as these are required to grow the body properly.The results indicated a highly significant effect (p < .01) of blends on the protein's % contents because sugar cane contains fewer proteins than whey.As the percentage of sugar cane was increased in the respective blends, protein content was reduced.Blend zero (WFDC; 0% sugarcane juice) had highest percentage of protein about 0.80% while Blend 5 (WFD (50%); 50% sugarcane juice) had minimum protein percentage; 0.70%, while other Blends; WFD (10%), WFD (20%), WFD (30%), and WFD (40%) had varying amounts of protein i.e. 0.78 ± 0.01%, 0.75 ± 0.02%, 0.73 ± 0.01% and 0.71 ± 0.00% respectively.On the other hand, the effect of storage on protein percentage of whey-based sugar cane beverage shows that the proteins % of selected sugar cane whey beverage remained constant during storage of 28 days and a non-significant effect of storage on proteins of the beverage were observed.

Physicochemical analysis of drink pH of beverage:
The results of the statistical analysis of this research indicated a highly significant effect (p < .01) of Blends and storage days on the pH of whey-based beverages.This is because sugar cane has less pH than the sweet whey used in this research.As the percentage of sugar cane juice increased, the pH decreased ultimately (Table 4).Blend zero (WFDC) had 5.64 ± 0.02 pH while blend 5 WFD (50%) exhibited a minimum of nearly 4.74 ± 0.03.While the effect on pH of selected blend WFD (30%) during storage showed that the level of pH decreased with time from day 0 to day 28; the pH of whey-based functional drinks on 0, 7, 14, 21 and 28 days of storage was observed 5.18 ± 0.00, 5.09 ± 0.01, 5.00 ± 0.01, 4.89 ± 0.02 and 4.75 ± 0.00 respectively.Different other studies also supported these findings.In the development of wheybased peach beverage, the pH was also decreased in all the respective blends and days.

Density of beverage:
The results of this research indicated that the density of whey-based sugar cane beverages remained constant during the storage and there was a non-significant effect (p > .05) of storage days on density.The blends had a highly significant (p < .01)effect on density because sugar cane had more density.Blend zero (WFDC) contained about 1.02 ± 0.04 (g/ml) density while blend 5 (WFD (50%)) contained maximum density which is about 1.02 ± 0.03 (g/ml) other blends; WFD (10%), WFD (20%), WFD (30%), and WFD (40%) had densities (g/ml); 1.02 ± 0.01, 1.02 ± 0.02, 1.02 ± 0.01 and 1.02 ± 0.01 respectively.While the density of selected blend WFD (30%) remained constant with time from day 0 to day 28 and there was non-significant effect of storage on the density of the beverage; the densities of whey-based functional drink of sugar cane juice were recorded 1.02 ± 0.01 (g/ml) during the entire storage period (Table 4).

Viscosity of beverage:
The results indicate a highly significant effect (p < .01) of blends and storage days on the viscosity of whey-based sugar cane beverages.There was a lowering trend of viscosity along the blends of this functional beverage.Blend zero (WFDC) had viscosity about 18 ± 0.09 (c.p) while blend 5 (WFD (50%)) had minimum viscosity which is about 11.22 ± 0.11 (c.p) while other blends; WFD (10%), WFD (20%), WFD (30%), and WFD (40%) had varying viscosities; 15.46 ± 0.12, 14.23 ± 0.07, 12.86 ± 0.21 and 12.08 ± 0.05 (c.p) respectively.Apart from that, the effect of storage on the viscosity of the selected blend (WFD (30%)) showed that the viscosity of sugar cane whey beverage increased with time from day 0 to day 28, and there was a highly significant effect of storage (p < .01) on the viscosity of the beverage; the viscosities of whey-based functional drink at day 0, day 7, day 14, day 21 and day 28 were; 12.86 ± 0.21, 13.49 ± 0.10, 14.23 ± 0.07, 13.84 ± 0.09 and 13.95 ± 0.05 (c.p) respectively (Table 4).The factors which affect the viscosity are mainly the total solids and fibrous materials of the beverage. [17]

Sedimentation of beverage:
Sedimentation is the term used to describe some settled down particles in the bottom of the container due to the high density of particles and low solubility.The results of this research indicate that there was a highly significant effect of blends on the sedimentation of wheybased sugar cane functional beverages; that is just because sugar cane contains more total solids, fiber, and suspended particles which interact with the whey proteins and increase sedimentation.In addition, sugar cane juice contains gummy substances like dextran which also lead to the increased sedimentation of this beverage.Blend zero (WFDC) had percentage of sedimentation about 3.03 ± 0.05% while blend 5 (WFD (50%)) had maximum sedimentation percentage; 12.86 ± 0.08%, on the other side blends; WFD (10%), WFD (20%), WFD (30%), and WFD (40%) had gradually increasing amounts of sedimentations; 5.50 ± 0.02, 7.42 ± 0.09, 8.83 ± 0.11, 10.66 ± 0.06 and 12.86 ± 0.08 (%) respectively.On the other hand, the effect of storage on sedimentation of wheybased sugar cane beverage in selected blend WFD (30%), shows that the sedimentation of sugar cane whey beverage increased with time from day 0 to day 28 and there was a highly significant effect (p < .01) of storage on sedimentation % of the beverage; the percentages of sedimentations of wheybased functional drink at day 0, day 7, day 14, day 21 and day 28 were; 8.83 ± 0.11, 9.66 ± 0.06, 10.47 ± 0.23, 10.63 ± 0.10 and 10.81 ± 0.09% respectively (Table 4).

Total Phenolic Contents (mg/L):
The results of this research demonstrated that the amount of total phenolic contents remained constant during the storage, and there was a non-significant effect (p > .05) of storage days on total phenolic contents.On the other hand, the effect of blends on the amount of total phenolic contents was highly significant (p < .0).Sugar cane contains more total phenolic contents; as the percentage of sugar cane juice was increased, the total phenolic contents were ultimately increased.Blend zero (WFDC) contained about 4.83 (μg/L) total phenolic contents, while blend 5 (50% sugar cane juice) contained the maximum amount of total phenolic contents, which is nearly 76.33 (μg/L).The other blends; WFD (10%) (10% juice), WFD (20%) (20% juice), WFD (30%) (30% juice) and WFD (40%) (40% juice) had the TPC levels; 14.9 μg/L, 33.1 μg/L, 51.03 μg/L, and 64.17 μg/L (Figure 1a).While the effect of storage on total phenolic contents of selected blend WFD (30%) was slightly decreased throughout the storage period and there was non-significant effect of storage on the total phenolic contents of the beverage; the total phenolic contents of selected whey-based functional drink of sugar cane juice on day 0, day 7, day 14, day 21, and day 28 were; 51.03, 51.04, 51.01, 51.01 and 51.00 (μg/L) respectively.These findings were also supported by different other studies. [18]ntioxidants Activity: The results of this research showed that the number of antioxidants remained constant during the storage and there was a non-significant effect (p > .05) of storage days on antioxidants.On the other hand, the effect of blends on the number of antioxidants was highly significant (p < .01)just because sugar cane contains more antioxidants.As the percentage of sugar cane juice was increased, the antioxidants were ultimately increased.Blend zero (control group) contained about 0.42 (μg/mL.)antioxidants, while blend 5 (50% sugar cane juice) contained the maximum amount of antioxidants which is nearly 30.73 μg/mL.The other Blends WFD (10%) had 5.6 μg/mL, WFD (20%) had 11.63 μg/mL, WFD (30%) showed 17.08 μg/mL and WFD (40%) depicted 23.46 μg/mL of antioxidants.While the effect of storage on antioxidants in the selected blend WFD (30%) shows that the level of antioxidants was reduced with time.The change in antioxidants with days was; 17.08 μg/mL on day 0, 16.96 μg/mL on day 7, 16.68 μg/mL on day 14, 16.57μg/mL on day 21 and the lowest amount of antioxidants was on day 28 which was 16.19 μg/mL (Figure 1b).

Sensory evaluation
In this research hedonic scale containing 9 points for marking the level of acceptance was used.In addition to that, five sensory parameters were selected to be tested by the panelists which were appearance, smell, taste, mouth feel, and overall acceptability.The sensory panelists were trained  judges who were experts in sensing the quality of various foods, especially functional food.The results demonstrated that all the parameters have been non-significantly affected by blends (Figure 2) and highly significant by the storage period in a selected beverage (Figure 3).

Discussion
Functional foods comprise a vital source of chemical compounds including nutrients, antioxidants and total phenolic compounds.The different part of fruits contains these chemicals in abundant quantities.The current results are according to the findings of Thakkar et al. [19] who indicated that sugarcane contains about 80% of water in which sucrose and other substances are held in solution It is formed about 88% by weight of juice in the stem.The remaining 12% represents the insoluble cane fiber component.The other tests included pH (5.6), acidity% (0.3), fat% (0.2), protein% (0.5), ash% (0.3), density g/mL (1.03), sedimentation % (5), viscosity c.p (3.8), antioxidants μg/mL (65) and total phenolic contents μg/L ( 16). [20]In another study, a blend of apple pulp and WPC was incorporated.
The outcomes showed a highly significant effect of blends on total solids. [21]Milk contains minerals that come into whey when it is drained from the curd and become part of liquid whey in dissolved form athanasiadis. [22] During storage, significant changes were found in the pH, acidity, total sugars, and reducing sugars in a whey-based guava functional drink. [23]In another research showed similar results in which pineapple juice was incorporated into whey. [24]Moreover, one of the latest variants of whey-based functional drinks has been formulated using plant-based herbal extractsthat enhance nutritional benefits. [25]The whey-based orange beverage had pH levels ranging from 3.7 to 3.8.In addition, the peer-whey beverage was identified with pH levels of 3.8 and 3.6 with sucrose contents of 4% and 6%, respectively.The whey-based peach drink contained 3.75-3.6pH, 4 and 6% sucrose.While the apple-containing whey drink had 3.95-3.8pH with 4% and 6% levels of sucrose, respectively. [26]he main reason behind this increase in acidity was the development of different organic acids while storing the beverage.These acids include carboxylic acid, lactic acid, pectinic acid, citric acid and acetic acid, which are responsible for the increase in titratable acidity. [24]The factors affecting the density are mainly the total solids and other beverage components.However, as it is previously demonstrated, there was a non-significant effect of storage days on the total solids; therefore, the density of the wheybased sugar cane beverage is less likely to be affected by storage days.These results are also according to Ogasawara et al. [27] Different other studies also supported these findings.In the development of whey-based peach beverages, the viscosity was decreased in the blends.The reason behind this was more solids in peach than sugarcane juice, which included glucides, mineral salts, vitamins, organic acids, pigments, and sugars.Similar results were obtained in several other studies; however, some studies also depicted the opposite results.For example, in a study of the development of whey-based papaya beverage the viscosity decreased during the storage, which could be due to the development of acids that affected the viscosity.In that study, the viscosity decreased from 20 c.p to 17 c.p after the storage study of 15 days. [28]The factors, which affect the sedimentation, are mainly the total solids and other components like complex carbohydrates, fibrous materials, and sugars of the beverage. [29]In the development of the whey mango beverage, the total phenolic contents were increased in the Blend.The reason behind this was more solids in mango than in sugarcane juice, which included glucides, mineral salts, vitamins, organic acids, pigments and sugars. [29]In research, the total phenolic contents identified in whey mango beverages were between 348 mg GAE/100 ml, which were positively influenced by the incorporation of fruit contents into the whey. [27]These findings were also supported by different other studies.In the development of whey mango beverage, the antioxidants were also increased in the blend.The reason behind this was the more contents of antioxidants in mango as compared to whey.In past studies on the analysis of antioxidant activity through different methodologies like phosphomolybdenum assay, which is demonstrated as a quantitative method to assess the difference in capacities of water-soluble and fat-soluble antioxidants, which is also called the total antioxidant capacity, it has been demonstrated that the antioxidants had electron giving capacity.They act as chain terminators for the free radical formation and convert the very reactive free radicals into the less reactive substances as demonstrated by Arslan and Ozcan. [14]Hence, the research has proven that the higher reducing ability of the sugarcane extracts may have contributed to the higher antioxidant activity.The study shows that the total antioxidant activity of leaf extract juices is much higher than the sugar cane stem juice, which ranges from 62.3 to 80.26 g/mL in stem juice and 70.43 and 99.9 g/mL in the leaves extract juice. [9]Sensory analysis has been the biggest challenge to the food technologist ever to cope with the challenges, which develop during storage.Similarly, in this research, all parameters were significantly changed during the storage.In a study of apple-whey based herbal functional beverage, different sensory attributes were discussed.It was deduced that there are several biochemical reactions that keep occurring during storage, like the Maillard reaction, which causes the drink's appearance to be dark. [30]Similarly, production of whey-based watermelon juice drink by an amalgamation of whey with watermelon juice at 10, 20, and 30% with sugar of 7% has been reported by Dande et al. [31] The results demonstrated that blend (1) and blend (2) had the highest overall acceptability with 10% and 20% watermelon juice.

Conclusion
It is concluded that whey-based sugar cane beverage is an innovative and newly emerged functional drink with a lot of nutrition and health-promoting effects.The effect of blends on physiochemical tests including pH, acidity (%), sedimentation (%), viscosity (c.p), and antioxidants (μg/mL) was highly significant.While blends had a substantial impact on density (g/mL) and total phenolic contents, but storage had little effect (days).However, the sensory analysis indicated that all the parameters were affected by both blends and storage (days), with a highly significant effect.Based on current lifestyle trends, these novel functional beverages, which are well received by potential customers, may be seen as a product that promotes health and convenience.These alternative products may help make the dairy industry more sustainable by making it possible to lower costs related to getting rid of whey while only adding small costs to the dairy plant.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 1 .Figure 2 .
Figure 1. a Effect of Blends and storage days on total phenolic content of WFD.b Effect of Blends and storage days on antioxidant activity of WFD.

Figure 3 .
Figure 3.Effect of storage in selected Blend on sensorial properties.

Table 1 .
Different Blends of whey functional drink WFD.

Table 2 .
Composition of whey and sugar cane.