Development and storage stability studies of functional fruit and vegetable-based drinks incorporated with polyphenols extracted from herbs and spices

ABSTRACT

The study was designed to develop functional cucumber and pomegranate based ready-to-serve (RTS) drink as thirst quencher. To boost antioxidant activity and to add natural preservation to the drink, microwave-assisted extracts of herbs and spices; mint, lemongrass, ginger, cinnamon were incorporated either individually or collective, at an equivalent dose of 2% for comparison. In this regard, ginger extract incorporated drink maintained its storage quality owing to highest free radical scavenging potential (49 to 67%) in relevance to highest polyphenol and flavonoid contents (1203 ± 64 mg GAE/L and 178 ± 8 mg TE/L, respectively). Whereas, ascorbic acid was maximally recorded in lemongrass-based drink 22 ± 1 mg/100 mL. During 30 days storage under refrigeration, significant decrease was recorded in pH of the RTS drink along with considerable increase in acidity and browning index however, total soluble solids demonstrated non-significant pattern. Sedimentation showed an inclining trend (p˂.05) with progression in storage but comparatively less sedimentation was attributed to lemongrass incorporated drink followed by cinnamon containing drink. Turbidity also indicated significant changes but showed inverse pattern with respect to sedimentation. Additionally, ginger-based drink expressed considerably low total plate count as 2.3 ± .1 log CFU/mL with nil yeast and mold growth by all the extracts throughout storage. Further, ginger portrayed highest rate for odor, taste and consistency though, mixed herbs and spices based drink attained the best color score. Conclusively, ginger-based drink portrayed maximum antioxidant and antimicrobial properties.

KEYWORDS:

• Physicochemical aspects
• mint
• lemongrass
• ginger
• cinnamon
• preservation

Introduction

Fruit and vegetable based drinks are gaining popularity as thirst quencher, especially in summer season that prevails for a long time in countries like Pakistan. Different combinations of fruits and vegetables like apple-carrot, orange-beetroot, etc. have already gained fame as fresh and processed juices. On the same line, combinations of cucumber with pineapple, carrot, and pomegranate have become a choice of the day rather the consumers are looking forward for their processed counterparts. Fruit- and vegetable-based drinks contain a lot of nutritive and bioactive molecules that promises overall health benefits^[1]. Besides, Pakistan ranks amongst the top producers of fruits and vegetables, though half of this produce goes wasted due to improper handling and poor processing procedures^[2];[4]. In developing countries, people have more tendencies for fruits than vegetables; therefore, fruits and predominantly vegetables need to be processed for economic gains.^[5–8]

Cucumber (Cucumis sativus L.) is a popular Indian origin vegetable of Cucurbitaceae family. It contains high water content, thus low in calories and possesses the ability to cleanse body wastes and toxins. Its fresh juice contains seeds which are having cooling and hydrating effects on the body. Further, it contains antioxidants like cucurbitacins, vitamin C, and flavonoids that have the potential to address diabetes, hyperlipidemia, and oxidative stress biomarkers.^[1–4;9] Furthermore, sweet pomegranate fruit (Punica granatum L.) belongs to family Punicaceae that also carries an excellent source of antioxidant vitamins, flavonoids, and ellagitannins. These polyphenols have the ability to fight against free radical chain reactions induced in the physiological system of the body hence prevent aging.^[10,11]

Currently, scientific fraternity is keen to develop beverages which are 100% natural in terms of ingredients. To fulfill the demand of flavor, antioxidant and antimicrobial characteristics in beverages, extracts of different herbs and spices are considered owing to their GRAS status.^[12,13] In this regard, polyphenols of mint (monoterpenoids and sesquiterpenoids), lemongrass (antioxidant vitamins and essential oils), ginger (gingerols and shogaols), and cinnamon (essential oils) have proved their efficacy to extend the storage stability in varied food products, apart from imparting particular flavors and boosting free-radical scavenging potential of its consumers.^[14–23]

The reason for this research activity was to develop a preservative-free drink based on cucumber and pomegranate to satisfy the thirst desire of consumers in summer season along with the provision of nutritive and bioactive ingredients from varied herbs and spices. Besides, in this study, antiradical compatibility of herbs and spices, either individually or in combination, was also observed in cucumber-pomegranate (C-P) drink. Further, the aforementioned extracts were tested in C-P drink for their sensory response and ability to delay deteriorative changes during storage.

Materials and methods

Reagents and samples

The research was conducted in the Food Processing and Preservation and Food Analytical Laboratory of Department of Food Science and Technology, Government College Women University Faisalabad, Pakistan. Purposely, fully mature and fresh cucumber, white and sweet pomegranate, ginger, fresh leaves of lemongrass and mint, and dried barks of cinnamon with no visible signs of physical injury or spoilage were procured from the local market of Faisalabad. The standards and reagents were procured from Sigma-Aldrich and Merck (Darmstadt, Germany). The key reagents include Folin-Ciocalteu phenol reagent, sodium carbonate (Na₂CO₃, CAS 497-19-8), aluminum chloride (AlCl₃, CAS-7446-70-0), Trolox 6-hydroxy-2,5,7, 8-tetramethylchroman-2-carboxylic acid (C₁₄H₁₈O₄, CAS 53,188-07-1), iron (II) sulfate heptahydrate (FeSO₄.7 H₂O, CAS Number: 7782-63-0), gallic acid ((HO)₃C₆H₂CO₂H, CAS: 149-91-7), quercetin (C₁₅H₁₀O₇, CAS: 6151-25-3). 2,2-diphenyl-1-picrylhydrazyl (DPPH, CAS: 1898- 66-4), methanol (CAS: 67-56-1), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, CAS: 30931-67-0), iron (III) chloride hexahydrate (FeCl₃.6 H₂O, CAS: 10025-77-1), and 2,4,6-tripyridyl-s-triazine (TPTZ, CAS number 3682-35-7).

Microwave-assisted extraction

The extract of herbs; mint, lemongrass, ginger and cinnamon was prepared using the protocol of Oualcadi et al.^[24] with some modifications. Intentionally, 10 g of each shade dried samples was treated with 100 mL of 95% aqueous ethanol and kept macerated for 3 hrs under ambient conditions. Then the extraction was carried out using microwave oven (Micrologic, France) at power of 400 W for 1 min. Afterwards, microwave treated sample was filtered using Whatman No.1 paper. Lastly, the obtained extract was separated from solvent using rotary evaporator (Eyela, Japan) at 40 ± 5°C. Finally, the liquid extract was transferred to dark sterile flacon tubes of 100 mL capacity followed by air tight closure and storage at 4°C prior further use.

Formulation plan of polyphenols supplemented cucumber-pomegranate drink

The formulation in expressed in Table 1 and preparation method is discussion in Figure 1.

Figure 1. Processing line of herb supplemented cucumber-pomegranate drink.

Table 1. Percent formulation of polyphenols supplemented cucumber-pomegranate drinks.

Ingredients	Treatments
	CP0	CP1	CP2	CP3	CP4	CP5
Cucumber juice	12.35	11.35	11.35	11.35	11.35	11.35
Pomegranate juice	12.35	11.35	11.35	11.35	11.35	11.35
Mint extract	-	2	-	-	-	0.5
Lemongrass extract	-	-	2	-	-	0.5
Ginger extract	-	-	-	2	-	0.5
Cinnamon extract	-	-	-	-	2	0.5
Sugar	10	10	10	10	10	10
Water	65	65	65	65	65	65
Citric acid	0.1	0.1	0.1	0.1	0.1	0.1
Carboxymethyl cellulose	0.2	0.2	0.2	0.2	0.2	0.2

†CP₀ = Cucumber-pomegranate drink free from herb extract, CP₁ = Cucumber-pomegranate drink supplemented with mint extract, CP₂ = Cucumber-pomegranate drink supplemented with lemongrass extract, CP₃ = Cucumber-pomegranate drink supplemented with ginger extract, CP₄ = Cucumber-pomegranate drink supplemented with cinnamon extract, CP₅ = Cucumber-pomegranate drink supplemented with mixed herb extracts

Storage study of the developed RTS drink

The prepared juice prototypes were analyzed using physicochemical tools throughout storage at varied intervals; 1^st, 10^th, 20^th, and 30^th day under refrigeration.

DPPH (1, 1-diphenyl-2-picrylhydrazyl) scavenging assay

DPPH free radical scavenging ability of extract was measured by the method proposed by Sarkar et al..^[25] Purposely, DPPH solution was prepared by mixing .004 g of DPPH radical in 100 mL of methanol. The resultant fresh DPPH solution (1 mL) was added to test tube followed by the inclusion of sample (25 µL). The reaction mixture was then vortexed and kept under dark for 15 min. Then, the optical density of the samples and control were recorded at 517 nm using UV/VIS Spectrophotometer (CECIL CE 7200). The findings of the test were expressed as % DPPH and calculated by adopting following equation:

$\mathrm{DPPH}\mathrm{radical}(\mathrm{\%})=\frac{\mathrm{Control}\mathrm{OD}-\mathrm{Sample}\mathrm{OD}}{\mathrm{Control}\mathrm{OD}}\times 100$

ABTS [2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) assay

The antioxidants in juice samples supplemented with herb extracts capture free radicals in ABTS reagent by donating electrons, this leads to decrease in green color of ABTS radical as measured by Ashfaq et al..^[26] In this test, 5 mL of ABTS solution was diluted with methanol until the attainment of absorbance i.e. .700 ± .005. The prepared sample (20 µL) was be treated with ABTS solution (5 mL), and the optical density was measured at 734 nm. The sample value was then obtained through standard curve of Trolox and expressed the findings in terms of Trolox Equivalent (TE).

FRAP (ferric reducing antioxidant power) assay

The antioxidants in the developed RTS drink reduced ferric ions (colorless) to ferrous ions (blue) hence raised the absorbance at 593 nm.^[27] Briefly, fresh FRAP reagent was prepared using 25 mL acetate buffer (300 mM), 2.5 mL TPTZ (10 mM) and 2.5 mL FeCl₃·6 H₂O solution (20 mM). The FRAP reagent was mixed thoroughly with sample. After 10 min (under dark), the optical density was recorded at 593 nm. The values were expressed in μM Fe (II)/g using linear standard curve on the basis of varied concentrations of FeSO₄.

Total polyphenols (TP)

Purposely, TP of the herbal supplemented drink was determined by following the Folin-Ciocalteu method as mentioned by Sattar et al..^[28] In this context, 30 µL of the C-P juice sample was mixed with 150 µL of Folin-Ciocalteu reagent and 120 µL of 7.5% sodium carbonate solution. Afterwards, the solution was properly vortexed (10 s) and kept at 40°C (under dark) for 30 min. Then, the optical density of all the treatments were noted at 765 nm using UV/VIS Spectrophotometer (CECIL CE 7200) and results were expressed as mg GAE/100 mL juice.

Total flavonoids (TF)

Total flavonoids were assessed using aluminum chloride colorimetric assay method as elaborated by Tarafdar et al..^[29] In this method, 25 µL of the sample was mixed with 10 µL of 5% aqueous sodium nitrite then kept at room temperature for 15 min. Afterwards, 1 M sodium hydroxide dissolved in 50 µL of distilled water along with 15 µL of 10% aqueous aluminum chloride were mixed into the whole mixture, and then optical density of the samples were measured at 430 nm. Finally, the attained standard curve was used to express the data as quercetin equivalents (QE).

Quantification of ascorbic acid (AA)

The ascorbic acid content of the juice samples was measured through titration using 2,6-dichlorophenol indophenol dye that is blue in color but changed to pink in the presence of ascorbic acid within a pH range of 1 to 3.5.^[30] Dye factor was determined by standardizing dye solution against standard ascorbic acid and 3% meta-phosphoric acid till light pink color achieved and titrate volume noted. The sample was taken and diluted with 3% meta-phosphoric acid and then filtered. Afterwards, an aliquot of sample was taken and titrated against dye till light pink color appeared. Note down the volume of dye used and concentration was expressed as mg ascorbic equivalent to mL of dye solution.

Storage stability aspects

pH

The pH of the developed juice samples was determined using a digital pH meter (Ino Lab 720, Germany). During testing, probe of calibrated pH meter was inserted into the sample and stable values were noted.^[31]

Total acidity (TA)

The percent total acidity of the polyphenols supplemented C-P drinks was assessed as per the procedure described by Nawaz et al.^[32] Intentionally, 10 mL of the drink was taken in a beaker along with 25 mL of distilled water. Afterwards, titration was carried out with .1 N sodium hydroxide, using two drops of phenolphthalein indicator. The volume of sodium hydroxide consumed in neutralizing the acidity of the drink was recorded in triplicates and expressed as % citric acid equivalent.

$\mathrm{Total}\mathrm{acidity}(\mathrm{\%})=\frac{\mathrm{Average}\mathrm{Vol}.\mathrm{of}\mathrm{NaOH}\times 0.1\mathrm{N}\mathrm{NaOH}\times \mathrm{Factor}\mathrm{miliequivalent}\mathrm{citric}\mathrm{acid}(0.064)}{\mathrm{Weight}\mathrm{of}\mathrm{sample}}\times 100$

Total soluble solids (TSS)

The soluble solids in the prepared juice formulations was quantified using hand held refractometer (TAMCO, Model No. 90021, Japan) as described by Qian et al..^[33] The correctness of refractometer was checked by computing the refractive index of distilled water. Then, the juice sample was placed on the lens of the refractometer and values were determined at 20°C and expressed as °Brix.

Sedimentation index

The sedimentation of the juice was assessed by transferring the juice into a graduated 100 mL cylinder and studied under ambient conditions at set intervals up to 30 days, determining shelf life of the developed drink.^[34] The volume of sediment was evaluated by subtracting total volume from serum volume.

$\mathrm{Sedimentation}\mathrm{index}=\frac{\mathrm{Sediment}\mathrm{volume}(\mathrm{mL})}{\mathrm{Total}\mathrm{sample}\mathrm{volume}(\mathrm{mL})}$

Turbidity

The stability of the developed juice was assessed by turbidity (serum cloudiness) as stated by Tiruneh et al.^[34] The juice sample was centrifuged at 4000 rpm for 15 min and absorbance was noted at 660 nm using a UV-Vis spectrophotometer (CECIL CE 7200) in order to determine cloud stability against distilled water. This absorbance was directly associated with turbidity of the juice and expressed in nephelometric turbidity units (NTU).

Color profile

The color tonality of the samples was studied as per the guidelines of Bhat et al.^[30] and Yüceer and Caner.^[36] The sample was placed in a plastic cup, and the light captures its color from the bottom of the cup to determine the values of L (lightness; 0 = an ideal black object, 100 = an ideal white object), a* (–a greenness; +a redness) and b* (–b blueness; +b yellowness) using CIE-Lab colorimeter (CIELAB SPACE, Color Tech-PCM, USA). These aspects were then employed to calculate color intensity (chroma or C*), hue angle, ΔE and browning index (BI) as expressed herein:

$\mathrm{Color}\mathrm{intensity}({\mathrm{C}}^{\ast })=[{\mathrm{a}}^{\ast }2+{\mathrm{b}}^{\ast }2]1/2$

$\mathrm{Hue}\mathrm{angle}=\tan -1({\mathrm{b}}^{\ast }/{\mathrm{a}}^{\ast })$

$\mathrm{\Delta E}=\sqrt{\mathrm{\Delta }\mathrm{CIEL}{ }^{\ast 2}+\mathrm{\Delta }\mathrm{CIEa}{ }^{\ast 2}+\mathrm{\Delta }\mathrm{CIEb}{ }^{\ast 2}}$

$\mathrm{Browning}\mathrm{Index}(\mathrm{BI})=100(\mathrm{x}-0.31)/0.172$

Where, $\mathrm{x}=\mathrm{CIE}{\mathrm{a}}^{\ast }+1.75(\mathrm{CIE}{\mathrm{L}}^{\ast })/5.645(\mathrm{CIE}{\mathrm{L}}^{\ast })+\mathrm{CIE}{\mathrm{a}}^{\ast }-3.012(\mathrm{CIE}{\mathrm{b}}^{\ast })$

Microbiological evaluations

To enumerate viable microbial count in varied treatments of cucumber-pomegranate juice, total viable count was determined using pour plate technique.^[35] Control and extract treated samples were serially diluted with sterile .85% sodium chloride solution. Afterwards, 1.0 mL of each dilution was plated into duplicate plates and plate count agar (Beijing Land Bridge Technology Co. Ltd., Beijing, China) was used for counting total aerobic bacteria (TAB) after incubation at 37°C for 48 ± 2 h. Further, total fungal (yeasts and molds) count was carried out via spread plate method using rose bengal agar (Beijing Land Bridge Technology Co. Ltd., Beijing, China) after incubation at 27°C for 72 ± 2 h. The viable colonies were counted and expressed as log CFU/mL.

Sensory acceptability

The juice sample was assessed through 15 evaluators who were experienced faculty members, aged between 25 to 45 years. Varied samples of juice were evaluated using nine-point hedonic scale; 1 as extremely disliked and 9 as extremely liked.^[37] The panelists were provided with the prepared juice (25 mL) in transparent plastic cups, labeled randomly and advised to rate each sample for color, taste, odor, and consistency. The juice was served at room temperature under fluorescent light in isolated sensory tasting booths to avoid biasness during evaluation session. Besides, assessors were provided with mineral water to cleanse their palate prior tasting and between the samples. Using the aforementioned data, acceptance index of the panelist was determined.

Statistical analysis

The attained data were subjected to statistical software using Statistix 8.1 to assess the significance of each aspect.^[38] Based on two variables, treatment and storage intervals, two-way ANOVA under completely randomized design was applied throughout the study. The variance amongst the means was assessed through Tukey’s honest significant difference (HSD) test. All the food analyses were recorded in triplicates except sensory studies (n = 15).

Results and discussion

Influence of herbs and spices extracts

Incorporation of microwave-assisted extracts of herbs and spices in C-P drink significantly (p˂.05) improved free-radical scavenging abilities, as assessed by DPPH-, ABTS-, and FRAP assays (Table 2). Apparently, it is because of the predominant existence of bioactive ingredients (polyphenols, flavonoids, and ascorbic acids) in the extracts. It is in agreement with Ashfaq et al.,^[27] who have confirmed direct association between functional ingredients and free-radical scavenging activity. With progression in storage, free radical scavenging activity and phytochemistry showed considerable (p˂.05) decrease from 1^st to 30^th day. Further, at termination of the study, polyphenol, flavonoid, and ascorbic acid contents of C-P drinks remained relatively stable in extract enriched formulations than control; therefore, inclusion of herbs and spices extracts improved the food quality by delaying negative deviations over the prolonged storage.

Table 2. Free radical scavenging activity of polyphenols supplemented cucumber-pomegranate drinks during storage.

Parameters	Treatments	Storage intervals (Days)				Means
		1^st	10^th	20^th	30^th
DPPH (%)	CP0	38 ± 0^ghij	31 ± 1^jkl	25 ± 0^lm	17 ± 1^m	28 ± 0^e
	CP1	57 ± 5^ab	53 ± 4^abcd	46 ± 4^defg	42 ± 3^efgh	50 ± 4^b
	CP2	49 ± 2^bcdef	42 ± 1^efghi	37 ± 2^ghij	25 ± 2^lm	38 ± 2^d
	CP3	61 ± 3^a	55 ± 4^abc	53 ± 3^abcd	50 ± 3^bcde	55 ± 3^a
	CP4	49 ± 5^bcdef	42 ± 2^efghi	35 ± 2^hijk	27 ± 1^kl	38 ± 2^d
	CP5	53 ± 0^abcd	47 ± 4^cdef	41 ± 3^fghi	33 ± 3^ijkl	44 ± 3^c
Means		51 ± 3^a	45 ± 3^b	40 ± 2^c	33 ± 2^d
ABTS (mg/100 mL)	CP0	256 ± 15^jkl	229 ± 12^kl	191 ± 3^lm	163 ± 10^m	210 ± 10^e
	CP1	571 ± 29^cd	520 ± 28^de	491 ± 25^e	483 ± 26^ef	517 ± 27^b
	CP2	369 ± 17^gh	339 ± 17^hi	303 ± 15^ij	285 ± 13^ijk	324 ± 15^d
	CP3	665 ± 32^a	640 ± 33^ab	614 ± 31^abc	597 ± 32^bc	629 ± 32^a
	CP4	462 ± 24^ef	428 ± 12^fg	385 ± 21^gh	373 ± 19^gh	412 ± 19^c
	CP5	559 ± 31^cd	520 ± 13^de	485 ± 25^ef	470 ± 16^ef	508 ± 21^b
Means		480 ± 24^a	446 ± 19^b	411 ± 20^c	395 ± 19^c
FRAP (mM/100 mL)	CP0	16 ± 1^hijk	13 ± 1^kl	12 ± 1^l	10 ± 0^l	13 ± 13^e
	CP1	23 ± 1^abc	22 ± 1^bcd	21 ± 1^cde	19 ± 1^defg	21 ± 1^b
	CP2	17 ± 0^fghi	16 ± 1^hijk	15 ± 1^ijkl	12 ± 1^lm	15 ± 1^d
	CP3	26 ± 1^a	25 ± 2^a	24 ± 1^ab	24 ± 1^ab	25 ± 1^a
	CP4	18 ± 1^efgh	17 ± 1^ghi	16 ± 1^hijk	14 ± 1^kl	16 ± 1^d
	CP5	22 ± 1^bcd	20 ± 1^def	18 ± 1^efgh	17 ± 0^ghij	19 ± 1^c
Means		20 ± 1^a	19 ± 1^b	18 ± 1^c	16 ± 1^d

^*Values containing different alphabets are significant (p < .05), ^**Two-way ANOVA followed by Tukey’s HSD multiple comparison tests, ^***Means±SD (n = 3)

†CP₀ = Cucumber-pomegranate drink free from herb extract, CP₁ = Cucumber-pomegranate drink supplemented with mint extract, CP₂ = Cucumber-pomegranate drink supplemented with lemongrass extract, CP₃ = Cucumber-pomegranate drink supplemented with ginger extract, CP₄ = Cucumber-pomegranate drink supplemented with cinnamon extract, CP₅ = Cucumber-pomegranate drink supplemented with mixed herb extracts

‡DPPH = 1, 1-diphenyl-2-picrylhydrazyl, ABTS = 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), FRAP = Ferric Reducing Antioxidant Power

In comparison to control treatment, maximum percent inhibition of DPPH and ABTS radicals was observed in ginger supplemented C-P juice; CP₃ (49.22 & 66.66%) followed by mint; CP₁ (43.90 & 59.40%), herbs and spices mix; CP₅ (36.30 & 58.75%), cinnamon; CP₄ (27.60 & 49.09%) and lemongrass; CP₂ (27.22 & 35.25%) containing drinks. Likewise, ferric reducing antioxidant power (FRAP) was maximally viewed in ginger supplemented C-P juice sample; CP₃ (48.07%) whilst, the minimum was found in lemongrass based C-P drink; CP₂ (14.56%). In regards to storage, the decrease in DPPH & ABTS radicals was recorded as 12.09 & 7.14% at 10^th day. Though, at 20^th and 30^th day, the percent reductions in the corresponding parameters were 22.75 & 14.34 and 36.23 & 17.74 in comparison to control. Similarly, the reduction in FRAP was viewed as 21.21% at 30^th day in contrast to control sample.

With regards to functional ingredients, maximum polyphenols (mg GAE/L) were recorded in ginger based drink; CP₃ i.e. 1203 ± 64 trailed by mint; CP₁ 1109 ± 52, herbs and spices mix; CP₅ 1072 ± 54, cinnamon; CP₄ 1033 ± 45 and lemongrass; and CP₂ 981 ± 48 incorporated C-P drinks. On the other hand, flavonoids (mg TE/L) were maximally reported in cinnamon enriched drink; CP₄ 178 ± 8 followed by mint; CP₁ 167 ± 7, herbs and spices mix; CP₅ 162 ± 8, ginger; CP₃ 152 ± 7 and lemongrass; CP₂ 144 ± 7 containing drinks. Whilst, the lowest polyphenols and flavonoids were noted in control i.e. 713 ± 33 mg GAE/L and 107 ± 5 mg TE/L, respectively (Table 3, Figure 2).

Figure 2. Storage stability of functional ingredients in herb supplemented cucumber-pomegranate drink.

† CP₀ = Cucumber-pomegranate drink free from herb extract, CP₁ = Cucumber-pomegranate drink supplemented with mint extract, CP₂ = Cucumber-pomegranate drink supplemented with lemongrass extract, CP₃ = Cucumber-pomegranate drink supplemented with ginger extract, CP₄ = Cucumber-pomegranate drink supplemented with cinnamon extract, CP₅ = Cucumber-pomegranate drink supplemented with mixed herb extracts.

Table 3. Functional characterization of polyphenols supplemented cucumber-pomegranate drinks during storage.

Parameters	Treatments	Storage intervals (Days)				Means
		1^st	10^th	20^th	30^th
TP (mg GAE/L)	CP0	824 ± 43^hi	742 ± 26^ij	667 ± 34^j	619 ± 31^j	713 ± 33^e
	CP1	1170 ± 57^abc	1130 ± 60^abcde	1081 ± 52^bcdef	1053 ± 37^cdef	1109 ± 52^b
	CP2	1061 ± 50^cdef	1021 ± 50^defg	960 ± 46^fgh	882 ± 44^ghi	981 ± 48^d
	CP3	1254 ± 65^a	1220 ± 59^ab	1185 ± 72^abc	1153 ± 60^abcd	1203 ± 64^a
	CP4	1127 ± 29^abcde	1042 ± 53^cdef	992 ± 50^efg	970 ± 48^fgh	1033 ± 45^cd
	CP5	1139 ± 57^abcd	1083 ± 60^bcdef	1051 ± 53^cdef	1014 ± 48^defg	1072 ± 54^bc
Means		1096 ± 50^a	1040 ± 51^b	989 ± 51^c	948 ± 45^c
TF (mg QE/L)	CP0	119 ± 6^ij	110 ± 6^j	102 ± 7^j	98 ± 2	107 ± 5^d
	CP1	170 ± 8^abcde	161 ± 9^abcdefg	160 ± 8^bcdefg	158 ± 8^bcdefg	162 ± 8^b
	CP2	154 ± 8^cdefgh	145 ± 7^fgh	142 ± 7^gh	134 ± 6^hi	144 ± 7^c
	CP3	183 ± 10^a	178 ± 9^ab	176 ± 9^abc	175 ± 3^abcd	178 ± 8^a
	CP4	162 ± 8^abcdefg	153 ± 8^defgh	150 ± 7^efgh	142 ± 5^gh	152 ± 7^c
	CP5	173 ± 9^abcd	169 ± 3^abcde	165 ± 8^abcdef	161 ± 10^abcdefg	167 ± 7^b
Means		160 ± 8^a	153 ± 7^b	149 ± 8^bc	1456 ± 6^c
AA (mg/100 mL)	CP0	19 ± 1^cdefg	17 ± 1^fghijk	14 ± 1^kl	11 ± 0^l	15 ± 1^d
	CP1	20 ± 1^bcde	18 ± 1^efghij	16 ± 1^hijk	15 ± 1^ijk	17 ± 1^c
	CP2	23 ± 2^a	23 ± 1^ab	22 ± 1^abc	20 ± 1^bcde	22 ± 1^a
	CP3	20 ± 1^bcde	20 ± 1^cdefg	17 ± 1^efghij	15 ± 1^ijk	18 ± 1^c
	CP4	20 ± 1^bcdef	18 ± 1^defghi	16 ± 1^ghijk	14 ± 1^jk	17 ± 1^c
	CP5	211^abcd	20 ± 1^bcdefg	19 ± 1^cdefgh	17 ± 1^efghij	19 ± 1^b
Means		21 ± 1^a	19 ± 1^b	17 ± 1^c	16 ± 1^d

^*Values containing different alphabets are significant (p < .05), ^**Two-way ANOVA followed by Tukey’s HSD multiple comparison tests, ^***Means±SD (n = 3)

†CP₀ = Cucumber-pomegranate drink free from herb extract, CP₁ = Cucumber-pomegranate drink supplemented with mint extract, CP₂ = Cucumber-pomegranate drink supplemented with lemongrass extract, CP₃ = Cucumber-pomegranate drink supplemented with ginger extract, CP₄ = Cucumber-pomegranate drink supplemented with cinnamon extract, CP₅ = Cucumber-pomegranate drink supplemented with mixed herb extracts

‡TP = Total polyphenol, TF = Total Flavonoids, AA = Ascorbic Acid

These outcomes were in agreement to Saad et al.^[9] who mentioned the highest flavonoids in cinnamon-cucumber drink 45 ± 0 µg QE/mL in relevance to other plant extracts. Furthermore, Imran et al.^[17] found that inclusion of mint extract improved the flavonoid profile of the C-P drink more than ginger i.e. in-harmony with the current findings. Moreover, maximum ascorbic acid (mg/100 mL) was viewed in lemongrass carrying drink (22 ± 1) followed by herbs and spices mix (19 ± 1), whereas ascorbic acid content of ginger (18 ± 1), mint (17 ± 1), and cinnamon (17 ± 1) enriched drinks were at par. However, minimum ascorbic acid was noted in control sample i.e. 15 ± 1 mg/100 mL (Table 3, Figure 2).

In the current study, lemongrass enriched C-P drink showed lower polyphenol and flavonoid contents than other extract enriched formulations, but its antioxidant activity was still comparable, it might be due to its higher ascorbic acid level, as endorsed by Ranjah et al.,^[39] who found that incorporation of lemongrass in RTS drink momentously improved DPPH radical scavenging potential up to 31 ± 2 as compared to control 16 ± 0%. Further, the scientist team noted reduction in ascorbic acid content of the drink by 33% during storage (at 30^th day); however, this reduction was still better than decrease faced by control treatment i.e. 52%. This could further be linked with another study conducted by Kieling & Prudencio,^[40] who developed lemongrass-lime drink and observed lemongrass as a significant source of ascorbic acid up to 33 ± 0 mg/100 mL. Further, Babajide et al.^[5] proved that addition of ginger in cucumber-pineapple drink significantly (p˂.05) improved its ascorbic acid content as compared to control, this is in-collaboration to the present research, indicating ginger best after lemongrass in case of ascorbic acid content.

Throughout storage, loss in polyphenols was maximally observed in control treatment i.e. 24.89%. On the other hand, percent decline in polyphenols was minimally noted for ginger carrying formulation (8.02) followed by mint (10.00), herbs and spices mix (10.96), cinnamon (14.00) and lemongrass (16.90) enriched drinks, at the end of this study. Likewise, maximum reduction in flavonoids was viewed in control sample (17.76%) during storage. Whilst, the lowest loss of flavonoids was noted in cinnamon incorporated C-P drink (4.27%) followed by mint (6.92%), herbs and spices mix (7.15%), ginger (12.16%), and lemongrass (13.05%) enriched samples.

Additionally, ascorbic acid portrayed substantial (p˂.05) loss over the storage nonetheless the decrease in ascorbic acid was delayed in treatments carrying herbs and spices extracts. The maximum delay in ascorbic acid degradation was noted in lemongrass incorporated C-P drink (14.40%) followed by herbs and spices mix (16.72%), mint (23.95%), ginger (24.65%), and cinnamon (27.42%) extract enriched C-P drinks. On the other hand, the highest loss of ascorbic acid was noticed in control sample i.e. 42.19% throughout storage period.

Earlier, Majumdar et al.^[41] developed ashgourd-mint juice and noted substantial loss in ascorbic acid content after six months. In another study, Ullah et al.^[42] found that ascorbic acid remained more stable in ginger juice than lemon juice. Recently, Saad et al.^[9] compared varied plant extracts in terms of retention capacity of ascorbic acid in RTS drink and found mint better than cinnamon over four-month storage. However, overall ascorbic acid loss recorded was between 40 and 55% in RTS drink, under ambient conditions that is in synchronization with the present study outcomes. In parallel to the present investigation, Saad et al.^[9] worked on cucumber juice and found significant decline in polyphenols and flavonoids.

Previous studies also presented that addition of pomegranate in RTS drinks enhanced the antioxidant potential as different varieties or cultivars of pomegranate or different mode of extraction depicted polyphenols in pomegranate in the range of 220–2931 mg/100 mL.^[10] Further, Hamad et al.^[11] found that inclusion of sweet pomegranate in RTS drink raised the ascorbic acid from 17.7 to 17.8 mg/100 mL. Besides, sweet, white pomegranate was found to possess more ascorbic acid then red varieties.^[43] Later, Moreira et al.^[44] demonstrated that microwave mode of extraction possesses higher polyphenol contents and antioxidant activity as compared to solvent extraction method. These researches endorsed the reason behind higher antioxidant potential of the developed drink in the current study.

Fluctuations in storage stability

pH, acidity, and TSS

Data in Table 4 revealed significant change in pH, acidity and TSS values with respect to samples. Likewise, storage impacted substantial impacts (p˂.05) on pH and acidity conversely, TSS behaved non-momentously during storage (Table 5). The pH and acidity of the prepared drinks were affected by herbs and spices extract. Incorporation of ginger extract based C-P drink; CP₃ retained pH (3.6 ± .3) during storage whereas, highest change in pH was noticed in control (3.1 ± .1). One the other hand, the lowest acidity was that of ginger; CP₃ .45 ± .02%, however, the highest acidity was of control sample (.58 ± .02%). The TSS of the control sample; CP₀ was the least (15 ± 1°B) because of the absence of extract associated soluble solids. Further, the highest TSS was that of cinnamon incorporated drink; CP₄ (16 ± 1°B).

Table 4. Effect of treatments on physicochemical attributes of polyphenols supplemented cucumber-pomegranate drinks.

	Parameters
Treatments	Total soluble solids (^o Brix)	pH	Titratable acidity (%)	Color intensity (C*)	Hue		ΔE	Browning Index
CP0	15 ± 1^b	3.1 ± .1	.58 ± .02^a	14 ± 1^e	77 ± 3^b		-	19 ± 1^e
CP1	16 ± 1^ab	3.2 ± .2^b	.51 ± .01^c	34 ± 2^c	96 ± 5^a		27 ± 1^c	55 ± 2^c
CP2	16 ± 1^ab	3.2 ± .2^b	.55 ± .02^b	32 ± 1^c	79 ± 4^b		22 ± 1^d	56 ± 3^c
CP3	15 ± 1^ab	3.6 ± .3^a	.45 ± .02^e	24 ± 1^d	80 ± 3^b		12 ± 1^e	36 ± 2^d
CP4	16 ± 1^a	3.4 ± .3^a	.48 ± .02^d	39 ± 2^a	58 ± 2^d		40 ± 2^a	102 ± 5^a
CP5	15 ± 1^b	3.2 ± .2^b	.46 ± .02^de	36 ± 1^b	65 ± 3^c		33 ± 2^b	83 ± 4^b

^*Values containing different alphabets are significant (p < .05), ^**Two-way ANOVA (treatments expressed in Table 4 and storage demonstrated in Table 5) followed by Tukey’s HSD multiple comparison tests, ^***Mentioned values are means of treatment means, ^****Means±SD (n = 3)

†CP₀ = Cucumber-pomegranate drink free from herb extract, CP₁ = Cucumber-pomegranate drink supplemented with mint extract, CP₂ = Cucumber-pomegranate drink supplemented with lemongrass extract, CP₃ = Cucumber-pomegranate drink supplemented with ginger extract, CP₄ = Cucumber-pomegranate drink supplemented with cinnamon extract, CP₅ = Cucumber-pomegranate drink supplemented with mixed herb extracts

Table 5. Effect of storage on physicochemical attributes of polyphenols supplemented cucumber-pomegranate drinks.

Parameters	Storage intervals (Days)
	1^st	10^th	20^th	30^th
Total soluble solids (^o Brix)	16 ± 1^a	16 ± 1^a	16 ± 1^a	16 ± 1^a
pH	3.5 ± .3^a	3.4 ± .2^b	3.2 ± .2^c	3.1 ± .1^d
Titratable acidity (%)	.47 ± .01^d	.49 ± .02^c	.51 ± .02^b	.53 ± .02^a
Color intensity (C*)	29 ± 1^b	29 ± 1^b	31 ± 1^a	32 ± 1^a
Hue	74 ± 3^b	75 ± 3^b	76 ± 3^ab	79 ± 4^a
ΔE	30 ± 1^a	27 ± 1^b	26 ± 1^b	26 ± 1^b
Browning Index	56 ± 2^c	57 ± 3^bc	59 ± 3^ab	60 ± 3^a

^*Values containing different alphabets are significant (p < .05), ^**Two-way ANOVA (treatments expressed in Table 4 and storage demonstrated in Table 5) followed by Tukey’s HSD multiple comparison tests, ^***Mentioned values are means of storage means, ^****Means±SD (n = 3)

During storage, pH showed a declining trend from 3.5 ± .3 to 3.1 ± .1 however, acidity, being inversely proportional, indicated inclination from .47 ± .01% (initiation) to .53 ± .02% (termination), respectively. Likewise, no change was noticed in TSS from Day 1 to Day 30, and the value remained as16 ± 1°B.

Normally, acidity of RTS drink enhances due to fermentative effect produced by acid producing bacteria that converts sugars to acid during storage. This effect could be minimized in the presence of polyphenolic extracts that are antimicrobial in nature as viewed in the current study.^[9] Another reason for increase in acidity during storage could be acid hydrolysis of polysaccharides, where acid is utilized in the conversion of non-reducing sugars to reducing ones as elaborated by Nawaz et al.^[31]

Recently, Ranjah et al.^[39] formulated lemongrass drink and observed increase in acidity and decrease in pH with progression in storage; however, total soluble solids portrayed inclination i.e. in-corroboration with the current study. Additionally, Majumdar et al.^[41] developed ashgourd-mint juice and found significant decrease in pH and increase in acidity during six-month storage whereas, TSS responded non-significantly during storage. Similarly, Kausar et al.^[8] formulated cucumber-melon drink and assessed increment in acidity (.4–.5%) and decrease in pH (4.9–4.8) during four-month storage; however, TSS showed an inverse trend in contrast to the current research (15.5–16.1%).

Sedimentation and turbidity

The extract incorporated C-P drinks demonstrated significant differences in sedimentation response with maximum sedimentation observed in mint drink; CP₁ (26.76%) followed by ginger; CP₃ (25.39%) and herbs and spices mix; CP₅ (23.39%) drink. On the other hand, cinnamon and lemongrass incorporated C-P drinks; CP₄ and CP₂ presented average sedimentation in relevance to other treatments as 21.42 and 17.60%, respectively. However, minimum sedimentation was noticed in control sample; CP₀ (12.18%). During storage, noticeable (p˂.05) increase in sedimentation was viewed i.e. 17.84, 21.23, and 25.57% at 10^th, 20^th, and 30^th day, correspondingly (Figure 3).

Figure 3. Effect of treatments and storage duration on sedimentation of herb supplemented cucumber-pomegranate drink.

† CP₀ = Cucumber-pomegranate drink free from herb extract, CP₁ = Cucumber-pomegranate drink supplemented with mint extract, CP₂ = Cucumber-pomegranate drink supplemented with lemongrass extract, CP₃ = Cucumber-pomegranate drink supplemented with ginger extract, CP₄ = Cucumber-pomegranate drink supplemented with cinnamon extract, CP₅ = Cucumber-pomegranate drink supplemented with mixed herb extracts.

Similarly, turbidity was significant varying amongst treatments, whereas considerable decrement in turbidity was witnessed throughout storage (Figure 4). The turbidity of the mint carrying RTS drink; CP₁ was more than the remaining treatments at initiation. However, on termination of the study, maximum decrease in turbidity of mint was observed (35.07%) followed by cinnamon; CP₄ (31.36%), herbs and spices mix; CP₅ (31.21%) and ginger; CP₃ (25.00%) incorporated drinks. Contrariwise, the lowest change in turbidity was found in control sample; CP₀ (19.07%).

Figure 4. Effect of treatments and storage duration on turbidity of herb supplemented cucumber-pomegranate drink.

† CP₀ = Cucumber-pomegranate drink free from herb extract, CP₁ = Cucumber-pomegranate drink supplemented with mint extract, CP₂ = Cucumber-pomegranate drink supplemented with lemongrass extract, CP₃ = Cucumber-pomegranate drink supplemented with ginger extract, CP₄ = Cucumber-pomegranate drink supplemented with cinnamon extract, CP₅ = Cucumber-pomegranate drink supplemented with mixed herb extracts.

Recently, Tiruneh et al.^[34] further elaborated that turbidity and sedimentation possesses inverse relationship, it is observed that sedimentation or separation of un-dissolved precipitates accelerates as storage duration proceeds, leading to reduced turbidity. That’s why, in the present study, mint incorporated drink demonstrated maximum sedimentation along with maximum decline in turbidity during storage, however, its turbidity was maximum on 1^st day, owing to its viscous nature.

Color profile

Statistical inference represented dramatic variations in CIEL* values with respect to treatments however, non-momentous changes were noted regarding storage. On the other hand, significant differences were observed in CIEa* and CIEb* values with respect to treatments and storage. The highest CIEL* value was recorded in control treatment; CP₀ (90 ± 4), indicating lighter color whilst the lowest CIEL* value was that of cinnamon-enriched C-P drinks; CP₄ (60 ± 2) that demonstrated darker color relative to other treatments. Nonetheless, ginger (CP₃), lemongrass (CP₂) and mint (CP₁) incorporated C-P drinks also showed somewhat lighter tones with CIEL* values as 84 ± 3, 78 ± 3 and 73 ± 3, accordingly, whereas herbs and spices mix supplemented C-P juice; CP₅ portrayed darker tone closer to cinnamon supplemented treatment; CP₄ (66 ± 3). During storage, slight increment in CIEL* value was noticed from 74 ± 3 (Day 1) to 76 ± 3 (Day 30).

The maximum value for CIEa* was found in cinnamon-based C-P drink, CP₄ (20 ± 1) followed by the herbs and spices mix drink; CP₅ (15 ± 1). Further, CIEa* values for control (CP₀), ginger (CP₃) and lemongrass (CP₂) based treatments were at par; 3 ± 0, 4 ± 0 and 6 ± 0, respectively. As per the rule, a higher positive value of CIEa* indicates more inclination toward red color. On the other hand, mint extract incorporated drink; CP₁ the depicted value for CIEa* was −3 ± 0, showing a greenish hue of mint extract. With progression in storage, CIEa* value declined from 9 ± 0 to 6 ± 0, indicating fading of color or may be associated with a decrease in pigmented polyphenols.

CIEb* value was maximally reported in mint supplemented treatment; CP₁ (34 ± 1) followed by cinnamon; CP₄ (33 ± 1), herbs and spices mix; CP₅ (33 ± 1) and lemongrass; CP₂ (32 ± 1) based drinks but lower values for CIEb* were noted in case of ginger based formulation; CP₃ (24 ± 1) followed by control treatment; CP₀ (14 ± 1). Throughout storage, CIEb* values improved notably from 26 ± 1 to 30 ± 1.

Using color tonality values; CIEL*, CIEa* and CIEb*, numerous parameters could be calculated that signifies food quality over the storage period. These aspects include chroma (clarity/intensity of color), hue (shade of color), ΔE (color difference with respect to control) and browning index. In the present study, all the aforementioned parameters demonstrated considerable change regarding treatments and storage.

The higher values for chroma (Tables 4 and 5) were observed in cinnamon; CP₄ (39 ± 2), herbs and spices mix; CP₅ (36 ± 1) mint; CP₁ (34 ± 2), lemongrass; CP₂ (32 ± 1) and ginger; CP₃ (24 ± 1) enriched juice samples whereas, the lowest chroma value was that of control; CP₀ i.e. 14 ± 1. During storage, chroma presented an inclining trend from 29 ± 1 to 32 ± 1, hence indicates more clarity in the drink.

The maximum hue angle (Tables 4 and 5) was recorded that of mint based treatment; CP₁ due to its greenish shade (96 ± 5), whereas minimum value was found for cinnamon containing drink; CP₄ (58 ± 2) as it was heading toward reddish shade followed by herbs and spices mix sample; CP₅ (65 ± 3). Nevertheless, remaining treatments; ginger (CP₃), lemongrass (CP₂) and control (CP₀) were having tendency toward yellow color with values found as 80 ± 3, 79 ± 4 and 77 ± 3. Over the storage, hue angle demonstrated increasing tendency from 74 ± 3 (Day 1) to 79 ± 4 (Day 30).

Delta E in Figure 5 showed minimum color difference with respect to ginger based treatment; CP₃ (12 ± 1) in comparison to control treatment; CP₀, followed by lemongrass; CP₂ (22 ± 1) and mint; CP₁ (27 ± 1) enriched samples, whereas the color difference was maximally varying in cinnamon based drink; CP₄ (40 ± 2) trailed by herbs and spices mix containing sample; CP₅ (33 ± 2). As the storage proceeds, less color difference was observed against control from 30 ± 1 (Day 1) to 26 ± 1 (Day 30).

Figure 5. Effect of treatments and storage duration on ΔE value of herb supplemented cucumber-pomegranate drink.

† CP₀ = Cucumber-pomegranate drink free from herb extract, CP₁ = Cucumber-pomegranate drink supplemented with mint extract, CP₂ = Cucumber-pomegranate drink supplemented with lemongrass extract, CP₃ = Cucumber-pomegranate drink supplemented with ginger extract, CP₄ = Cucumber-pomegranate drink supplemented with cinnamon extract, CP₅ = Cucumber-pomegranate drink supplemented with mixed herb extracts.

Further, browning index (BI) showed substantial variance between treatments owing to the existence of brown pigmented substances in herbal extracts for instance cinnamon extract carrying sample; CP₃ with BI reported as 102 ± 5 followed by herbs and spices mix drink; CP₅ (83 ± 4). Additionally, BI of lemongrass (CP₂) and mint (CP₁) supplemented treatments were recorded as 56 ± 3 and 55 ± 2, accordingly. However, lower BI was viewed in ginger containing treatment; CP₃ (36 ± 2), that might be due to the presence of the highest phenolic compounds the retards the enzymatic or non-enzymatic browning reactions to arise during storage. On the other hand, the least browning was noted in control; CP₀ (19 ± 1) that might be due to absence of any brown-pigmented substances. An increase in browning was observed as the storage progressed from Day 1 (56 ± 2) to Day 30 (60 ± 3), possibly linked with chemical and biochemical reactions that are inevitable during storage.

Currently, Saad et al.^[9] determined the CIEL*, CIEa* and CIEb* values of cucumber drink as 21 ± 1, −3 ± 0 and 10 ± 0, respectively. Earlier, a group of scientists, Opara et al.^[43] measured the CIEL*, CIEa* and CIEb* and C* values of white pomegranate as 66 ± 1, 22 ± 1, 42 ± 1, and 47 ± 2, respectively. Hence, it could be deduced that presence of cucumber and pomegranate along with herbs and spices extract in specified doses might impact on color differently.

Microbial analysis

Cucumber-pomegranate drink formulations were analyzed for total plate count and yeast and mold count at interval of 10 days during storage under refrigerated conditions (Table 6). Analysis of variance pertaining to total plate count (TPC) of functional drinks indicated significant effect of treatments, storage and their interactions during storage. The maximum TPC was viewed in control treatment; T₀ 1.8 ± .1 log CFU/mL whereas, the minimum TPC was reported in CP₃ as 1.3 ± .1 log CFU/mL at 1^st day that reached to 3.0 ± .2 and 2.7 ± .2 log CFU/mL on 30^th day, respectively. However, momentous change was observed over the storage amongst all the treatments, varying from 1.6 ± .1 to 2.9 ± .2 log CFU/mL. The count of yeast and mold revealed nil growth during storage.

Table 6. Microbiological evaluation of polyphenols supplemented cucumber-pomegranate drinks during storage.

Parameters	Treatments	Storage intervals (Days)				Means
		1^st	10^th	20^th	30^th
TAB (log CFU/mL)	CP0	1.8 ± .1^hij	2.5 ± .1^f	2.8 ± .2^c	3.0 ± .2^a	2.7 ± .1^a
	CP1	1.5 ± .1^j	2.0 ± .1^hi	2.5 ± .1^f	2.8 ± .2^bc	2.5 ± .1^d
	CP2	1.7 ± .1^ij	2.0 ± .1^h	2.8 ± .2^d	2.9 ± .2^a	2.6 ± .1^b
	CP3	1.3 ± .1^j	1.9 ± .1^hij	2.3 ± .1^g	2.7 ± .2^e	2.3 ± .1^e
	CP4	1.7 ± .1^ij	2.0 ± .1^hi	2.7 ± .1^e	2.9 ± .2^a	2.6 ± .1^c
	CP5	1.7 ± .1^ij	2.0 ± .1^hi	2.6 ± .1^e	2.9 ± .2^b	2.5 ± .1^d
Means		1.6 ± .1^d	2.1 ± .1^c	2.6 ± .1^b	2.9 ± .2^a
Y&M (log CFU/mL)	CP0	ND	ND	ND	ND	-
	CP1	ND	ND	ND	ND	-
	CP2	ND	ND	ND	ND	-
	CP3	ND	ND	ND	ND	-
	CP4	ND	ND	ND	ND	-
	CP5	ND	ND	ND	ND	-
Means		-	-	-	-

^*Values containing different alphabets are significant (p < .05), ^**Two-way ANOVA followed by Tukey’s HSD multiple comparison tests, ^***Means±SD (n = 3)

†CP₀ = Cucumber-pomegranate drink free from herb extract, CP₁ = Cucumber-pomegranate drink supplemented with mint extract, CP₂ = Cucumber-pomegranate drink supplemented with lemongrass extract, CP₃ = Cucumber-pomegranate drink supplemented with ginger extract, CP₄ = Cucumber-pomegranate drink supplemented with cinnamon extract, CP₅ = Cucumber-pomegranate drink supplemented with mixed herb extracts

In accordance to FDA Philippines, 2013, total plate count is permissible in beverage < 1 × 10³ CFU/mL and the same was noticed in the prepared drinks hence considered safe for consumption under refrigeration storage. In functional beverages, the microbial activity was lower as compared to the control that might be due to less water activity. These outcomes were in line with Akhtar et al.,^[45] who reported that total plate count increases gradually with the progression in storage. Similar trend was noted by Nawaz et al.,^[32] who found that fruit-based drinks remained safe microbiologically for 28 day under refrigeration conditions.

A group of scientists El-Saadony et al.^[6] found that extracts carrying more polyphenols and flavonoids have more capacity to control detrimental changes in cucumber juice. In varied other studies, cinnamon, ginger, lemongrass, and pomegranate demonstrated antimicrobial effects.^{[14,21,46,47]} Moreover, Shi et al.^[48] found natural preservatives like tree tea oil effective against microbial invasion in fresh cucumber juice.

Changes in sensory perspectives

Sensory characteristics give an indication of consumer preference toward a particular food. Statistical analysis depicted that herbs and spices extracts in C-P drinks impacted substantially (p < .05) on sensory attributes like color, odor taste, consistency, and acceptance index with regards to treatments and storage (Table 7). The maximum score for color was attained by herbs and spices mix C-P drink; CP₅ (7.7 ± .5) followed by cinnamon; CP₄ (7.6 ± .5), i.e. in par with lemongrass; CP₂ (7.6 ± .4), ginger; CP₃ (7.4 ± .7), control; herb free; CP₀ (7.1 ± .5) and mint; CP₁ (7.1 ± .4) incorporated C-P drink samples. From odor point of view, the maximum score was allocated to ginger-based treatment; CP₃ (8.0 ± .6) trailed by mint-based C-P drink; CP₁ (7.9 ± .5). Whereas, cinnamon; CP₄ and lemongrass; CP₄ based drinks were rated at par by the judges i.e. 7.4 ± .6 and 7.4 ± .4. Further, the herbs and spices mix drink; CP₅ (7.1 ± .4) and control formulation; CP₀ (6.8 ± .6) were given the minimum score with respect to odor. Hedonic rating for taste demonstrated maximum scores for ginger; CP₃ 7.8 ± .6 and minimum for control; CP₀ 6.9 ± .8. Besides, taste scores for cinnamon; CP₄, mint; CP₁, herbs and spices mix; CP₅ and lemongrass; CP₂ based juice samples were 7.3 ± .9, 7.2 ± .5, 7.1 ± .6 and 6.7 ± .5, respectively. Scores for consistency responded considerable variance with maximum value viewed in ginger; CP₃ (6.9 ± .4) followed by cinnamon; CP₄ (6.7 ± .4), control; CP₀ (6.6 ± .4), herbs and spices mix; CP₅ (6.5 ± .4), mint; CP₁ (6.3 ± .4) and lemongrass; CP₂ (6.1 ± .4) enriched RTS drinks.

Table 7. Sensory aspects of polyphenols supplemented cucumber-pomegranate drinks during storage.

Parameters	Treatments	Storage intervals (Days)				Means
		1^st	10^th	20^th	30^th
Color	CP0	7.3 ± .4^cde	7.1 ± .7^cde	7.1 ± .4^cde	7.0 ± .4^de	7.1 ± .5^c
	CP1	7.4 ± .4^cde	7.2 ± .4^cde	7.1 ± .4^cde	6.7 ± .4^e	7.1 ± .4^c
	CP2	8.1 ± .5^ab	7.6 ± .4^bcd	7.4 ± .4^cde	7.2 ± .4^cde	7.6 ± .4^ab
	CP3	7.8 ± .6^bc	7.5 ± .8^bcd	7.3 ± .5^cde	7.1 ± .9^cde	7.4 ± .7^b
	CP4	8.5 ± .8^a	7.5 ± .5^bcd	7.3 ± .4^cde	7.2 ± .5^cde	7.6 ± .5^ab
	CP5	8.7 ± .5^a	7.6 ± .5^bcd	7.3 ± .4^cde	7.2 ± .4^cde	7.7 ± .5^a
Means		8.0 ± .5^a	7.4 ± .5^b	7.3 ± .4^bc	7.1 ± .5^c
Odor	CP0	7.2 ± .7^defghij	7.0 ± .8^ghij	6.7 ± .6^jk	6.2 ± .5^k	6.8 ± .6^d
	CP1	8.2 ± .7^abc	8.2 ± .5^ab	7.8 ± .5^abcde	7.3 ± .4^defghij	7.9 ± .5^a
	CP2	7.8 ± .5^abcdef	7.4 ± .4^cdefghi	7.3 ± .4^defghij	7.1 ± .4^fghij	7.4 ± .4^b
	CP3	8.3 ± .4^a	8.2 ± .9^ab	7.9 ± .7^abcd	7.7 ± .4^abcdefg	8.0 ± .6^a
	CP4	7.8 ± .6^bcdefgh	7.6 ± .4^defghij	7.2 ± .6^hij	6.9 ± .6^defghij	7.4 ± .6^bc
	CP5	7.4 ± .4^defghij	7.2 ± .4^fghij	7.1 ± .4^fghij	6.7 ± .4^ijk	7.1 ± .4^cd
Means		7.8 ± .6^a	7.6 ± .6^ab	7.3 ± .5^b	7.0 ± .5^b
Taste	CP0	6.8 ± .8^cdefg	6.6 ± .9^efg	6.5 ± .8^fg	6.4 ± .7^g	6.6 ± .8^c
	CP1	7.5 ± .5^abcde	7.2 ± .4^bcdefg	7.1 ± .5^bcdefg	7.0 ± .5^cdefg	7.2 ± .5^b
	CP2	7.1 ± .9^bcdefg	6.7 ± .4^defg	6.6 ± .4^efg	6.6 ± .4^efg	6.7 ± .5^c
	CP3	8.2 ± .6^a	7.9 ± .5^ab	7.7 ± .5^abc	7.4 ± .8^abcdef	7.8 ± .6^a
	CP4	7.6 ± .7^abcd	7.4 ± .8^abcedef	7.2 ± 1.1^bcdefg	7.2 ± 1.1^bcdefg	7.3 ± .9^b
	CP5	7.2 ± .6^bcdefg	7.2 ± .9^bcdefg	7.1 ± .5^bcdefg	7.1 ± .4^bcdefg	7.1 ± .6^b
Means		7.4 ± .7^a	7.1 ± .7^ab	7.1 ± .6^b	6.9 ± .7^b
Consistency	CP0	7.0 ± .5^abcd	7.0 ± .5^abcd	6.6 ± .4^cdefgh	6.0 ± .4^ij	6.6 ± .4^bc
	CP1	6.8 ± .4^abcdef	6.5 ± .4^defghi	6.1 ± .4^ghij	5.8 ± .4^j	6.3 ± .4^d
	CP2	6.7 ± .4^bcdefg	6.2 ± .4^abcdef	6.0 ± .4^fghij	5.5 ± .4^hij	6.1 ± .4^cd
	CP3	7.3 ± .5^a	7.1 ± .5^abc	6.8 ± .4^abcdef	6.3 ± .4^efghij	6.9 ± .4^a
	CP4	7.2 ± .5^ab	6.9 ± .4^abcde	6.6 ± .4^cdefgh	6.2 ± .4^ghij	6.7 ± .4^ab
	CP5	6.8 ± .4^abcde	6.8 ± .4^abcdef	6.3 ± .4^efghi	6.1 ± .4^hij	6.5 ± .4^bcd
Means		7.0 ± .5^a	6.7 ± .4^a	6.4 ± .4^b	6.0 ± .4^c

^*Values containing different alphabets are significant (p < .05), ^**Two-way ANOVA followed by Tukey’s HSD multiple comparison tests, ^***Means±SD (n = 15)

†CP₀ = Cucumber-pomegranate drink free from herb extract, CP₁ = Cucumber-pomegranate drink supplemented with mint extract, CP₂ = Cucumber-pomegranate drink supplemented with lemongrass extract, CP₃ = Cucumber-pomegranate drink supplemented with ginger extract, CP₄ = Cucumber-pomegranate drink supplemented with cinnamon extract, CP₅ = Cucumber-pomegranate drink supplemented with mixed herb extracts

Concisely, amongst extracts, ginger extract enriched C-P drink; CP₃ indicated maximum acceptance index i.e. 83.7 ± 3.5%, followed by cinnamon supplemented C-P juice; CP₄ (80.6 ± 3.1%), whereas mint; CP₁ (79.1 ± 3.0%) and herbs and spices mix; CP₅ (79.0 ± 2.7%) drink samples showed almost similar acceptance response. On the other hand, minimum acceptability scores were achieved by lemongrass; CP_2, and control treatments; CP₀ as 77.1 ± 2.7 and 75.3 ± 2.1%, correspondingly. In regards to storage, the maximum score for all sensory traits was observed at 1^st day whilst, minimum scores were allotted at 30^th day. At the termination of the storage period, the average decrease in scores of color, odor, taste, consistency, and overall acceptance was 11.07, 10.27, 6.08, 14.37, and 10.41%, respectively.

The outcomes of the present study are in agreement with Saad et al.^[9] who found that RTS drinks possessing higher functional ingredients have more chances to prevent deteriorative changes during storage, leading to higher acceptability. Similar effect is endorsed by the current study, where ginger and cinnamon, being significant sources of polyphenols and flavonoids showed better consumer acceptability during storage. Earlier, Ullah et al.^[42] also explicated that the presence of ginger extract highly improves the sensory acceptability of carrot-kinnow juice. Further, varied researchers confirmed that the incorporation of mint and lemongrass in RTS drinks improves the sensory characteristics in comparison to control sample.^[39,40,49] Additionally, Kasim et al.^[7] prepared inulin enriched cucumber drink and found its enhanced consumer liking 6.0 ± .8 against control 5.5 ± 1.1. Likewise, inclusion of sweet and sour pomegranate juice in RTS drinks was linked with better taste, color and clarity of the developed drink.^[50]

Conclusion

The tested herbs in this study confirmed their antimicrobial activity at the rate of 2% in RTS cucumber-pomegranate drink. Specifically, ginger extract maintained optimal storage quality of the prepared drink under refrigeration for 30 days owing to its highest free-radical scavenging potential. Apart from this, incorporation of ginger and herb and spices mix in the prepared drinks portrayed highest organoleptic aspects amongst all other drinks, including control. Consequently, it could be deduced that incorporation of extracts of herbs and spices offers a complementary approach to improve the storage stability of the RTS fruit- and vegetable-based drinks. For future works, the major bioactive compounds in each drink formulation will be assessed in-depth to understand their fate throughout storage.

Acknowledgments

Authors extend their appreciation to Government College Women University Faisalabad, Pakistan.

Disclosure statement

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

Data availability statement

All the assessed data of this study is compiled in this article along with its supplementary material.