Effects of Chemical and Natural Additives on Cucumber Juice’s Quality, Shelf Life, and Safety

Microbial contamination affects beverages’ lifetime, quality, and safety. Cucumber crops are seasonally spoiled because of the overproduction. The current study aimed to maximize the importance of natural preservatives and reduce the usage of artificial ones to prolong the cucumber juice’s storage life, enhance flavor, and control the microorganisms after protein isolate and organic acids supplementation. The additions included control (no addition), citric, benzoic acid, sodium salts, kidney bean pepsin hydrolysate (KPH), chicken egg protein isolate (CEPI), duck egg protein isolate (DEPI), and quail egg protein isolate (QEPI) as J-Control, J-Citric, J-Benzoic, J-sod. Citrate, J-sod. Benzoate, J-KPH, J-CEPI, J-DEPI, and J-QEPI, respectively. The antioxidant activity of these additives and juices was evaluated by DPPH radical scavenging activity. The antimicrobial activity, including antibacterial and antifungal activities, was evaluated by using disc assay and the radial growth of fungal mycelium, respectively. The phenolic compounds and flavonoids were estimated by a spectrophotometer as Gallic acid equivalent (GAE) and quercetin equivalent (QE), respectively. Moreover, chemical parameters such as pH, total soluble solids (TSS), Titratable acidity (TTA), and Vitamin C were evaluated by AOAC. Finally, the color properties were estimated by a spectrophotometer, using the Hunter method. KPH had higher significant (p ≤ 0.05) antioxidant activity (88%), along with antimicrobial activity. It significantly (p ≤ 0.05) reduced the growth of G+ and G− bacteria by 71–97% and 58–66% respectively. Furthermore, it significantly (p ≤ 0.05) inhibited the tested fungi growth by 70–88% and the other additives less than that. During the storage of cucumber juice for an interval of zero, two, four, and six months, the phenolic compounds and flavonoids were significantly (p ≤ 0.05) decreased. Consequently, the potential activity of the juice was reduced; in addition, pH and vitamin C were significantly (p ≤ 0.05) decreased during the storage period. Meanwhile, the TSS and Titratable acidity were significantly raised. As for color and sensory properties, J-sod. Benzoate, J-KPH, J-CEPI, and J-DEPI had significantly (p ≤ 0.05) high scores in color, taste, and flavor against the control. Generally, the usage of natural additives extends the cucumber juice’s lifetime and increased the manufacture of high-quality and valuable juice.

The chicken, quail, and duck egg whites were diluted [29] with some modification, with water (1:3 w/v), and were stirred for thirty minutes and centrifuged for 20 min undercooling (15,000× g). The obtained supernatant was precipitated with 10% polyethylene glycol (PEG) 4000 and centrifuged under cooling at 15,000× g for twenty minutes. The residues were dissolved in 50 mM Tris-HCl, 200 mM NaCl, and 5 mM CaCl 2 , pH 7.8 (TBS-Ca). After leaving at 4 • C overnight, the mixture was centrifuged (4 • C, 15,000× g, 20 min). The precipitates were washed with TBS-Ca, homogenized with TBS buffer with ten mM EDTA, pH 7.8, and kept for thirty minutes at 4 • C. Then they were centrifuged at (4 • C, 1400× g, 20 min), and the supernatant was collected and adjusted to pH 5 with HCl. Following further centrifugation (4 • C, 1400× g, 20 min), it was dialyzed against 20 mM Tris-HCl, 50 mM NaCl (pH 8.0). The dialysate fractions were eluted on the Q Sepharose column by the gradual concentration of NaCl solution (0.1 to 0.6 molar). The chicken, duck, and quail egg protein's isolates were diluted with NaCl (0.35 to 0.45 M) and then freeze-dried.

Processing of Cucumber Juice Supplemented by Preservatives
The fresh cucumber was washed, cleaned, and processed in a Braun blender (Blender mixer Type 441), resulting in juice. The juice was heated at 83 • C for 2-3 min, with a few pressures 75 MPa "hyperbaric preservation" [17,19,30,31] in HIRAYAMA HG-SERIES autoclave (Concord, CA 94520, USA), and immediately cooled-down. Table 1 shows the juices constitutes by the Abbe Refractometer. The prepared juices were packed into sterilized bottles (350 mL) and divided into four groups, each of which included nine bottles, one for control and the others for juices supplemented with chemical and natural additives. The bottles were capped and tightly sealed and stored at room temperature for six months. The following analyses were carried out at intervals of preservation (0, 2, 4, and 6 months).

Estimation of Physiochemical Parameters
Titratable acidity of juices was calculated as citric acid (mg/mL) at the storage period of 0-6 months, at room temperature, according to standard method 942. 15. Additionally, juice pH was assessed for the same samples by pH meter. Total soluble solids (TSS %) was determined by using an Abbe Refractometer (WZS portable refractometer, China). A few drops of the juice were mounted on the tip of the refractometer, and readings were taken [32]. Vitamin C was determined according to Ranganna [33]. A total carbohydrate was estimated according to the Chaplin [34] method. Then, 200 µL of hydrolysate sample and glucose standard (0, 20-100 µg/mL) was added to 200 µL of phenol (5%) and 1 mL of concentrated sulfuric acid. After thirty minutes, the OD was estimated at Foods 2020, 9, x FOR PEER REVIEW 4 of 18 for six months. The following analyses were carried out at intervals of preservation (0, 2, 4, and 6 months).

Estimation of Physiochemical Parameters
Titratable acidity of juices was calculated as citric acid (mg/mL) at the storage period of 0-6 months, at room temperature, according to standard method 942.15. Additionally, juice pH was assessed for the same samples by pH meter. Total soluble solids (TSS %) was determined by using an Abbe Refractometer (WZS portable refractometer, China). A few drops of the juice were mounted on the tip of the refractometer, and readings were taken [32]. Vitamin C was determined according to Ranganna [33]. A total carbohydrate was estimated according to the Chaplin [34] method. Then, 200 μL of hydrolysate sample and glucose standard (0, 20-100 μg/mL) was added to 200 μL of phenol (5%) and 1 mL of concentrated sulfuric acid. After thirty minutes, the OD was estimated at ƛ 490 nm. The concentration of total sugars in cucumber juices was calculated by using the linear equation in the glucose standard curve: where y is the absorbance, and x is glucose concentration (μg/ml).

R² = 0.9987
where y is the absorbance, and x is Gallic acid concentration (μg/mL). 490 nm. The concentration of total sugars in cucumber juices was calculated by using the linear equation in the glucose standard curve: y = 0.0053x − 0.0193 where y is the absorbance, and x is glucose concentration (µg/ml).

Total Phenolic Compounds (TPC)
Total phenolic compounds (TPC) was assessed in cucumber juices supplemented with chemical preservatives and isolated proteins as GAE (0, 200-1000 µg/mL), following the Folin-Ciocalteu method [35], according to the equation of the Gallic acid standard curve: where y is the absorbance, and x is Gallic acid concentration (µg/mL). Total Flavonoids Three mL aliquot of 10 g/L AlCl 3 ethanoic solution was added to 0.5 mL of each juice supplemented with chemical preservatives and isolated proteins, the mixtures were then incubated for an hour, at room conditions [36]. The absorbance was estimated at 430 nm. Total flavonoids in samples was measured as QE (0, 20-100µg/mL), using the quercetin acid standard curve equation.
where y is the absorbance, and x is the quercetin concentration (µg/mL).
DPPH Radical-Scavenging Activity DPPH radical-scavenging activity was followed in milk samples, as an indicator of antioxidant activity [37]. An aliquot (100 µL of each sample) was added to 1 mL of 1 mL DPPH in ethanol and incubated at room temperature for thirty minutes [38], before measuring the color absorbance at 517 nm against a control. The percentage of antioxidant activity of free radical DPPH was calculated as follows: where A control is the control absorbance, and A sample is sample absorbance, i.e., DPPH reaction absorbance.

Color Measurements
The color of cucumber juices was measured by using a spectrophotometer (Hunter Lab, Color

Sensory Evaluation
Eighty members (faculty staff and students) from the Food Science Department, Faculty of Agriculture, Zagazig University, Egypt, evaluated the sensory properties of cucumber juices, (control+ eight juice with different additives), using a scorecard for each sensory attributes (color, odor, flavor, taste, and overall acceptability), using a 9-point Hedonic scale, whereby the scores ranged from dislike extremely (1) to like extremely (9) [40]. The room was illuminated with white light, and each session continued for two hours. Water was provided to each panelist for mouth-rinsing after testing each product, to avoid the carry-over effect.

Antibacterial Activity
Antibacterial activity was estimated [41,42]. Paper discs saturated with 30 µL of each additive at different concentrations (0, 50, 100, and 200-1000 mg/mL) were then added to Petri dishes containing nutrient agar infected with pathogenic microorganisms, the G+ bacteria (L. monocytogenes and B. cereus), Foods 2020, 9, 639 6 of 17 and G− bacteria (E. coli and Ps. aeruginosa) incubated at 37 • C for 24 h. The developed inhibition zones (mm) were manually measured by using a transparent ruler. The negative control was disc-saturated with distilled water. Minimum inhibitory concentration (MIC) was estimated as the lowest concentration and showed a clear zone on MHA plates. Turbidity (A600) assay was used to determine the extent of the bacterial growth in nutrient broth media suspensions during 24 h of incubation. The MIC of each sample was added to a tube containing 100 µL of pathogenic bacteria in 10 mL nutrient broth, incubated at 37 • C and measured every six hours, before recording the turbidity compared with control.

Microbial Count
Total viable count and coliform bacterial count in cucumber juices supplemented with chemical preservatives and isolated proteins at 0.2% (w/v) were performed during preservation periods (0-6 months), at room temperature, by using the pour plate technique [43]. First, 1 mL (v/v) of the sample was diluted with one-fold of 2% sterile sodium citrate solution, to prepare a suspension. Then, 1 mL of the suspension was used for the serial dilution of between 10 −1 and 10 −5 . After that, 1 mL of each dilution was placed in sterile disposable Petri dishes (sterilin) in triplicates. At about 44 to 50 • C, the number of different bacteria was determined by using specified media [44][45][46]. Colon bacteria were counted on MacConkey agar and brooded at 37 • C for 24 h. The total viable count (TVC) on Agar media was counted and incubated at 25 m, for a period of 72 h. Microbiological results were converted to logarithms (CFU/g).

Antifungal Activity
The inhibition action of the chemical and natural additives against three fungal species were obtained from the Department of Agricultural Microbiology, Faculty of Agriculture, Zagazig University, Egypt [47,48]. First, 5 mL of each additive, at different concentrations (0, 50, 100, 200, 400, 800, and 1000 µg/mL), was poured into potato dextrose agar (PDA) medium in Petri dishes and then protected at 28 • C for seven days. After 24 h of incubation, mycelia disk (5 mm) was carefully picked from the edge of fungal cultures and placed in the center of each Petri dish containing the additives. The PDA plates without any addition or water were prepared as negative and positive controls, respectively. The fungal mycelium's radial growth was recorded (cm). The minimum fungal concentration was estimated according to [49], by inoculating the contents of all the prepared fungi combined with the additives' concentrations in test tubes, as prepared in case of an MIC test on new PDA tubes. All test tubes were nurtured at 28 • C, for 48 to 72 h.

Statistical Analysis
The obtained data means were statistically analyzed by using Microsoft Office Excel (version 2019) and ANOVA variance single factor, at a probability level of p ≤ 0.05; multiple comparisons were carried out, applying the least significant difference (LSD).

Results and Discussion
Cucumber juice is considered a valuable and therapeutic beverage. It possesses different medicinal properties, such as antimicrobial, antioxidant, and anticancer properties [50].

Physiochemical Changes Analysis
3.1.1. DPPH Radical-Scavenging Activity of Additives Figure 1 shows the antioxidant activity of chemical additives from citric acid, benzoic acid, sodium citrate, and sodium benzoate, as well as natural ones from KPH to QEPI, pepsin kidney bean peptide, chicken egg protein isolate, duck egg protein isolate, and quail egg protein isolate. All preservatives were added at a concentration of 0.2% (w/v). KBH significantly (p ≤ 0.05) exhibits the highest radical-scavenging activity, with 88% compared to TBHQ 93%, with a relative increase ranging from 5% to 9% from DEPI, QEPI, and sodium benzoate.
The antioxidant activity of organic acid and their salts depends on the carboxyl group giving an electron to free radical and converting to less-reactive (stable) acyl free radical that can be reduced into organic acid again or oxidized to dehydro-organic acid [3]. Sarmadi and Ismail [51] and Afandi et al. [52] investigated the free-radical-scavenging action of protein isolates and hydrolysate with two mechanisms, hydrogen atom transfer (HAT) and single electron transfer (SET). They may act in parallel or with one dominating, according to protein isolate structure, wherein the aromatic amino acids convert radicals to stable molecules by donating electrons, but keeping their own stability. Hydrophobic amino acids improve peptides' solubility in lipids through hydrophobic side chains. Meanwhile, essential and acidic amino acids act as metal ion chelators and proton donners through their COOH and NH 2 side chain.
Foods 2020, 9, x FOR PEER REVIEW 7 of 18 et al. [52] investigated the free-radical-scavenging action of protein isolates and hydrolysate with two mechanisms, hydrogen atom transfer (HAT) and single electron transfer (SET). They may act in parallel or with one dominating, according to protein isolate structure, wherein the aromatic amino acids convert radicals to stable molecules by donating electrons, but keeping their own stability. Hydrophobic amino acids improve peptides' solubility in lipids through hydrophobic side chains. Meanwhile, essential and acidic amino acids act as metal ion chelators and proton donners through their COOH and NH2 side chain.  Figure 2 shows the antioxidant activity changes of cucumber juices during the storage period of zero, two, four, and six months period. The antioxidant activity of cucumber juices significantly (p ≤ 0.05) decreased. There was no significant decrement in J-KPH, with the smallest relative decrease about 1.5%; the antioxidant activity relatively reduced from 7% to 14% for juices from J-QEPI to Jcontrol. Klimczak et al. [53] reported that the decrease in antioxidant activity might be linked to a reduction in total phenolic content and vitamin C during storage. Protein-phenolic complexes' formation may affect the physical and chemical properties of the protein. Moreover, the phenolicsproteins binding due to blocking some amino acid side chains possibly increased the activity. Furthermore, the protein-phenolic complex may also increase the bioaccessibility and activity of phenolics [54][55][56][57][58].  Figure 2 shows the antioxidant activity changes of cucumber juices during the storage period of zero, two, four, and six months period. The antioxidant activity of cucumber juices significantly (p ≤ 0.05) decreased. There was no significant decrement in J-KPH, with the smallest relative decrease about 1.5%; the antioxidant activity relatively reduced from 7% to 14% for juices from J-QEPI to J-control. Klimczak et al. [53] reported that the decrease in antioxidant activity might be linked to a reduction in total phenolic content and vitamin C during storage. Protein-phenolic complexes' formation may affect the physical and chemical properties of the protein. Moreover, the phenolics-proteins binding due to blocking some amino acid side chains possibly increased the activity. Furthermore, the protein-phenolic complex may also increase the bioaccessibility and activity of phenolics [54][55][56][57][58]. Table 2 shows that the significant (p ≤ 0.05) relative decrease of total phenolic content ranged from 9% to 23% GAE µg/mL after six months. Besides, flavonoids were significantly (p ≤ 0.05) decreased from 38.515-29.727 to 29.764−22.212 QE µg/mL after six months, for juices. Vallverdu-Queralt et al. [59] found a decrease in total polyphenol content of tomato juices after three, six, and nine months of storage. Consequently, Kaur et al. [60] showed a significant reduction of phenolic compounds during the six-month storage of cucumber juice supplemented with chemical additives. The protein isolates significantly (p ≤ 0.05) maintained the fluids more than the chemically treated sample. The lowest decrement was found in J-KPH, with about 9% relative decrease.  Table 2 shows that the significant (p ≤ 0.05) relative decrease of total phenolic content ranged from 9% to 23% GAE μg/mL after six months. Besides, flavonoids were significantly (p ≤ 0.05) decreased from 38.515-29.727 to 29.764−22.212 QE μg/mL after six months, for juices. Vallverdu-Queralt et al. [59] found a decrease in total polyphenol content of tomato juices after three, six, and nine months of storage. Consequently, Kaur et al. [60] showed a significant reduction of phenolic compounds during the six-month storage of cucumber juice supplemented with chemical additives. The protein isolates significantly (p ≤ 0.05) maintained the fluids more than the chemically treated sample. The lowest decrement was found in J-KPH, with about 9% relative decrease.    Table 3 shows that total sugars significantly (p ≤ 0.05) decreased from the range of 278.81-170.70 mg for J-control-J-QEPI, on the day of preparation, to the range of 95.79-52.40 mg after six months, a relative decrease of 65-70%. Juice acidity was increased because of total sugar in juice analyzed into simple sugar by the fermentative effect of acid-producing bacteria in agreement with Sivakumar [61]. Although TSS not significantly increase because of increments of simple sugars ( Figure 3D), Kausar et al. [62] observed that TSS increased (15.49-16.09%) during the storage of cucumber-melon drink [50] and cucumber-mint drink. Similar results were noticed in watermelon juice blended with ginger obtained Foods 2020, 9, 639 9 of 17 by [63]. Kinh et al. [64] reported an increase in soluble content of apple pulp during storage when preserved with chemical preservatives. Figure 3A shows significant (p ≤ 0.05) decreasing of pH value, from 4.4 to 3.6, in all samples after six months, with a relative decrease of about 25-30%. Besides that, the results indicated that pH changes occur less in natural additives than in chemical and control. The pH decreased because of increasing in acidity. Aderinola et al. [65] observed a decrease in pH values and an increase in TTA during the storage of carrot-cucumber juice; these changes might occur due to the fermentation of sugar present in the juice. The acidity in juices J-control to J-Sod-Benz significantly (p ≤ 0.05) increased more than fluids supplemented with protein isolates (Figure 3C), and the changes were significant at least in J-KPH, J-CEPI, and J-DEPI. As per results, natural additives > chemical additives have a significant (p ≤ 0.05) effect on Vitamin C content of cucumber juice. On the storage debut, the Vitamin C content in cucumber juices ranged from 5 to 5.3 mg/100 g, respectively. The values significantly (p ≤ 0.05) faded, as heat treatment destroys, at the end of the storage period to 2.5-2.3 ( Figure 3B). Francis et al. [66] detected the decrement in vitamin C of watermelon juices blended with sodium benzoate and lime; this degradation might be due to the high sensitivity of light, oxygen, heat, and enzymatic or non-enzymatic oxidation.  Table 4 showed the juices color parameters, where the L* non-significant decrease from 29.24 for control to 28.7 at the end of storage, with low relative decrease 3%, nearly no change in a, and slightly increase in b from 11.27 to 11.29. The significant (p ≤ 0.05) best color, according to L* results, was juice five, supplemented with sodium benzoate. Besides, J-KPH increased in L* from 28.05 to 29.03 with a  Table 4 showed the juices color parameters, where the L* non-significant decrease from 29.24 for control to 28.7 at the end of storage, with low relative decrease 3%, nearly no change in a, and slightly increase in b from 11.27 to 11.29. The significant (p ≤ 0.05) best color, according to L* results, was juice five, supplemented with sodium benzoate. Besides, J-KPH increased in L* from 28.05 to 29.03 with a relative increase of 4%, decreased in a* from −3.2 to −3.4, and showed no change in b*. J-sod-citrate was significantly less white, and other color parameters, L* 22.8, a* 0.44, and b* 11, increased after six months to 23, 0.76, and 12. Tomato juice with Na benzoate seems to be more stable than the other preservatives during six months of storage and was less off-color and developed less turbidity [60,67]. Means in the same column with different letters are significantly different (p ≤ 0.05). L * (lightness-darkness), a * (redness-greenness), b * (yellowness-blueness), H (Hue angle), C * (Chroma), WI (whiteness index.

Bacterial Growth Curve
The growth curves of the tested bacteria reached the highest turbidity after about 16 h at 37 • C in E. coli and Ps. aeruginosa, but after 12 h in B. cereus and L. monocytogenes. KBH had significant (p ≤ 0.05) values in reducing the growth of Gram-positive bacteria by 71% and 79%, as well as Gram-negative bacteria by 58% and 66%, for B. cereus, L. monocytogenes, E. coli, and Ps. aeruginosa, respectively. Generally, chemical additives reduced the growth of B. cereus by 10-45%, L. monocytogenes by 18-58%, E. coli by 10-38%, and Ps. aeruginosa by 20-44%.However, the natural ones reduced the G+ bacteria growth by 55-71% and 53-79%, as well as by 41-58% and 49-66% for G− bacteria, as shown in (Figure 4). The antimicrobial mechanism of organic acid penetrated the microorganisms' cell membrane, decreased cell pH, and controlled the processes of metabolism, especially the synthesis of ATP, RNA, protein, and DNA replication [4]. Most of the positively charged peptides are electrostatically bound to negatively charged compounds on the bacterial cell wall, leading to cell wall destruction [71][72][73]. Furthermore, the peptide hydrophobicity plays an essential role in disturbing the bacterial cell membrane and cell wall. MIC was determined from chemical and natural additives against all experimental bacterial strains.
respectively. Generally, chemical additives reduced the growth of B. cereus by 10%-45%, L. monocytogenes by 18%-58%, E. coli by 10%-38%, and Ps. aeruginosa by 20%-44%. However, the natural ones reduced the G+ bacteria growth by 55%-71% and 53%-79%, as well as by 41%-58% and 49%-66% for G− bacteria, as shown in (Figure 4). The antimicrobial mechanism of organic acid penetrated the microorganisms' cell membrane, decreased cell pH, and controlled the processes of metabolism, especially the synthesis of ATP, RNA, protein, and DNA replication [4]. Most of the positively charged peptides are electrostatically bound to negatively charged compounds on the bacterial cell wall, leading to cell wall destruction [71][72][73]. Furthermore, the peptide hydrophobicity plays an essential role in disturbing the bacterial cell membrane and cell wall. MIC was determined from chemical and natural additives against all experimental bacterial strains.  Table 7 showed that the highest total bacterial count obtained in control during storage ranged from 2.7 to 5 (log CFU/mL) at room temperature, from two to six months. Meanwhile, the bacterial load significantly (p ≤ 0.05) decreased in J-citric and J-benzoic decreased from 2.7 to 2.5, and 2.6 respectively, with about 50%. On the other hand, the bacterial load of J-KPH, J-CEPI, and J-DEPI significantly (p ≤ 0.05) decreased with a relative increase of about 60%, because of the antimicrobial potential of protein isolates [71][72][73]. Similar results were obtained by Habib and Iqbal [30], who observed the least TVC in mixed cucumber-tomato-pumpkin juice, especially in the blend percentage 5%-7%-9%.

Conclusions
The study was able to establish the KBH, and Sod-Benzoate has a significant effect on the lifetime and sensory properties of the treated juice samples. It could be concluded that cucumber juice blended/enhanced with KPH and sodium benzoate (0.2%) was rated higher when compared to other samples. The addition of KBH to the cucumber juice maintained the storage life of the juice for six months, at room temperature, with 50-75 MPa, thus making it an available meal, ready to serve, and a refreshing drink with a good nutritional, medicinal, and caloric value. The results of this research work confirmed both the vulnerability of pure cucumber juice to a microbial attack due to its high moisture content and the preservative potentials of natural additives, especially KBH, because of their antioxidant and antimicrobial activity. It was observed that KBH had two significant roles in the juice samples; that of a flavor (natural) and a preservative. We recommended the utilization of natural additives because of their safety, unlike chemical ones. Finally, this preservation method protected the cucumber crop from spoilage and increased the manufacture of high-quality and valuable juice.