Comparative Effects of Hibiscus Leaves and Potato Peel Extracts on Characteristics of Fermented Orange Juice

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of flavonoids, antioxidant, anticancer, as well as anti-inflammatory properties (Benavente-García and Castillo, 2008).World production of oranges is estimated approximately 73,298,838 tons annually.An alternative to waste post-harvest is to process the fruit and produce industrial products such as juices, jams, wines, etc.The use of the fermentation of orange fruits as a substrate for producing high-quality value products has been accomplished; an example is wines obtained by the fermentation of fruit (González et al., 2010).
Lactic Acid Bacteria (LAB) have positive healthpromoting effects, e.g.regulating the intestinal flora, decreasing cholesterol level in the blood, strengthening the immune system, and ameliorating oral diseases (Hashemi et al., 2017;Kun et al., 2008).The fermented food with LAB also can prolong its shelf life, enhance the nutritional and organoleptic qualities, improve the sensory properties, and remove undesirable compounds (Kaprasob et al., 2017).The traditional study of LAB fermentation focuses on dairy products, which are unsuitable for vegans, lactose, and casein intolerant individuals; and high cholesterol risk individuals (Wu et al., 2020).Thus, the request for the development of nondairy LAB fermentation food products was pointedly increasing.Fruit juices have been proposed as an alternative vehicle for LAB fermentation due to the high contents of minerals, vitamins, dietary fiber, and antioxidant compounds (Costa et al., 2013;Wu et al., 2020).El-Hadary et al. (2022) mentioned that pomace and peels (food manufactories by-products) are abundant sources of bioactive compounds.Generally, the byproducts of food manufactories did no t use, however transaction food manufactories process to environmentally friendly ways becoming a highly important issue.The processing of by-products can generate high food additives value products such as flavonoids (rutin and luteolin), polyphenols (mainly oleuropein and hydroxytyrosol), etc. use in food preparation and manufacturing that increased the shelf life and oxidative stability of stored food products (Canabarro et al., 2019).
The antioxidants are one of most important food additives which used to elongate the shelf life of food products and preserve the nutritional quality of lipid and modify the consequences of oxidative damage in the human body (Canabarro et al., 2019).Natural antioxidants are used instead of synthetic ones to inhibit lipid oxidation in foods to improve their quality and nutritional value.In recent years, several natural antioxidants were used in food preparation and manufacturing which have increased the shelf life and oxidative stability of stored food products (El-Hadary et al., 2022).
Hibiscus (Hibiscus sabdariffa) is an important medicinal plant widely distributed in many several areas around the world.Hibiscus characterizes by the presence of phytochemical compounds like flavonoids, phenolic acids, and polysaccharides (Vasudeva and Sharma, 2008).Preliminary phytochemical screening revealed the presence of polyphenols in the leaves extract of hibiscus; also, a recent study indicated that leaves extract of hibiscus had high antioxidant effects (Nade et al., 2010).In addition, potatoes (Solanum tuberosum) are peeled during processing.The potato peels are a good source of natural antioxidants.Polyphenols are considered as important groups of natural antioxidants, which are present in potatoes (El-Hadary et al., 2022).
The aims of the present investigation were to the enhancement physical, chemical, and microbiological properties, sensory evaluation, and shelf life of fermented orange juice by hibiscus leaves and potato peel extracts additives.

Materials
Orange fruits (Citrus xsinensis) were obtained from a private farm from Ismailia governorate, Egypt.Hibiscus leaves were obtained from a private farm from Sharqia governorate, Egypt.Potato wastes (S. tuberosum) were obtained from farm frites factory.

Preparation of samples
Firstly, the different parts of potato factories' wastes (30 kg; water, starch, and peels) were separated.Then, the potato peels were transferred and washed well with [ DOI: 10.18502/jfqhc.10.1.11988 ] [ Downloaded from jfqhc.ssu.ac.ir on 2024-03-07 ] tap water to get rid of the remnants of starch sticking out, and stacked on trays for a 30 min to get rid of excess water before drying.The hibiscus leaves were examined to eliminate the undesirable parts and stacked on trays.Both samples were dried in an oven-dryer at 37 °C for 48 h.The samples were flipping once every hour in the first 4 h.Then, the dried samples were ground to a fine powder, place in plastic bags, wrapped with aluminum foil, and stored at -20 °C until the next procedures (El-Hadary et al., 2022).

Extraction of the antioxidants extracts
The extraction of hibiscus leaves and potato peel extracts of dried samples was conducted using the difference in pressure in 70% concentrations of ethanoic alcohol (El-Hadary et al., 2022).One hundred g of dried weight of hibiscus leaves and potato peel were soaked in 1,000 ml of 70% concentrations of ethanoic alcohol in 2,000 ml conical flask for 6 h with decrease the pressure to 0.6 Pa every 30 min in the sample flask during extraction.The obtained extracts were filtered using paper (Whatman No. 1, England), concentrated using a rotary evaporator (EYELA, Japan) until disappearing the alcoholic odor.Then, they were stored at 4±2 °C until the next procedures (El-Hadary et al., 2022).

Preparation of orange juice and inoculum
The orange fruit was washed with tap water, the pulp was chopped, and the seeds were removed.Then, the orange pulp was crushed using a food-grade juicer and filtered through 80 mesh gauze.Orange juice was divided into five parts.The first part was orange juice with 5% of Hibiscus Leaves extracts (HL5), the second part was orange juice with 10% of Hibiscus Leaves extracts (HL10), the third part was orange juice with 5% of Potato Peel extracts (PP5), the fourth part was orange juice with 10% of Potato Peel extracts (PP10), while the Fifth part was orange juice without any additives (PC).Then, the samples were pasteurized at 80 °C for 15 min and cooled to room temperature (25 °C) (Burca-Busaga et al., 2022).
The used cultures were grown at 37 °C separately for 24 h in de Man Rogosa Sharpe (MRS) broth (Difco Laboratories, Detroit, MI, USA) in order to attain approximately 106 Colony-Forming Units (CFU)/ml as inoculating before inoculation into orange juice as 0.5% (V/V).
Enumeration of the cells was performed by plating serial dilutions of bacterial suspensions on MRS agar plates, and incubating at 37 ºC, and counting the colonies after 48 h.The inoculation by L. plantarum and L. bulgaricus was done in the range of CFU/ml, and then incubated at 37 ºC for 24 h to make sure the fermentation operation (Mousavi et al., 2011).After 24 h, the analysis was estimated at zero time.Then, the samples were storaged at 4±2 ºC for 21 st days and analyzed every 7 days.

Determination carbohydrates
Total crude carbohydrates, reduced sugar, and nonreduced sugar of various samples were measured per the methods recommended in AOAC (2019).

Determination of pH
The pH of all samples was measured with a glass electrode of a digital pH meter (Model Mettler Toledo, Switzerland) (AOAC, 2019).

Determination of titratable acidity
The acidity of samples was evaluated by the overall titration method supported the carboxylic acid percentage (AOAC, 2019).A few drops of phenolphthalein were added to 20 ml of homogenized samples in a titration flask.Then, titration with 0.1 M NaOH until a faint pink color persists for 30 s.The concentration of citric acid calculated using the official equation depended on 1 ml of 0.1 M NaOH corresponding to 0.064 mg of citric acid.

Determination of Total Soluble Solids (TSSs)
TSSs and also the index of refraction were assayed using the refractometric method, with an Abbe refractometer and corrected to the equivalent reading at 20 °C (AOAC, 2019).

Determination of viscosity
The viscosity of every sample was resoluted at temperature by employing a Brookfield digital viscometer (NDJ-85, Niryn Intelligent Company limited, Shanghai).An acceptable spindle (spindle 2) and rotational speed (60 rpm) were selected for this study (Sun et al., 2006).

Determination of color
Orange juice color was measured using the Hunter-Lab (Hunter Lab Color Flex EZ, USA).Color parameter (L*) indicated the degree of lightness to darkness, (a*) indicated the degree of redness to greenness, and (b*) indicated the degree of yellowness to blueness (Zaki, 2022).

Determination of Total Phenolic Contents (TPC)
The content of total phenol concentration of various samples was measured by a UV spectrophotometer (Jenway-UV-VIS Spectrophotometer, UK), supported a [ DOI: 10.18502/jfqhc.10.1.11988 ] [ Downloaded from jfqhc.ssu.ac.ir on 2024-03-07 ] colorimetric oxidation/reduction reaction, that described by Škerget et al. (2005).The reagent of oxidizing was Folin-Ciocalteu reagent consistent with AOAC (2019).Five tenths ml of diluted extract (10 mg in 10 ml solvent), 2.5 ml of Folin-Ciocalteu reagent (diluted 10 times with distilled water), and a pair of ml of Na2CO3 (75 g/L) were added.The mixture was incubated for five min at 50 ˚C, and then cooled.For an effect sample, 0.5 ml of water was used.The absorbance was measured at 760 nm.TPC expressed as acid equivalent (Gallic Acid Equivalents (GAE)/g of dried extract) was calculated using the subsequent equation supported the calibration curve (Figure 1) by use the different concentrations of gallic acid (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 mg GAE/ml): =0.2269x+0.4847R²=0.992Where y is that the absorbance; x is that the concentration (mg GAE/g of dried extract); R 2 is correlation coefficient.

Determination of Total Flavonoids Contents (TFCs)
The concentration of TFCs of various samples was measured according Ordoñez et al. (2006) with some modification description by El-Hadary et al. (2022).One point five ml of AlCl3 ethanolic solution (20 g/L) was added to 0.5 ml of each extract of samples (10 mg in 10 ml solvent), separately and incubated for 1 h at room temperature.The absorbance was measured at 420 nm at room temperature and therefore the yellow color indicated the presence of flavonoids.TFC expressed as Quercetin equivalent (mg QE/g of dried extract) was calculated using the subsequent equation supported the calibration curve (Figure 2) by use the different concentrations of quercetin (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 mg QE/ml): y=0.3531x+0.8191R²=0.992Where x is that the absorbance; y is that the concentration (mg QE/g of dried extract); R 2 is correlation coefficient.

DPPH atom scavenging assay
The decolorization of deep purple color of methanolic DPPH˙ solution by various samples was measured according to Gülçin et al. (2004).One tenth ml of every extract (10 mg in 10 ml methanol solvent) was added to 3 ml of 0.1 mM DPPH˙ dissolved within the methanol solvent to every extract, separately, and measured for 2 h every 30 min at room temperature.The control of the assay was prepared consistent with usage to negative control from DPPH˙ solution and only solvent without extracts, and therefore the positive control by exchange the extracts by BHA synthetic antioxidants.The absorbance resolves against a negative control at 517 nm for each period, separately.Percentage of antioxidant activity of DPPH˙ radical was calculated using the subsequent equation: Where, A c is the absorbance of the negative control; A t is the absorbance of the sample and/or positive control.IC 50 is the antioxidant concentration that inhibits the DPPH • reaction by 50% under experimental conditions.

Determination of ascorbic acid
Vitamin C (ascorbic acid) content resolves using 2,6dichlorophenol indophenol reagent (Fluka, Deisehofen, Germany) in step with the strategy described by AOAC (2019).One ml of sample was added to 1 ml of 50 mM solution of oxalic acid (1:1, v/v), mix thoroughly, and leave at room temperature for 30 min.Centrifugation of the tube at 4,000 rpm for 10 min, then the whole of the separated supernatant was collected with a pipette.The standard sample was prepared using 1 ml of the standard solution instead of the analyzed liquid without centrifugation.The absorbance measurements of the test sample (Ax) and standard sample (As) were determined at 700 nm against the mixture of 50 mM solution of oxalic acid (1:1, v/v) as a reference sample.The concentration of vitamin C (Cx: µM) in the liquid of different samples was calculated using the formula: Where, Cs is the concentration of vitamin C in standard solution.

Microbiological analysis
Viable cell counts were determined by serial dilutions and standard plate method after incubation.Dilutions of 10 -7 and 10 -8 CFU/ml were prepared of the fermented samples and plated in double plates.Then, sterilized MRS agar (Merck, Germany) medium was poured on them (standard plate count method) (Mousavi et al., 2011).The plates were incubated at 30 °C for 48 h.Then plates containing 30-300 colonies were counted and recorded as CFU per ml of solution (Vinderola et al., 2000).Additionally, the viability of lactic acid cultures was resoluted during the cold storage period using the mentioned method and expressed as CFU/ml (AOAC, 2019).

Sensory evaluation
Sensory evaluation was done per Min et al. (2003).Ten panelists were selected (Staff of Food Science Department, Faculty of Agriculture, Zagazig University, Egypt) without care old or sex.The panelists were asked to point their preference on a 9-point Hedonic scale with a degree of liking: 1=dislike extremely, 2=dislike greatly, 3=dislike moderately, 4=dislike slightly, 5=neither like nor dislike, 6=like slightly, 7=like moderately, 8=like a great deal, and 9=like extremely.In each session, five different samples got to rate the color, test, flavour, and overall acceptability of the samples.

Statistical analysis
The tests were exhausted triplicate in line with Steel et al. (1997), and therefore the data were analyzed using the means, variance by Microsoft Office Excel (2016), paired sample t-test, and one-way ANOVA variance analysis by IBM SPSS version 25.0 software (SPSS Inc., Chicago, IL, USA) at the extent of probability of (p<0.05).

Acidity
The acidity of fermented orange juices were shown in Table 1.The results showed no significance means differences (p>0.05) between samples, and no significance means differences (p>0.05) between the values to the same sample during the time of storage.All samples showed low increases in acidity by progress in storage time.The highest value of acidity was 10% of Fermented Hibiscus Leave extracts (FHL10), followed by other samples at 21 st days of storage time.While, the lowest value of acidity was 10% of Fermented Potato Peels extracts (FPP 10) at zero time of storage.

Effect on TSS and viscosity
The results of TSS assay showed a significant means difference (p<0.05) between almost samples.The highest value of TSS was FHL 10 at 21 st days of storage time with a significant mean difference (p<0.05) between other samples with no significant mean difference (p>0.05) between FPP5 and FPP 10 , and the highest value was FHL 10 after both 14 th and 21 st days of storage time (22.3±0.2 and 22.6±0.1,respectively) followed by FPP 5 after both 14 th and 21 st days of storage time (21.3±0.3 and 22.1±0.1,respectively).While, the lowest values were all samples at zero time of storage with a significant means difference (p<0.05) between PC and other samples (16.36±0.1);and no significant means difference (p>0.05) between FHL 5 and FPP 5 (15.82±0.12 and 15.85±0.1,respectively); and no significant means difference (p>0.05) between FHL 10 and FPP 10 (15.35±0.2 and 15.4±0.1,respectively).The results also showed the positive effect of the additives hibiscus leaves and potato peel extracts in TSS values of fermented orange juices during the time of storage.
All the samples registered the lowest viscosity values at zero time of storage with no significant means difference (p>0.05) between samples.The results also showed the increases in viscosity values by the progress in storage time with clear improvement in values of samples which have hibiscus leaves and potato peel extracts.All samples registered the highest value of viscosity at 21 st days of storage time, and the highest value was FPP 10 followed by FHL 10 with no significant means difference (p>0.05) between both of them (14.5±0.3 and 14.3±0.2,respectively), while the lowest value was PC at zero time of storage (10.57±0.57)with no significant means difference (p>0.05) between PC and other samples.

Effect on vitamin C
The impact on the contents of vitamin C during the time of storage was measured and the results are shown in Table 2.The results showed little decrease in values of vitamin C by the progress of storage time to all the samples.The data at 21 st registered the lowest value to all the samples and PC was the lowest (51.81±0.21mg/g of wet weight) with significant mean difference (p<0.05)just with FHL 10 (52.87±0.07mg/g of wet weight).The results also showed a clear effect hibiscus leaves extracts additives during all the time of storage which showed significant mean difference (p<0.05) between FHL 10 and all other samples during storage time.

Effect on carbohydrates
Data in Table 3 show the effect on reducing sugar, nonreducing sugar, and total carbohydrates; respectively.The results showed a decrease in reducing sugar, nonreducing sugar, and total carbohydrates values by progress in time of storage.The results also showed no significant means difference (p>0.05) between all the samples at all the time of storage, and no significance in means differences (p>0.05) between the values to the same sample during the time of storage to reducing sugar, non-reducing sugar, and total carbohydrates.The data at 21 st days of storage time registered the lowest values of both reducing sugar, non-reducing sugar, and total carbohydrates to all the samples, while data at zero time was the highest.The reducing sugar represented a high percentage of total carbohydrates, which give 61.68% at the highest value and 60.91% at the lowest value.

Effect on antioxidants activities
The antioxidant activities of different fermented orange  4. TPCs, TFCs, and DPPH˙scavenging radical activity (IC 50 ) were estimated.The results showed significance in means differences (p<0.05) between samples, and significance in means differences (p<0.05) between the values to the same sample during the time of storage.The results also showed a clear positive effect of hibiscus leaves and potato peel extracts additives by the decreases in IC 50 value during storage time to all samples, however, the decreases in TPC and TFC values.The highest values of antioxidants activities were at zero time of storage and the highest values were FHL 10 to both TPCs and TFCs (448.15±1.15mg GAE/g and 115.2±0.2 mg QE/g, respectively), and PC in IC 50 estimated (43.56±0.56µg/g of wet weight).

Effect on color
The impact of ethanolic hibiscus leaves and potato peel extracts on the color of different fermented orange juices was measured.Table 5 showed values of lightness (L*), redness (a*), and yellowness (b*), respectively.The results showed decreases in lightness (L*), redness (a*), and yellowness (b*) by the progress in time of storage.However, there was the little significant mean differences (p<0.05) between samples in lightness (L*) values during storage time, while there was no significant mean differences (p>0.05) between samples at 7 th days of storage time.The results registered a clear effect to additives hibiscus leaves extracts on redness (a*) values which FHL 10 gave the highest values of redness (a*) during storage time (62.62±0.12,61.95±0.45,61.1±0.1, and 60.44±0.44 at zero, 7 th , 14 th , and 21 st days of storage time, respectively), and positive effect to additives potato peel extracts on yellowness (b*) values which FPP 10 gave the highest values of yellowness (b*) during storage time (82.87±0.37,80.73±0.23,79.52±0.52,and 78.37±0.37 at zero, 7 th , 14 th , and 21 st days of storage time, respectively).

Effect on the total microbial count
The total microbial count of different fermented orange juices was estimated and the results are shown in Table 6.The results showed a few significant mean differences (p<0.05) between samples during the time of storage with no significant mean differences (p>0.05) between the values to the same sample during storage time, however, the increases in total microbial count values.The results also showed the positive effect to additives hibiscus leaves and potato peel extracts, while PC gave the lowest values of total microbial count with clear significant mean differences (p<0.05) between samples during the time of storage.All the samples showed no significant mean differences (p> 0.05) during storage time accepted PC which gave significant mean differences (p<0.05) between the values at 14 th and 21 st days of storage time.

Effect on sensory evaluation
Sensory evaluation of different fermented orange juices was estimated and the results are shown in Table 7.The results showed the effect on color, tastes, flavour, and overall acceptability.Potato peel extracts were a positive effect on all sensory evaluations, while additives hibiscus leaves extracts had a negative effect on color evaluation at 10% during all the time of storage.In general, the data showed more acceptability to the FPP10>FPP5>FHL10>FHL5>PC to all sensory evaluations during all the time of storage, and all the degree was ≥7.Almost all panelists were attributing positive effect of potato peel extracts additives to enhance the appearance and flavour, in addition to the decreases in sensory evaluations to PC was attributing to the little bitter taste to the control sample in the last time of storage.

Discussion
The increases in acidity by adding hibiscus leaves and potato peels extracts and also the progress in time of storage may be because of the positive of these extracts on protect citric acid from metabolized by bacterial after fermentation starts, which also explains the decreases of total carbohydrates (both reduce and non-reduce sugar).The appropriate explanation for this phenomenon is the availability of carbohydrates in high concentrations in orange juice.Mousavi et al. (2011) had different results when fermenting a pomegranate juice by probiotic bacteria due to the considerable amounts of organic acid i.e., citric acid in pomegranate juice that was used.
The effect on viscosity, TSS, and a lightness of juice color may be due to the volume of bacterial growth.The spoilage and bacterial growth of food bring about physical changes such as an increase in the viscosity, gelation, sedimentation, and/or color change of the food.First of place, the increases in viscosity by the progress in time of storage and by adding the extracts due to the enhancement of the bacterial growth that has acted to generate the products which have a viscous effect.Also, the increase in TSS by adding the hibiscus leaves and potato peels extracts compared with the control sample attributed to the increases in metabolized resulting from the high volume of bacterial growth.In addition, Quan et al. (2022) attributed the increase in TSS to the ability of bacteria on metabolize organic compounds in fermented orange juices.
[ DOI: 10.18502/jfqhc.10.1.11988 ] [ Downloaded from jfqhc.ssu.ac.ir on 2024-03-07 ]      The different concentrations of vitamin C between the control sample and the samples which had hibiscus leaves and potato peels extracts may be attributed to the concentrations of vitamin C in these extracts.In addition, the decrease in vitamin C values during storage time may be attributed to the consumption in the nurture of the microorganisms.Also, the increase in vitamin C concentration of samples that had hibiscus leaves and potato peels extracts is due to the protective effect of these extracts on vitamin C consumption by oxidative process and/or bacterial metabolized.Quan et al. (2022) attributed the increase in vitamin C concentration in samples of fermented orange juice to the ability of probiotic bacteria to generation vitamin C.
The results of antioxidants activity by different methods shown the hibiscus leaves and potato peels extracts exhibited high potential antioxidant.In addition, fermentation has a positive affect that shown from the increase in DPPH˙ scavenging radical activities (IC 50 ) during storage time.The data was similar to a previous study on the effects of L. plantarum fermentation on phenolic characteristics and various polyphenol compounds, even though different raw materials (Li et al., 2021).The increases in DPPH˙ scavenging radical activities (IC 50 ) during storage time, however the decreases in TPC may be because of production enzymes or some other bioactive compounds which an antioxidants effect or by the decreases in TFC which had a negative effect on antioxidants activities.Orange juice in different fermentation stages had different antioxidant activities, but overall, the antioxidant activity continued to increase.
The turbidity of juice during the progress in time of storage, and the difference of color by the different additives of extracts may be attributed to the effect of these extracts on bacterial growth.The decreases in lightness (L*) values by the progress in time of storage may be because of the effect of bacterial cells, while the increases in redness (a*) values by additives hibiscus leave extracts, and yellowness (b*) values by additives potato peel extracts because the red color to hibiscus leaves extracts and yellow color to potato peel extracts.The data was similar to a previous study on the effects of L. plantarum fermentation on color characteristics which found a positive effect on turbidity by adding L. plantarum to mulberry juice, also the fermentation effect on both of redness and yellowness (Kwaw et al., 2018).
The little increase in the total microbial count during storage time indicated the positive effect of hibiscus leaves and potato peel extracts additives due to the contents of these extracts from important compounds use in microbial metabolized.Also, the protective effect of these extracts gave more validity to the products during the time of storage and more quality not only to microbial growth but also protected the important compounds which had a positive effect on human health.

Table 1 :
Acid value of different fermented orange juice samples treated with hibiscus leaves and potato peel extracts

Table 2 :
The contents of vitamin C of different fermented orange juice samples treated with hibiscus leaves and potato peel extracts

Table 3 :
The contents of carbohydrates of different fermented orange juice samples treated with hibiscus leaves and potato peel extracts

Table 4 :
The antioxidants activities-Total Phenolic Contents (TPCs; concentration mg GAE/g of wet weight) and Total Flavonoids Contents (TFCs; concentration mg QE/g of wet weight) of different fermented orange juice samples treated with hibiscus leaves and potato peel extracts

Table 5 :
Color of different fermented orange juice samples treated with hibiscus leaves and potato peel extracts

Table 6 :
Total microbial counts of different fermented orange juice samples treated with hibiscus leaves and potato peel extracts

Table 7 :
Sensory evaluation of different fermented orange juice samples treated with hibiscus leaves and potato peel extracts