Efficent Use of Antioxidants to Preserve Fruit Juice

In order to follow up spoilage of fruit juice: apple and orange juice, treated or non-treated with synthesis antioxidants and powder natural extracts, the samples have been analysed during production process and after storage for 5 days at a temperature of 50 0 C (ASLD method - accelerated shelf life determination), by determining the vitamin C content, the clarity and colour, and also the water activity. Previously, antioxidants activity was calculated for the synthesized compounds and powder natural extracts.

Many epidemiological studies validate a healthy effect of fruits daily intake and also for derivate products.The biologically active components contained -antioxidants: polyphenols, carotenoids and vitamins -exhibit protective effects against cell oxidation.They reduced appearance of cancers, heart disease and other degenerative diseases [1][2][3][4].
By the nutrients-rich content, the fruit juice is indicated for everyday consumption.During the industrial processing activities, in order to obtain high quality products, they are treated with antioxidants able to destroy or inhibit the oxidising enzymes.
The extracts of vegetable origin, in pure form or alcoholic extracts, contain compounds with protective role preventing oxidation of fruit juice.This justifies the interest expressed in literature for the substitution of synthetic antioxidants with natural ones [5].
The antioxidant activity of functional ingredients used the radical DPPH (2,2-diphenyl-1-picrylhydrazyl) method to estimate the antioxidant capacity to act as direct trapping of free radicals or as hydrogen donors.
The DPPH is a stable free radical with the unpair electron placed at nitrogen atom of hydrazil group.Its working wavelength of maximum absorbance, λ max to be used for the absorbance measurements is given as 517 nm and gives rise to the deep violet colour.The violet colour of DPPH faints into the yellow colour, when the unpaired electron of DPPH is mixed with a hydrogen atom from the antioxidant with can collect the free radicals [2].Thus it is obtained the DPPH-H, the reduced form (2) [6].
Representing the DPPH radical by Z* and the donor molecule by AH, the first reaction is: where ZH is the reduced form, and A* is the free radical produced in this first step.This latter radical will then undergo further reactions according to stoichiometry, which gives the number of molecules of DPPH reduced (decolorized) by one molecule of the reacting substance.
In case of ascorbic acid, there are two adjacent sites facilitating hydrogen abstraction in reaction with the free DPPH radical, leading to a 2:1 stoichiometry, that is, 2 molecules of DPPH are reduced by one molecule of ascorbic acid [7]. (2)
The antioxidant activity can be estimated by the indirect spectophotometric method that uses 2,2-diphenyl-1picrylhydrazyl as generating system for the stable free radical DPPH.When the violet solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then gives rise to the reduced form (2) with the loss of this violet colour.From the picryl group still present, it can be noticed a residual dull yellow colour [6].The reaction that has just taken place involves a fainting of the violet colour into the yellow colour.
The antioxidant activity of vegetable extracts and antioxidants is calculated according to relation 6 [9]: (6) where: A 0 = sample absorbance at t = 0 min A 60 = sample absorbance at t = 60 min The results obtained from previous studies [10,11] have suggested the antioxidants utilization in studies and researches regarding the spoilage of protected or nonprotected fruit juice by addition of the antioxidants in study.This way, it's was obtained drinks with functional ingredients, how is a new concept on soft drinks market.
There have been used the following natural extracts: Teavigo green tea extract (E1) -(DSM Nutritional Products) and black tea extract (E2), extra green tea extract (E3), rooibos extract (E4) -(Herbal Teea International).The powder extracts are obtained by replacing the humidity from liquid extract (30% water) with an equivalent quantity of calcium phosphate, starch and maltodextrin substratum.The humidity is lost using vacuum to avoid excessive thermo-degradation of extract [5].The commercial products obtained as powder extracts from various types of tea contain, as antioxidant active principles: (-)-Epigallocatechin-3-gallate in E1, E2, E3 extracts and quercetin, luteolin, rutin and isoquercetin in E4.
According to the technical specifications, the recommended antioxidants dosage is 0.1 g/L, and 0.01 g/L in case of lutein.Higher concentrations of lutein cause an undesired bright red colouring of samples.

Determination of antioxidant activity using the DPPH method:
In an eprouvette, are introduced 4 mL DPPH methanol solution (Merck) with a concentration of 20 mg/L and 0.01 ml test solution: tocopheryl acetate, taxifolin, ascorbic acid, lutein, coenzyme Q10, E1, E2, E3 and E4 (concentration 0.01%).The variation of the extinction coefficient was determined spectrophotometrically at wavelength 517 nm during 15 min exposure at room temperature.
The juice samples treated with antioxidants have been pasteurized at 90 0 C for 30s.From the 2 L of apple juice, respectively orange juice, 20 small samples were taken, 100 ml each.They have been treated with the antioxidants in study and packed in PET packaging cases. 2 antioxidants free witness samples were kept.The first 10 samples were analysed immediately after preparation, the remaining samples after 5-day storage at 50 0 C (ASLD method).
In order to estimate the shelf life of the juice, which normally needs long study periods and physico-chemical analyses, the accelerated shelf life determination method (ASLD) was used [12].
In order to evaluate antioxidants protected and nonprotected juice degradation, the following operations were carried out: -the content of C vitamin [mg/L] was determined.The extraction of ascorbic acid from the sample to be analysed was made with oxalic acid solution 1‰ and titration with 2.6 dichlorofenolindofenol [13].
-at Perkin Elmer UV/VIS, Lambda 20 spectrophotometer has been measured: • absorbance at wavelength of 420 nm for clear juice (apples) and 640 nm for opalescent juice, with pulp particles (orange) in order to assess the product colour; • transmittance at wavelength of 625 nm to determine the clarity, respectively 440 nm to determine the colour.Evaluation: A 420 apple juice: 0.440 -0.460 nm; A 640 orange juice (in order to evaluate the colour of opalescent juice, the samples are diluted with water at 3 0 Brix, using the Pearson's least square method): 1.650 -1.750 nm [14,15] -it has been determined the water activity, a w by means of Aquaspector AQS-2-TC device, NAGY using kiselgur adsorbing environment.

Results and discussions
Figure 1 below represents graphically the absorbance variation of solutions with addition of synthesised antioxides during a certain period of time.
In figure 1 it can be noticed that the adsorption process aims to constant values so that the kinetic curves indicate the DPPH radicals capture ratio for the antioxidants in study.The antioxidant efficiency of dihydroquercetin (93.69%) and ascorbic acid (94.25%) can be noticed from both the calculated antioxidant activity values and the fact that the DPPH solution with dihydroquercetin, respectively ascorbic acid addition has faded from violet to dull yellow relatively rapid (aprox.1 min.).
Figure 2 below represents graphically the absorbance variation in time of solutions with addition of natural antioxidant (E1-E4).The antioxidant activity of roiboos extracts was compared with that of the green and black tea.For this specific purpose, DPPH was used to capture free radicals.The antioxidant activity of vegetable extracts estimated with the DPPH method decreases in the following order: green tea (Teavigo) >rooibos>black tea> extra green tea [16][17][18][19].

Antioxidant
Table 1 shows the chart represented by the antioxidant activities parameters of the substances in study.
The results of the analyses made for apple juice processed with or without the addition of synthesized antioxidants, immediately after preparation and kept at 50 0 C during 5 days, equivalent to 6-month storage at room temperature are presented in table 2.
The apple juice 100% obtained from apple juice clear concentrate is a clear product, with golden-brownish colour and a low content of Vitamin C, max 3.00 mg/L.The addition of antioxidants prevents, to a certain extent, spoilage.Due to the high temperature (90 o C) used for pasteurization and the relatively high storage temperature (50 o C), the content of vitamin C decreases considerably during pasteurization.If ascorbic acid is added to act as antioxidant, from 100 mg/L initially added, after 5-day storage at 50 o C temperature, the product remains with only 19.571 mg/L.
As regards the product clarity, there can be noticed the correlated values of transmittance (wavelength of absorption read at 625 nm) of the products kept in the drying chamber in comparison with the moment of preparation, for the samples treated with vitamin E, dihydroquercetin and ascorbic acid.Higher values of absorption show browning of fruit kept in the drying chamber, unlike the fresh ones, due to high storage temperature.The absorbance values of samples studied have shown an increase between 2.77 and 15%, in comparison with the sample non-treated with antioxidants, whose absorbance has modified with 10.57% after the same period of time.
In table 3 are presented the experimental results obtained for the apple juice with or without addition of vegetable extracts with antioxidant properties, immediately after processing and after 6-month storage at room temperature (kept at 50 0 C during 5 days).
In case of natural antioxidants, browning of apple juice kept in the drying chamber is more evident, fact also confirmed by the low values of transmittance, respectively high values of absorbance.From the extracts used, E3 and E4 have preserved the juice colour and clarity, the transmittance values being higher than in case of the witness sample, without addition of antioxidants.The samples absorbance values presented a variation between 5.3 and 33%, in comparison with 10.57% for the sample without addition of antioxidants.
The experimental results for orange juice with or without addition of synthesized antioxidants immediately after processing and kept at 50 0 C during 5 days, echivalentul at 6-month storage at room temperature are presented in table 4.
The addition of antioxidants does not prevent degradation of vitamin C in the orange juice stored at high temperatures.By comparing the absorbance values of samples kept in the drying chamber with the initial ones and the witness sample, it can be noticed the antioxidant activity of dihydroquercetin and coenzyme Q10.
In table 5 are presented the experimental results obtained for the orange juice with or without addition of vegetable extracts with antioxidant properties immediately after processing and after 6-month storage at room temperature.
The addition of vegetable extracts with antioxidant properties does not prevent the vitamin C degradation and orange juice colour during storage at high temperatures.
The studies results confirm the protective function of antioxidants against juice oxidation.Fruit nectars being more readily digestible than the other foods, are good source of antioxidants and the antioxidant capacities of them are comparable with the other research finding [20][21][22][23].
Due to -OH groups present in the antioxidants molecules, respectively the extracts molecules, part of the amount of water available to micro-organisms is binded, so that the levels of water activity are generally smaller then the levels of the witness sample.

Conclusions
The extracts of green tea (Teavigo) and rooibos, and dihydroquercetin have a high antioxidant capacity similar to ascorbic acid, used as reference standard (in juice industry).
The use of natural antioxidants, especially rooibos and green tea (Teavigo) extract has proved to be efficient for preserving fruit juice colour from undesired effects of spoilage that occurs during processing and storage.
Water activity is a predictive indicator for control of microbial growth in juice protected by treatment with antioxidant agents.As the antioxidants molecules contain various free -OH groups, by forming hydrogen bonds, the amount of water available to micro-organisms is reduced, thus the water activity also decreases.

Fig. 1 .
Fig. 1.Absorbance variation in time of DPPH solutions with addition of synthesized antioxidants

Fig. 2 .
Fig. 2. Absorbance variation in time of DPPH solutions with addition of vegetal extracts powder Figure 2 above shows that the adsorption aims to constant values, the kinetic curves representing the DPPH radical capture capacity for powder vegetable extracts with antioxidant properties.From graph 2 showing the absorption of the DPPH solution with addition of vegetable extracts, it can be noticed that the green tea extract (E1) (95.68%) and the rooibos extract (E4) (89.23%) have the highest antioxidant capacity.The antioxidant activity of roiboos extracts was compared with that of the green and black tea.For this specific purpose, DPPH was used to capture free radicals.The antioxidant activity of vegetable extracts estimated with the DPPH method decreases in the following order: green tea (Teavigo) >rooibos>black tea> extra green tea[16][17][18][19].

Table 2
EXPERIMENTAL VALUES FOR APPLE JUICE SAMPLES PROCESSED WITH THE ADDITION OF SYNTHESIZED A NTIOXIDANTS, IMMEDIATELY AFTER PREPARATION -(a), COMPARATIVE WITH SAMPLES KEPT AT 50 0 C DURING 5 DAYS -(b)

Table 3
EXPERIMENTAL VALUES FOR APPLE JUICE SAMPLES WITH THE ADDITION OF VEGETABLE EXTRACTS, IMMEDIATELY AFTER PREPARATION -(a), COMPARATIVE WITH SAMPLES KEPT AT 50 0 C DURING 5 DAYS -(b) Table 5 EXPERIMENTAL VALUES FOR ORANGE JUICE SAMPLES WITH THE ADDITION OF VEGETABLE EXTRACTS, IMMEDIATELY AFTER PREPARATION -(a), COMPARATIVE WITH SAMPLES KEPT AT 50 0 C DURING 5 DAYS -(b) Table 4 EXPERIMENTAL VALUES FOR ORANGE JUICE SAMPLES WITH THE ADDITION OF SYNTHESIZED ANTIOXIDANTS, IMMEDIATELY AFTER PREPARATION -(a), COMPARATIVE WITH SAMPLES KEPT AT 50 0 C DURING 5 DAYS -(b)