Gamma irradiation and subsequent storage reduce patulin content in apple juice

Patulin standard and chemical reagents, such as acetonitrile, ethyl acetate, formic acid, toluene, and Folin-Ciocalteau reagent, were procured from Sigma Chemical Co. (St. Louis, MO, USA). The stock solution of patulin (1,000 ppb in distilled water) was prepared and diluted for the calibration curve. Apple juice (N Co., Seoul, Korea) was purchased from a supermarket in Jeongeup City, Korea.

To monitor the conditions for both reducing patulin content and minimizing the changes in the functional components of juice samples, RSM (Gontard et al., 1992) was used to observe the changes in the corresponding parameters (dependent variables) as affected by treatment conditions (independent variables), which were based on a central composite design (Kim et al., 2005; Lee et al., 2000) and the statistical analysis system (SAS, 1990). Independent variables, including storage temperature (X₁, varying between −20°C and 20°C,), irradiation dose (X₂, varying between 0 and 2 kGy), and storage period (X₃, varying between 0 and 20 days), were coded at five levels (−2, −1, 0, 1, 2), and their values were chosen based on experiments for dependent variables (Yn), such as residual patulin content and polyphenolics content (Table 1). The central complete design consisted of 16 experimental points, including two points of center position (Kim et al., 2005; Lee et al., 2000).

Apple juice sample (50 mL) of a commercial product (N company) was bottled in a conical tube (polyethylene, PE), patulin (675 ppb) was injected and then irradiated with a cobalt-60 gamma irradiator (point source, AECL, IR-79, Nordion, Canada) at absorbed doses of 0, 0.5, 1.0, 1.5, and 2.0 kGy. The cobalt-60 source strength was approximately 100 kCi with 70 Gy/min dose rate at ambient conditions. The absorbed doses were monitored using free-radical and ceric/cerous dosimeters, and the actual dose ranges were within ±5.4% of the target doses. Apple juice samples were stored at −20, −10, 0, 10, and 20°C for 2 h before irradiation treatment, and then stored at the same temperature for 20 days after irradiation treatment and used in the experiment.

Analysis of patulin was carried out using the AOAC method (1996). In a tube, each sample from the gamma-irradiated and control groups was extracted by adding 50 mL of ethyl acetate. The extracts were mixed with 20 g of anhydrous sodium sulfate, allowed to stand at room temperature for 30 min, and filtered using a Whatman No. 4 filter paper (Sigma-Aldrich). The filtrate was concentrated at 40°C under a nitrogen stream, cooled at room temperature, and stored at −70°C in distilled water (pH 4.0).

Quantitative analysis of patulin was performed by high-performance liquid chromatography (HPLC) using a Waters Alliance 2690 (Waters Co., Milford, MA, USA) system equipped with a photodiode array detector (PAD, Waters 996, Waters Co.) HPLC was performed using a Shiseido column (3.9 mm i.d., 300 mm length), and the extracts were separated using a distilled water–acetonitrile (95:5) solvent at a flow rate of 0.7 mL/min. The column temperature was maintained constant at 25°C. The patulin peak was identified using a patulin standard by comparing its retention time. Detection was conducted by acquiring the PAD spectra of eluted compounds (200-400 nm). Based on chromatograms obtained at 276 nm. The entire UV spectra for all positive samples were compared with those of the external patulin standard solution analyzed under the same conditions. Control samples, along with the samples and calibration standards, were analyzed for each matrix.

The polyphenolic content was determined by the Folin-Ciocalteau colorimetric method (Gao et al., 2000). The extracts (0.9 mL) were mixed with 0.1 mL of 50 units/mL of ascorbic oxidase and then incubated for 90 min at 23°C to remove ascorbic acid. Next, the ascorbic acid-free extract (0.1 mL) was mixed with 0.2 mL of Folin-Ciocalteau reagent and incubated for 1 min at 23°C. After adding 3 mL of 5% Na₂CO₃ and incubating for 2 h at 23°C, the absorbance of the mixtures was recorded at 765 nm. The polyphenolic content was expressed as gallic acid equivalents.

Triplicate determinations were conducted at all design points in a randomized order. The corresponding extracts were subjected to analysis for dependent variables (responses), such as residual patulin content (Y₁) and polyphenolics content (Y₂). The mean values of triplicate determinations were analyzed to fit the following second-order polynomial models to all dependent Y variables. The following model was proposed for each response of Y:

where X₁, X₂, and X₃ correspond to independent variables, i.e., irradiation/storage temperature, irradiation dose, and storage period; b₀ is the intercept; and bn represents the corresponding regression coefficients (Gontard, 1992). A statistical analysis system from the SAS Institute (Cary, NC, USA) (SAS, 1990) was applied to predict models through regression analysis and analysis of variance. Response surfaces were developed using fitted polynomial equations. When the results demonstrated a saddle point in the response surfaces, the optimal conditions were determined using ridge analysis. Four-dimensional response surfaces were obtained using the Mathematica software (Wolfram Research, Champaign, IL, USA, 17).

Table 1 shows the residual patulin content under the 16 experimental conditions set by the central composite design. The regression equations for the response variables (Yn) are listed in Table 2. R² for the regression equation of Y₁ was 0.9163, with a significance of <5% being recognized (p<0.05). The predicted peak point resulted in the lowest residual patulin content of 58.42 ppb with the corresponding independent parameters conditions, such as 18.19°C of storage temperature, 1.24 kGy of irradiation dose, and 13.42 days of storage period (Table 3). Patulin content of 58 ppb was desirable, representing a 91% decrease compared to the prediction of 675 ppb from the nonirradiated control sample (Table 3). Four-dimensional response surfaces for residual patulin content as affected by treatment conditions are depicted in Fig. 1, indicating that the residual patulin content decreased by the irradiation dose with the most predominant effect (p<0.01), whereas the effects of storage temperature and period on its reduction were nonsignificant (Table 4).

Fig. 1. Four-dimensional response surface for patulin content in apple juice as affected by storage temperature (°C), irradiation dose (kGy), and storage period (day).

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Patulin has been detected in apples and other fruits, such as strawberries, mangoes, grapes, bananas, and tomatoes. Damage to fruit skin, rotten raw fruits, or inappropriate technological processing steps results in fungal growth and the consequent patulin production. Physicochemical methods, including clarification, washing, filtration, and chemical treatment, were reported to reduce patulin content in apple products, including apple juice, effectively. However, gamma irradiation and subsequent storage is one of the most efficient methods to reduce patulin content in apple juice because it can be applied to the final packaged products in an automatic conveyor system without further contamination during the drying and/or packaging processes of the treated products. UV radiation inactivates primary microorganisms and reduces patulin content (Kitipong et al., 2012). If the initial patulin contamination level is approximately 1,000 ppb (mg/liter), the UV exposure, ranging from 14.2 mJ/cm² (one pass) to 99.4 mJ/cm² (seven passes), was successful in reducing patulin levels by 72.57% to 5.14%, respectively.

Polyphenolic compounds in apples are health-beneficial ingredients with antioxidant and antibacterial functions (Gardner et al., 2000). The conditions for minimizing the changes in polyphenolic content were investigated when applying gamma irradiation and subsequent storage at various temperature conditions.

The polyphenolic content of apple juice according to the designed conditions is shown in Table 1. The changes in polyphenolic content under three different treatment conditions are illustrated in Fig. 2. The regression equation for polyphenolic content (Y₂) revealed an R² of 0.9026; its significance was confirmed within a level of 5% (Table 2). The maximum polyphenolics content of apple juice was 11.98 mg/g under irradiation/storage temperature of 14.40°C, irradiation dose of 0.78 kGy, and storage period of 3.4 days (Table 3). The polyphenolic content was reduced to 9.79 mg/g when the storage period exceeded 15.13 days, even at a low gamma irradiation dose of 0.22 kGy (Table 3). The polyphenolic content of apple juice was significantly influenced by the irradiation dose and storage period (p<0.05, Table 4).

Fig. 2. Four-dimensional response surfaces for polyphenolic content in apple juice as affected by storage temperature (°C), irradiation dose (kGy), and storage period (day).

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Possible mechanisms of the effect of radiation processing on phenolic compounds in mature, dry seeds of cereal and legumes are proposed, and higher irradiation doses also cause the breakage of the glycosidic bonds of phenolic compounds with carbohydrates (Shi et al., 2022). However, applied doses for apples or apple juices are too low to affect the glycosidic bonds of phenolic compounds.