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Keywords:

  • Aqueous chlorine dioxide;
  • buckwheat sprout;
  • fumaric acid;
  • hurdle technology;
  • optimisation;
  • response surface methodology;
  • ultraviolet-C

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. References
  8. Supporting Information

The effects of the combined treatments with aqueous chlorine dioxide (ClO2), fumaric acid and ultraviolet-C (UV-C) on the microbial quality of common buckwheat sprouts were examined using a response surface methodology. The populations of total aerobic bacteria, yeast and mould, and coliform decreased with increasing aqueous ClO2 and fumaric acid concentrations and increasing UV-C irradiation dose. However, the increase in the UV-C irradiation dose had a negative effect on the sensory quality. Therefore, the optimal combined treatment condition of 100 ppm aqueous ClO2, 0.31% fumaric acid and 1.9 kJ m−2 UV-C was selected for the buckwheat sprouts by providing reductions of 3.9, 1.8 and 2.4 log CFU g−1 on the populations of total aerobic bacteria, yeast and mould, and coliform, respectively. The combined treatment also maintained an acceptable sensory quality. These results suggest that the optimised combined treatment can be used as a microbial inactivation method for buckwheat sprouts.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. References
  8. Supporting Information

Sprouts, such as alfalfa, buckwheat, clover and mung bean, have been popular as a functional product containing vitamins, flavonoids and polyphenols (Paśko et al., 2009) and consumed as a raw form in salad and Bibimbap, which is a typical Korean food, in Korea (Waje & Kwon, 2007). In particular, buckwheat sprouts have a high nutritional value because the main functional components are rutin, orientin and isovitexin, which have antioxidant, antihypertensive and antidiabetic properties (Kim et al., 2008a). Therefore, the production of buckwheat sprouts in Korea increases c. 24% annually (Jang et al., 2010).

However, despite the beneficial nutritional aspects, commercially grown sprouts have been associated with microbiological hazards. In particular, the consumption of sprouts has caused many outbreaks of foodborne diseases, and Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp. have been isolated from mung bean and alfalfa sprouts (Singla et al., 2011). In addition, sprouts have been easily contaminated by microorganisms because the growth conditions of the sprouts are similar to those favourable to bacterial proliferation (Peñas et al., 2010). There have been several reports (Kim et al., 2009a; Waje et al., 2009) that showed high levels of total aerobic bacteria (7–8 log CFU g−1), yeast and mould (5–7 log CFU g−1), and coliform (4–8 log CFU g−1) in commercial sprouts, such as alfalfa, broccoli, buckwheat, kale, radish, soya bean and red cabbage. High microbial populations on the sprouts can cause a short shelf life of the sprout products and increase the consumers' concern about sprout-related illness outbreaks.

Thus, consumers demand the microbiological safety of sprouts; to meet the market demand for high quality, microbial growth in the sprouts should be controlled. If pathogenic microorganisms in the sprouts are not completely removed through the sanitising process, the sprouts can become a vehicle for pathogens. Therefore, as microbial inactivation methods, chlorine (Gandhi & Matthews, 2003), electrolysed water (Zhang et al., 2011), ozone (Singla et al., 2011) and gamma radiation (Waje et al., 2009) treatments have been used to reduce the bacterial counts on the sprouts.

Although chlorine is commonly used as a sanitising agent, there have been some concerns about its health hazards due to the trihalomethanes and chlorophenols generated during chlorination, which are known mutagenic and carcinogenic compounds (Keskinen et al., 2009). Ozone may also cause adverse effects on the fruit aroma due to the oxidation of volatile compounds (Nadas et al., 2003). Therefore, there is a need for an alternative microbial inactivation method that is effective, nontoxic and easily applicable.

As an effective alternative to chlorine or ozone, there have been many studies on aqueous chlorine dioxide (ClO2), which is a surface disinfectant (Vandekinderen et al., 2009; López-Velasco et al., 2012; Shin et al., 2012). Due to its strong sterilising power, aqueous ClO2 has been shown to improve the microbiological safety of vegetables including sprouts (Gómez-López et al., 2009; Kim et al., 2009a). In addition, weak organic acids such as fumaric acid, malic acid and acetic acid have been studied for their antimicrobial effects on foods. In particular, fumaric acid has been used as a food preservative with bactericidal activity (Comes & Beelman, 2002; Kim et al., 2009b).

Ultraviolet-C (UV-C) irradiation approved by the Food and Drug Administration (FDA) has numerous advantages over other sanitising methods because it can be used to control surface microorganisms on vegetables and fruits without additional chemicals or heat (Unluturk et al., 2008; Chun et al., 2010; Gómez et al., 2010).

Hurdle technology, which is the combination of chemical and physical disinfection treatments and packaging methods, has been introduced to improve microbial safety and quality of sprouts. Singla et al. (2011) reported that the combined treatment of malic acid and ozone was more effective in the inactivation of Shigella spp. on radish and mung bean sprouts than a single disinfectant treatment. In addition, the combined treatment of 250 MPa high pressure and 18 000 ppm hypochlorite or 1500 ppm carvacrol resulted in the significant inactivation of native microbial flora on mung bean seeds (Peñas et al., 2010). However, few studies have been conducted on the combined treatment of aqueous ClO2, fumaric acid and UV-C for sprouts.

Response surface methodology (RSM) is a statistical method based on the multivariate nonlinear model, which has been widely used for optimal processing in the food industry (Gupta et al., 2012). RSM is one of the most popular optimisation techniques due to being a less laborious method than other approaches (Nwabueze, 2010; Prasad et al., 2011). Thus, RSM can be used to optimise the combined nonthermal treatment by varying aqueous ClO2 concentration, fumaric acid concentration and UV-C dose, based on the criteria of microbial count and sensory quality of buckwheat sprouts in the present investigation. Therefore, the objective of this study was to obtain the optimum condition of the combined treatment of aqueous ClO2, fumaric acid and UV-C dose on the inactivation of pre-existing microorganisms and the maintenance of the sensory quality of common buckwheat sprouts using a RSM.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. References
  8. Supporting Information

Experimental material

The commercially produced common buckwheat (Fagopyrum esculentum Moench) sprouts were purchased from a local farm in Gyeonggi-do, Korea. Common buckwheat seeds were soaked in water at room temperature for 12 h, grown in a dark chamber at 25 °C for 7 days and harvested. The buckwheat sprouts were then packed with polyethylene terephthalate containers and stored at 4 °C overnight prior to the experiment. Buckwheat sprouts with a uniform length (13 ± 2 cm) were selected and used for the experiment. Regarding the native microbial flora of the nontreated buckwheat sprouts used in this study, the initial populations of total aerobic bacteria, yeast and mould, and coliform were 7.13, 5.78 and 6.97 log CFU g−1, respectively.

Experimental design

Response surface methodology was used to analyse the effects of three independent variables (aqueous ClO2 concentration, fumaric acid concentration and UV-C irradiation dose) on six response variables: the reduction in the populations of total aerobic bacteria, yeast and mould, and coliform, and the change in the appearance, colour and overall acceptability of the buckwheat sprout. The experiments were performed according to the Box–Behnken experimental design described by Peñas et al. (2010) with some modifications. The range and the level of each variable in the present investigation are summarised in Table S1. The ranges of the experimental parameters were selected based on the preliminary experiments. Seventeen combinations (including three replicates of the centre point) of three independent variables were performed following the design (Table S2). Three replications of each experiment were conducted.

Aqueous ClO2 and fumaric acid treatment

Aqueous ClO2 was prepared using a chlorine dioxide-generating system (CH2O Inc., Olympia, WA, USA) according to the method described by Kim et al. (2009b) and Shin et al. (2012). Aqueous chlorine dioxide generated by the system did not contain detectable free or total chlorine. The ClO2 concentration was measured according to the iodometry method (APHA, 1995). Aqueous ClO2 concentrations of 50 ppm and 100 ppm were made by dilution with distilled water. Fumaric acid powders (Sigma Aldrich, St. Louis, MO, USA) were dissolved in 50 ppm or 100 ppm aqueous ClO2 solutions or distilled water to obtain concentrations of 0.2% and 0.4%. All solutions were freshly made on the day of the experiment. The buckwheat sprout samples were treated by dipping into seventeen solutions comprising a combination of 0–100 ppm ClO2 and 0–0.4% fumaric acid at a ratio of 1:10 (w/v) with gentle agitation for 5 min and were then air-dried in a laminar flow biosafety hood for 60 min.

Ultraviolet-C irradiation

After the aqueous ClO2 and fumaric acid treatment, the UV-C irradiation was performed using unfiltered germicidal light-emitting lamps (Sylvania, G15T8, Phillips, Eindhoven, The Netherlands) located in a metal cabinet. The UV lamps were warmed up for 30 min before the irradiation to achieve reproducible results. The buckwheat sprouts were placed on a tray and irradiated with eight germicidal light-emitting lamps on both the upper and lower surfaces at a distance of 18 cm. The UV-C intensity was determined using a UV radiometer (UV-340, Lutron Electronic Ent. Co., Ltd. Taipei, Taiwan) calibrated at 254 nm, and the UV-C irradiation dose was changed by altering the exposure time (dose rate; 12 W m−2). The buckwheat sprouts were exposed to 2 and 4 kJ m−2, based on a previous study (Kim et al., 2009a) and a preliminary study using buckwheat sprout samples. The UV-C doses of 2 and 4 kJ m−2 were delivered for 166 and 332 s, respectively. After UV-C irradiation, the buckwheat sprout samples were immediately subjected to microbiological analysis and sensory evaluation without packaging and storage.

Microbiological analysis

The procedure of microbial enumeration described by Chun et al. (2010) was used with some modifications. The buckwheat sprout samples (20 g) were placed into 180 mL of peptone water (0.1% sterile peptone, w/v) in a sterile stomacher bag. The samples were then homogenised using a stomacher blender (MIX 2, AES Laboratoire, Combourg, France) for 3 min, filtered through sterile cheese cloth and diluted with peptone water for the microbial count. Serial dilutions were performed in triplicate. The total aerobic bacterial counts were determined by plating appropriately diluted samples onto plate count agar (PCA, Difco Co., Detroit, MI, USA). Yeast and mould were plated on potato dextrose agar (PDA, Difco Co.). Both plates were incubated at 37 °C for 48 h and 72 h, respectively. The coliform counts were determined by plating appropriately diluted samples onto 3 m Petrifilm Ecoli/Coliform Count Plate (Petrifilm EC, 3M Co., St. Paul, MN, USA). The coliform plates were incubated at 37 °C for 24 h. All experiments were performed in triplicate with three observations per trial. Each microbial count was reported as the mean of three determinations and expressed as the log CFU g−1.

Sensory evaluation

The buckwheat sprouts were analysed for their appearance, colour and overall acceptability by fifteen trained panellists (eight men and seven women; age range, 23–32) who had more experience in sensory evaluation of food products. Sample evaluation was carried out in triplicate for three sessions, and each panellist evaluated three samples per session. The buckwheat sprout samples (20 g) were presented on plastic dishes under normal light conditions. Compared with the control (nontreated samples), the sensory qualities of the treated samples were evaluated using a 9-point scoring method as follows: 8–9, very good; 6–7, good; 4–5, fair; 2–3, poor; and 1, very poor.

Statistical analysis

The statistical analysis was performed using Design-Expert version 8 software (Stat-Ease Inc., Minneapolis, MN, USA), and the results were the average of three independent determinations. The response was related to a function of independent variables by a quadratic polynomial model as follows:

  • display math

where Y is a dependent variable, and β0 is a constant coefficient. The coefficients (β1, β2 and β3), (β11, β22 and β33) and (β12, β13 and β23) represent the linear, quadratic and interaction coefficients, respectively, of the model. X1, X2 and X3 represent the independent variables in coded values (−1, 0 and 1). The goodness of fit of the model was evaluated by the coefficients of determination, R2 and inline image (adjusted determination coefficient). The significance of all the terms in the polynomial equation was evaluated by computing F-values and was analysed at a probability level of < 0.01 or 0.05.

Optimisation

Numerical and graphical optimisations were conducted to improve the microbial safety and maintain the sensory quality of buckwheat sprouts by analysing superimposed contour plots of the response variables using Design-Expert version 8 software (Stat-Ease Inc.).

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. References
  8. Supporting Information

Effects of combined treatments of aqueous ClO2, fumaric acid and UV-C on the microbial reduction in buckwheat sprouts

To evaluate the goodness of fit of the quadratic model applied for the microbial reduction in the buckwheat sprouts treated with the combinations of aqueous ClO2, fumaric acid and UV-C to determine the experimental results, the coefficients of determination (R2) were calculated (Table S3). In general, the value of R2 or inline image being close to 1 would indicate a goodness of fit of the quadratic model to the experimental results (Chauhan & Gupta, 2004; Peñas et al., 2010). The high value of R2 obtained in this study indicates that none of the predicted values had a significant lack of fit (Peñas et al., 2010).

The analysis of variance (anova) showed that the quadratic polynomial model adequately represented the experimental data, with the R2 being 0.9757, 0.9672 and 0.9778 for the reductions in the populations of total aerobic bacteria, yeast and mould, and coliform, respectively. The adjusted value of R2 (inline image) corrects R2 according to the sample size and the number of terms in the model. If there are too many terms in the model and the sample size is small, inline image will be less than R2, indicating that the model contains too many terms (Lahlali et al., 2008). The inline image of the total aerobic bacteria, yeast and mould, and coliform was 0.9444, 0.9250 and 0.9492, respectively, for the model. These values are similar to the R2 values, indicating that there is a good agreement between the experimental and predicted values for the reductions in the populations of total aerobic bacteria, yeast and mould, and coliform in the buckwheat sprouts.

The regression coefficients in Table S3 showed that all the linear terms of aqueous ClO2, fumaric acid and UV-C had significant (< 0.01) effects on the microbial inactivation in the buckwheat sprouts. The quadratic term of aqueous ClO2 indicated a significant (< 0.05) effect on the reduction in the yeast and mould and in the coliform. In addition, the fumaric acid treatment had a significant (< 0.05) quadratic impact on the inactivation of the total aerobic bacteria and the yeast and mould. The interactions of aqueous ClO2 and UV-C or fumaric acid and UV-C had significant (< 0.05) effects on reducing the populations of yeast and mould. Notably, the effectiveness of either aqueous ClO2 or fumaric acid increased when combined with UV-C irradiation. Our results also confirmed the results obtained by Kim et al. (2009a), in which the combination of 0.5% fumaric acid and 1 kJ m−2 UV-C reduced the populations of Ecoli O157:H7, Styphimurium and Lmonocytogenes inoculated on clover sprouts by 3.02, 2.88 and 2.35 log CFU g−1, respectively, and the combined treatment of 0.5% fumaric acid and 50 ppm aqueous ClO2 reduced the populations of total aerobic bacteria on alfalfa sprouts by 3.18 log CFU g−1. Kim et al. (2009b) also reported that the combined treatment of broccoli sprouts with 0.5% fumaric acid and 50 ppm aqueous ClO2 was more effective than that of either 50 ppm aqueous ClO2 or 0.5% fumaric acid alone, reducing the total aerobic bacteria and coliform by 2.70 and 1.17 log CFU g−1, respectively.

The antimicrobial effects of ClO2 and fumaric acid are primarily due to oxidation against cell surface membrane proteins and decrease in intracellular pH and enzyme inhibition, respectively (Comes & Beelman, 2002; Vandekinderen et al., 2009; Smigic et al., 2010). UV-C irradiation causes cross-links between neighbouring pyrimidine bases of cytosine and thymine in DNA, blocking microbial cell replication, and eventual cell death (Artés et al., 2009). A combination of these hurdles can have a synergistic effect on reducing microorganisms.

Fett (2002) reported that the addition of H2O2, acidified NaClO2, Na3PO4 and NaOCl to irrigation water did not reduce the levels of native microorganisms associated with alfalfa sprouts by more than 1 log cycle. Compared with this report, our study clearly indicates that the combined treatment of aqueous ClO2, fumaric acid and UV-C more effectively reduced the population of microorganisms on buckwheat sprouts, resulting in improved microbial safety.

The response surface plots were generated to better visualise the combined effects of the three independent variables on the microbial reduction (Figs S1 and S2). Figure S1a, b and c are response surface plots showing the reductions in the total aerobic bacteria, yeast and mould, and coliform on the buckwheat sprouts treated with different aqueous ClO2 and fumaric acid concentrations at a fixed UV-C dose of 2 kJ m−2. Figure S2a, b and c are response surface plots showing the reductions in the total aerobic bacteria, yeast and mould, and coliform on the buckwheat sprouts treated with different fumaric acid concentrations and UV-C doses at a fixed aqueous ClO2 concentration of 50 ppm. Based on Figs S1 and S2, the reductions in the populations of total aerobic bacteria and coliform increased as the aqueous ClO2 and fumaric acid concentrations or UV-C irradiation dose increased. However, it should be noted that the reduction in the population of yeast and mould was less than those in the total aerobic bacteria and coliform. This difference might be explained by the different optimal growth conditions of the microorganisms, depending on the species of microorganism. Bacteria have fastidious growth requirements and prefer to grow at a pH near neutral (pH 6.5–7.5), whereas yeast and mould are more tolerant of lower pH values than bacteria (Doores, 2005). Vandekinderen et al. (2009) reported that ClO2 treatment was effective against the growth of Gram-negative and Gram-positive bacteria, but that yeast and mould were more resistant. Similarly, Gabriel (2012) reported that yeast species had greater resistance to UV-C irradiation than Ecoli O157:H7. Therefore, further study is needed to determine the most appropriate processing condition to improve the reduction in the yeast and mould in buckwheat sprouts.

Change in sensory qualities of buckwheat sprouts

In a preliminary experiment (data not shown), fumaric acid was more effective than acetic acid, citric acid, malic acid or lactic acid at the same concentration for the reduction in the pre-existing microorganisms on buckwheat sprouts. In addition, all the organic acid treatments, such as fumaric acid, acetic acid, citric acid, malic acid and lactic acid, at more than 0.5% caused surface discolouration of the sprout due to the degradation of chlorophyll. Therefore, in the present investigation, fumaric acid in a range of concentrations <0.5% was applied.

To evaluate the goodness of fit of the quadratic model for the sensory qualities of the buckwheat sprouts treated with the combinations of aqueous ClO2, fumaric acid and UV-C to the experimental results, the R2 values were calculated (Table S3). The pattern of the goodness of fit of the quadratic model for the sensory scores of the buckwheat sprouts was similar to that of the microbial reduction. The anova showed that the resultant polynomial model adequately represented the experimental data, with the R2 coefficients of 0.9494, 0.9609 and 0.9651 for the appearance, colour and overall acceptability, respectively. These results indicated that the quadratic model accounted for more than 95% of the variation in the experimental data. The inline image of the appearance, colour and overall acceptability was 0.8843, 0.9105 and 0.9201, respectively, for the model, indicating that the model had a goodness of fit.

The coefficients of the regression equation describing the change in the sensory quality of the buckwheat sprouts treated with the combinations of aqueous ClO2, fumaric acid and UV-C are shown in Table S3. The sensory qualities, such as appearance, colour and overall acceptability, of the buckwheat sprouts were primarily affected by the linear term of UV-C (< 0.01). The quadratic term of UV-C also showed a significantly (< 0.01) negative effect on the sensory quality. However, the interaction between aqueous ClO2 and UV-C was significant only for the colour of buckwheat sprouts (< 0.05). Bermúdez-Aguirre & Barbosa-Cánovas (2013) reported that UV-C irradiation negatively affected the colour of lettuce, although UV-C was more effective in the inactivation of microorganisms when the UV-C dose was higher. Similar results were also observed in the present investigation. For aqueous ClO2 and fumaric acid, there were no significant (> 0.05) effects on the appearance, colour and overall acceptability of the treated buckwheat sprouts (Table S3). Therefore, the optimisation of the combined treatments is needed to minimise the changes in the sensory quality while maximising the microbial safety.

The response surface plots obtained for the maintenance of the sensory quality of the buckwheat sprouts treated with the combinations of aqueous ClO2, fumaric acid and UV-C are shown in Figs S3 and S4. The sensory scores for the appearance, colour and overall acceptability of the buckwheat sprouts decreased as the UV-C dose increased (Fig S4a, b and c), whereas the aqueous ClO2 (<100 ppm) and fumaric acid (<0.4%) concentrations did not significantly affect the sensory quality (Fig. S3a, b and c). Based on these results, a UV-C dose higher than 2 kJ m−2 to increase the microbial reduction caused a deterioration in the sensory quality, resulting in the discolouration and wrinkling of the leaves of the buckwheat sprouts. In addition, UV-C irradiation >2 kJ m−2 was unacceptable in terms of the sensory quality. Therefore, it should be noted that UV-C irradiation treatment for commercial use in fresh produce, including sprouts, should depend on the capability to reduce pre-existing microorganisms without causing undesirable quality changes. However, 100 ppm aqueous ClO2 had no significant impact on the sensory quality of the buckwheat sprouts. Our results are in good agreement with those of the other reports (Kim et al., 2008b), in which the aqueous ClO2 treatment did not affect the colour of iceberg lettuce during storage. Therefore, the hurdle technology in the present investigation should have a synergistic effect with respect to microbial inactivation with a minimal negative effect on the sensory quality of the buckwheat sprouts.

Overall, RSM can be used to optimise the combined treatment conditions of aqueous ClO2, fumaric acid and UV-C for the microbial reduction and maintenance of the sensory quality of buckwheat sprouts.

Optimisation

The optimisation of the combined treatment was obtained by analysing superimposed contours (Fig. S5), which were used to determine the best combination of aqueous ClO2, fumaric acid and UV-C. The optimal treatment condition was designed for reductions of more than 3.9, 1.8 and 2.4 log CFU g−1 in the populations of total aerobic bacteria, yeast and mould, and coliform, respectively, and for sensory quality scores >8.5. The superimposed contours of all responses for aqueous ClO2 and UV-C (Fig. S5a) or fumaric acid and UV-C (Fig. S5b) and their intersection zones indicated the ranges of the variables for improving the microbial safety and maintaining the sensory quality of the buckwheat sprouts. The optimum ranges of the variables obtained from the superimposed contours are as follows: aqueous ClO2, 96.5–99.8 ppm; fumaric acid, 0.23–0.35%; and UV-C, 1.8–2.1 kJ m−2. In addition, the optimum results were obtained by the numerical optimisation method at the following level of the treatment condition: aqueous ClO2, 100 ppm; fumaric acid, 0.31%; and UV-C, 1.9 kJ m−2. In the present investigation, hurdle technology was applied to reduce pre-existing microorganisms without affecting the sensory quality of buckwheat sprouts.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. References
  8. Supporting Information

Response surface methodology was used to optimise the combined treatments of aqueous ClO2, fumaric acid and UV-C to reduce the native microbial flora on buckwheat sprouts. The present investigation suggests that combination of aqueous ClO2, fumaric acid and UV-C can be used as an effective hurdle to inactivate microbial populations in buckwheat sprouts. In particular, the optimal condition to achieve microbial safety and maintain the sensory quality of buckwheat sprouts was the combination of 100 ppm aqueous ClO2, 0.31% fumaric acid and 1.9 kJ m−2 UV-C. Based on this finding, this hurdle technique can be used for industrial application without affecting sensory quality of buckwheat sprouts.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. References
  8. Supporting Information
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ijfs12283-sup-0001-FigureS1-S5-TableS1-S3.pdfapplication/PDF622K

Figure S1. Response surface plots for the reduction in microbial populations in the buckwheat sprouts treated with aqueous chlorine dioxide (ClO2) and fumaric acid at fixed ultraviolet-C (UV-C) irradiation dose (2 kJ m2). (a) Total aerobic bacteria, (b) yeast and mould, and (c) coliform.

Figure S2. Response surface plots for the reduction in microbial populations in buckwheat sprouts treated with fumaric acid and ultraviolet-C (UV-C) at fixed aqueous chlorine dioxide (ClO2) concentration (50 ppm). (a) Total aerobic bacteria, (b) yeast and mould, and (c) coliform.

Figure S3. Response surface plots for the change in sensory scores of the buckwheat sprouts treated with aqueous chlorine dioxide (ClO2) and fumaric acid at fixed ultraviolet-C (UV-C) irradiation dose (2 kJ m2).(a) Appearance, (b) colour and (c) overall acceptability.

Figure S4. Response surface plots for the change in sensory scores of the buckwheat sprouts treated with fumaric acid and ultraviolet-C (UV-C) at fixed aqueous chlorine dioxide (ClO2) concentration (50 ppm). (a) Appearance, (b) colour and (c) overall acceptability.

Figure S5. Superimposed contours for the reduction in microbial populations and change in sensory scores of the buckwheat sprouts at varying a aqueous chlorine dioxide (ClO2) concentration, fumaric acid concentration and ultraviolet-C (UV-C) irradiation dose. (a) Aqueous ClO2 and fumaric acid, and (b) fumaric acid and UVC.

Table S1. Levels of independent variables used in the Box–Behnken design.

Table S2. Box–Behnken design with coded and uncoded values and experimental results for response variables of buckwheat sprouts.

Table S3. Regression coefficients of the second-order polynomials and significance of each model for dependent response variables in buckwheat sprouts treated with combinations of aqueous chlorine dioxide (ClO2), fumaric acid and ultraviolet-C (UV-C) in actual level of variables.

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