Optimization of supplementation with maltodextrin and grape seed extract for improving quality of shredded Korean cabbage (Brassica rapa L. ssp. Pekinensis) during salting process

Korean cabbage (B. rapa L. ssp. Pekinensis cv. Hwiparam) was cultivated in the field and harvested in early November, 2015 in Haenam-gun, Korea. The outer leaves and foreign substances were removed, and the remaining leaves were evenly shredded into pieces of approximately 4×4 cm using a knife sterilized before use by dipping into 70% alcohol and flaming.

RSM was used to analyze the effects of four independent variables (NaCl concentration, salting time, MD concentration, and GSE concentration) on the following response variables: reduction in total aerobic bacteria, yeast, and mold counts; weight loss and salt content; and taste, texture, and overall acceptability of the salted shredded Korean cabbage. The range and levels of each variable are summarized in Table 1. The ranges of the experimental parameters were selected based on preliminary experiments.

The experiments were performed according to a previously described central composite design (25), with some modifications. In this study, the experimental combinations were at the midpoints of edges and at the center of the process space. In total, 20 combinations of four independent variables were used (Table 2). Each experiment was performed in triplicate.

Brine as a salting solution (5~15% NaCl, w/v) was prepared by dissolving commercially refined salt (purity: >99.5%; Hanju Co., Ulsan, Korea) in tap water. MD (dextrose equivalent: 15~20; Daesang Co., Gunsan, Korea) solution at concentrations of 5 and 10% was prepared by diluting with 5, 10, and 15% brine. The salting solution was agitated for 30 min, and GSE (DF-100; Food Additive Bank Co., Anseong, Korea) was dissolved in the solution with or without MD to final concentrations of 150 and 300 ppm. All solutions were freshly prepared on the day of the experiment. Shredded Korean cabbage samples were treated by immersion into the 20 solutions comprising different combinations of 5~15% NaCl, 0~10% MD, and 0~300 ppm GSE at a ratio of 1:5 (w/v) for a salting time of 2~8 hr. After salting, each cabbage sample was washed twice with distilled water for subsequent experiments.

For microbiological analysis, samples (25 g) were first mixed with 225 mL peptone water (0.1% sterile peptone, w/v) in a sterile Stomacher bag with a polyethylene filter layer. They were then homogenized using a Stomacher MIX 2 instrument (AES Laboratoire, Combourg, France) for 3 min, and diluted with peptone water for microbial counts. Serial dilutions were prepared in triplicate. For total aerobic bacterial counts, samples were spread on 3M Petrifilm Aerobic Count Plates (3M Co., St. Paul, MN, USA) and incubated at 37℃ for 48 hr, whereas yeast/molds were spread on 3M Petrifilm Yeast and Molds Count Plates (3M Co.) and incubated at 25℃ for 72 hr. Three replicates were prepared for each plate, and counts are expressed as log CFU/g.

Salted shredded Korean cabbage was analyzed for taste, texture, and overall acceptability by 30 trained panelists (15 men and 15 women; age range, 25~45 years) at room temperature (22℃±1℃). Samples (100 g) were presented on a plastic dish under normal light conditions. The sensory qualities of the samples were evaluated according to a 9-point scale as follows: 8 or 9, very good; 6 or 7, good; 4 or 5, fair; 2 or 3, poor; and 1, very poor. All experiments were performed in triplicate with three observations per trial.

Weight loss results from water loss and solid gain during osmotic dehydration. After the salting process, the weight loss of shredded Korean cabbage was calculated as follows:

Weight loss (%)=[fresh Korean cabbage weight (g)-salted and dehydrated Korean cabbage weight (g)]/fresh Korean cabbage weight (g)×100.

The salt content of each sample was measured using Mohr’s titration method (4). Samples (1 g) were homogenized in 99 mL distilled water and then passed through Whatman No. 2 filter paper (Whatman, Springfield, UK). A 1-mL volume of 2% potassium chromate indicator was added to 10 mL of the filtered sample solution, and this mixture was titrated against 0.02 N AgNO₃ (Daejung Chemicals & Metals Co., Siheung, Korea) until a red-brown color (i.e., the end point) was observed.

Statistical analysis was performed using Design-Expert v.10 software (Stat-Ease, Minneapolis, MN, USA); results represent the average of four independent measurements. The response was expressed as a function of the independent variables defined by the following quadratic polynomial model:

$\begin{array}{l}{\text{Y = b}}_{\text{0}}{\text{ + b}}_{\text{1}}{\text{X}}_{\text{1}}{\text{ + b}}_{\text{2}}{\text{X}}_{\text{2}}{\text{ + b}}_{\text{3}}{\text{X}}_{\text{3}}{\text{ + b}}_{\text{4}}{\text{X}}_{\text{4}}{\text{ + b}}_{\text{12}}{\text{X}}_{\text{1}}{\text{X}}_{\text{2}}{\text{ + b}}_{\text{13}}{\text{X}}_{\text{1}}\text{X3}\\ {\text{+ b}}_{\text{14}}{\text{X}}_{\text{1}}{\text{X}}_{\text{4}}{\text{ + b}}_{\text{23}}{\text{X}}_{\text{2}}{\text{X}}_{\text{3}}{\text{ + b}}_{\text{24}}{\text{X}}_{\text{2}}{\text{X}}_{\text{4}}{\text{ + b}}_{\text{34}}{\text{X}}_{\text{3}}{\text{X}}_{\text{4}}{\text{ + b}}_{\text{11}}{\text{X}}_{\text{12}}{\text{ + b}}_{\text{22}}{\text{X}}_{\text{22}}\\ {\text{+ b}}_{\text{33}}{\text{X}}_{\text{32}}{\text{ + b}}_{\text{44}}{\text{X}}_{\text{42}}\end{array}$

where Y is a dependent variable and b₀ is a constant. The coefficients (b₁, b₂, b₃, and b₄), (b₁₂, b₁₃, b₁₄, b₂₃, b₂₄, and b₃₄), and (b₁₁, b₂₂, b₃₃, and b₄₄) represent the linear, interaction, and quadratic coefficients, respectively, of the model, and X₁, X₂, X₃, and X₄ represent the independent variables. The goodness of fit of the model was evaluated based on the coefficient of determination (R²) and adjusted R² (R²_adj). The significance of all terms in the polynomial equation was assessed by computing F values and was analyzed at probability levels of p<0.05, 0.01, or 0.001.

Numerical optimization was performed to improve the microbiological safety and quality of salted shredded Korean cabbage through analysis of superimposed contour plots of the response variables using Design-Expert v.10 software.

The initial counts of total aerobic bacteria and yeast/molds on salted shredded Korean cabbage were 6.38 and 3.87 log CFU/g, respectively. These results are similar to those from a previous study that found high levels of total aerobic bacteria (5.2~7.6 log CFU/g) and yeast/molds (2.5~4.3 log CFU/g) in Korean cabbage (5,10). Such cabbage can be contaminated by various microorganisms, including pathogenic bacteria, during the salting process (9). High microbial counts on salted Korean cabbage can reduce the shelf life of kimchi products and increase consumer concerns regarding kimchi-related illness. Therefore, sterilizing salted Korean cabbages is necessary to ensure long shelf life and consumer safety.

We evaluated the goodness of fit of the quadratic model applied to microbial reduction in Korean cabbage salted under different conditions by calculating R² (Table 3). In general, R² or R²_adj values close to 1 indicate goodness of fit of the quadratic model to the experimental results (25,26).

The high R² value obtained here indicates that all of the predicted values had a good fit (26).

The analysis of variance (ANOVA) showed that the quadratic polynomial model adequately represented the experimental data, with R² values of 0.955 and 0.809 for the reductions in total aerobic bacteria and yeast/mold counts, respectively. R²_adj corrects R² according to sample size and number of terms in the model; in cases where there are many terms in the model and the sample size is small, R²_adj is less than R², indicating that the model is inadequate (26). The R²_adj of total aerobic bacteria and yeast/molds was 0.903 and 0.770, respectively, for the model; this was similar to the R² values, indicating good agreement between the experimental and predicted reductions in total aerobic bacteria and yeast/molds in shredded Korean cabbage during the salting process.

The p value was used to assess the significance of each term representing the interactions among variables (25); smaller p values indicate greater significance of the corresponding coefficient (25-27). The regression coefficients in Table 3 show that the linear terms of the GSE concentration had significant effects on microbial inactivation in salted shredded Korean cabbage (p<0.001).

NaCl and GSE concentrations had significant positive effects on reducing the counts of total aerobic bacteria (p<0.05). The quadratic term of the NaCl concentration indicated a significant positive effect on the reduction of total aerobic bacteria and yeast/mold counts (p<0.05). However, the quadratic terms of salting time, MD concentration, and GSE concentration did not significantly influence reductions in total aerobic bacteria or yeast/mold counts.

Response surface plots were generated to better visualize the combined effects of the four independent variables on the reduction in the amount of total aerobic bacteria (Fig. 1a) and yeast/molds (Fig. 1b) on shredded Korean cabbage treated with different NaCl and GSE concentrations at a fixed salting time of 5 hr and MD concentration of 5%. The magnitude of the reduction in total aerobic bacteria count increased as the GSE and NaCl concentrations increased, whereas the change in the reduction in yeast/mold count was less obvious, implying that the effect of GSE and NaCl on reducing the counts of preexisting microorganisms depends on the type of microbe present. It was previously shown that GSE treatment suppressed growth in gram-negative and -positive bacteria but was less effective against yeast and molds (14). Yeast showed greater resistance to NaCl (water activity) than bacteria (27). Additionally, the minimum inhibitory concentration of GSE was previously reported to be 850~1,000 and 1,250~1,500 ppm for gram-positive and -negative bacteria, respectively (28). However, we found that a GSE concentration of 300 ppm effectively inactivated native microbial flora in salted shredded Korean cabbages. This discrepancy can be explained by differences in treatment time, salt concentration, and surface topography in our study as compared to the previous reports. The use of chemical sanitizers is linked to the generation of carcinogenic compounds (29); as such, alternative methods to reduce microorganism contamination of processed salted Korean cabbage by non-thermal means are desired. GSE is favored as a preservative for various foods owing to its low toxicity (LD50=2,900 mg/kg) to humans (30). Indeed, in the present study GSE supplementation of brine reduced the total aerobic bacteria and yeast/mold counts on shredded Korean cabbage during salting.

Fig. 1. Response sur face plots for reduction of microbial counts in salted shredded Korean cabbage at var ious NaCl and GSE concentrations and fixed MD concentration (5%) and salting time (5 hr). GSE, grape seed extract; MD, maltodextrin. (a), Total aerobic bacteria; (b), yeast/molds.

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We calculated R² values to evaluate the goodness of fit of the quadratic model for sensory qualities of salted shredded Korean cabbage processed under different salting conditions to the experimental results (Table 3). The ANOVA showed that the resultant polynomial model adequately fit the experimental data, with R² coefficients of 0.960, 0.975, and 0.984 for taste, texture, and overall acceptability, respectively. These results indicate that the quadratic model accounts for >95% of the variation in the experimental data. In addition, the R²_adj values for taste, texture, and overall acceptability were 0.934, 0.955, and 0.970, respectively, indicating a good fit for the model.

The coefficients of the regression equation describing changes in the sensory quality of salted shredded Korean cabbage under different salting conditions are shown in Table 3. Sensory qualities including taste, texture, and overall acceptability of the cabbage were primarily affected by the linear terms of NaCl concentration and salting time (p<0.01 or p<0.001); moreover, the interaction between these two parameters was significant (p<0.001). The quadratic terms of NaCl concentration also had a significant negative effect on sensory quality (p<0.001), whereas salting time and MD and GSE concentrations did not affect these responses. It has been reported that hyper-salting negatively affects the sensory quality of salted Korean cabbage, although it was more effective in reducing salting time (4). Similar observations were made in the present investigation. MD and GSE supplementation had no significant effects on the taste, texture, or overall acceptability of the cabbage (p>0.05).

Response surface plots for the improvement of sensory quality of salted shredded Korean cabbage processed under various salting conditions revealed that sensory scores for taste, texture, and overall acceptability decreased when the NaCl concentration was >10% (Fig. 2a-c). In contrast, MD and GSE concentrations did not affect the sensory quality (p>0.05). It should thus be noted that binary or ternary salting solutions are capable of reducing the amount of preexisting microorganisms without undermining product quality.

Fig. 2. Response surface plots for changes in sensory qualities of salted shredded Korean cabbage at var ious NaCl and MD concentrations and fixed salting time (5 hr) and GSE concentration (150 ppm). MD, maltodextrin; GSE, grape seed extract. (a), Taste; (b), texture; (c), overall acceptability.

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Being an osmotic agent, salt suppresses oxidative or non-enzymatic browning and surface shrinkage, providing the driving force for mass transfer as well as a salty taste (31). In a preliminary study, >500 ppm GSE in the salting solution caused discoloration of the cabbage surface because of chlorophyll degradation. We therefore used GSE concentrations <300 ppm based on another report in which GSE did not affect the color of lettuce leaves and jalapeno peppers during storage (32). We expect the combination of MD and GSE in this study to have dual effects on microbial inactivation and salting time with minimal negative effects on the sensory quality of the product.

Weight loss is the most critical attribute for directly evaluating the degree of dehydration of Korean cabbage during the salting process. Additionally, salt content is an important determinant of gustatory appeal.

The ANOVA showed that the quadratic polynomial model adequately fit the experimental data, with R²=0.981 and 0.992 and R²_adj=0.958 and 0.989 for changes in weight loss and salt content, respectively, indicating good agreement between the experimental and predicted values.

The regression coefficients in Table 3 show that the linear terms of NaCl concentration, salting time, and MD concentration had significant positive effects on reductions in weight loss in salted shredded Korean cabbage (p<0.001). NaCl concentration and salting time had significant positive effects (p<0.001), whereas MD concentration had a significant negative effect (p<0.001) on salt content. In contrast, GSE concentration did not affect changes in weight loss and salt content. The interaction of NaCl and MD concentrations significantly influenced the reduction in weight loss (p<0.05). Notably, the effect of NaCl concentration and salting time on weight loss during the salting process increased as the MD concentration increased. The quadratic term of the four independent variables did not significantly affect the changes in weight loss and salt content (p>0.05).

Response surface plots were generated to visualize the combined effects of the four independent variables on changes in weight loss (Fig. 3a) and salt content (Fig. 3b). The samples were shredded Korean cabbage treated with different concentrations of NaCl and MD at a fixed salting time of 5 hr and GSE concentration of 150 ppm. Weight loss increased as a function of NaCl and MD concentrations. Salt penetrates into the cabbage via a dialysis mechanism, whereas water diffuses out by osmotic pressure. The rates of salt uptake and water release depend on the difference between NaCl and MD concentrations in the salting solution as well as salting time. The salt content of the cabbage increased with the concentration of NaCl used during salting; for instance, it was higher in cabbage salted with 15% brine as compared to 5 or 10% brine in the first 2~5 hr of the process. This effect can be explained by the difference in salt concentration inside and outside the cabbage, which leads to a higher penetration rate of salt into the cabbage during hyper-salting with 15% brine. Additionally, use of 10% MD as a supplementary salting medium reduced the rate of salt uptake. Thus, the salt content of shredded Korean cabbage can be controlled by MD supplementation of the salting solution. In addition, 10% MD supplementation during salting increased weight loss as compared to cabbage salted with brine but without MD for up to 5 hr.

Fig. 3. Response sur face plots for changes in salting characteristics of salted shredded Korean cabbage at var ious NaCl and MD concentrations and fixed salting time (5 hr) and GSE concentration (150 ppm). MD, maltodextrin; GSE, grape seed extract. (a), Weight loss; (b), salt content.

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The osmotic pressure gradient that drives mass transfer depends on the concentration of the salting solution (31). Low-molecular-weight osmotic agents such as salt can readily penetrate into cabbage. The moisture loss in onion and tomato samples subjected to osmotic dehydration in salt solution was reported to be higher than that in sucrose solution. This is because of the passage of the smaller salt molecules through cell membranes and the resultant pressure gradient in the cytoplasm and vacuoles, which increases water loss from the cells (33). In addition, maximum dehydration of cherry tomatoes was achieved using a mixed solution of salt and sucrose that increased the concentration gradient (34). Similarly, MD has a higher molecular weight than other frequently used osmotic agents (e.g., salt, glucose, sucrose, and maltose) (31) and thus does not penetrate the cell membrane, which likely led to its enhancement of dehydration in the present study.

We optimized the salting conditions by analyzing numerical simulations of the best combination of NaCl, MD, and GSE concentration and salting time. The optimal conditions were those that minimized total aerobic bacteria and yeast/mold counts, with weight loss in the range of 15~23%, salt content in the range of 1.5~2.0%, and sensory quality scores >6. Based on this analysis, the optimum values were: an NaCl concentration of 10.09~10.32%, MD concentration of 9.45~10.00%, GSE concentration of 234~300 ppm, and salting time of 5.68~5.94 hr.

In summary, this study established the ideal salting conditions with NaCl, MD, and GSE for improving the microbiological safety and quality of salted shredded Korean cabbage used for kimchi production. This new formulation involves no additional expense or specialized facilities, as MD and GSE are simply added during the salting process. In addition, the use of MD shortens the salting time and consequently decreases weight loss. Large-scale experiments are needed to verify that the optimal conditions determined here can be applied to commercial kimchi manufacturing.