Effect of clove powder on quality characteristics and shelf life of kimchi paste

Abstract Clove has been shown to extend the shelf life of various foods. This study investigated whether it can prolong the shelf life of kimchi paste. Clove powder at concentrations of 0%, 0.5%, 1%, 1.5%, and 2% was added to kimchi paste, which was then sealed and stored at 10°C for 20 days. Changes in microbial counts, gas composition, sugar and organic acid contents, pH, titratable acidity, and reducing sugar content were evaluated. Adding clove powder inhibited the growth of total aerobic and lactic acid bacteria and delayed changes in O2 and CO 2 concentration and sugar and organic acid contents. It also slowed the decrease in pH, increase in titratable acidity, and changes in reducing sugar content. These results indicate that clove powder effectively prolongs the quality attributes and thus extends the shelf life of kimchi paste.


| INTRODUC TI ON
Kimchi is a widely consumed traditional Korean food that is produced by blending pastes and several vegetables such as cabbage, radish/young radish, green onion, mustard leaf, cucumber, garlic chive, and perilla leaf, followed by fermentation by natural lactic acid bacteria during storage. Kimchi pastes have many flavors-including pungent, sweet, sour and/or spicy-that are conferred by specific ingredients, including red pepper powder, salt, sugar, fermented and salted seafood, and starch paste Park et al., 2012).
Recently, demands for distinct kimchi pastes and salted cabbages have sharply increased in Korea due to consumer interest in making kimchi at home according to personal preferences. As such, commercial production of kimchi paste has increased. However, its short shelf life is an obstacle for mass production since kimchi is manufactured through a nonthermal process that can lead to the growth of pre-existing microorganisms and thereby present a risk to consumer health. In particular, microbiological and enzymatic activities after production decrease pH, leading to formation of excessive organic acids and CO 2 and consequent deterioration of kimchi paste and packaging (Cheon, Seo, Chung, & Chun, 2016;Kang, Jung, & Seo, 2015;Park et al., 2008).
However, these approaches have met with limited success.
Cloves are used as a topical analgesic that has been used to promote healing and prevent aging, and treat cardiovascular disease, arthritis, infections, digestive problems, skin cancer, and thyroid dysfunction; it is also used in fragrances and flavorings (Chaieb et al., 2007;Fu et al., 2007;Nassar et al., 2007). Clove extracts in the form of essential oil and powder also exhibit a wide range of biological effects, including antioxidant and antibacterial activities (Menon & Garg, 2001;Sultana, Anwar, Mushtaq, Aslam, & Ijaz1, 2014). Cloves may extend the shelf life of various foods (meat, baked goods, etc.) by suppressing the growth of foodborne pathogens while adding a characteristic flavor (Ibrahium, Abd El-Ghany, & Ammar, 2013;Khaleque et al., 2016;Kumar & Tanwar, 2011;Tajik, Farhangfar, Moradi, & Rohani, 2014). However, no studies to date have investigated the effects of clove powder on the microbiological and physiochemical qualities of kimchi paste.
The present study addressed this issue by adding clove powder to kimchi paste and evaluating microbial count, headspace composition including CO 2 , free sugar and organic acid contents, pH, titratable acidity, and reducing sugar content during fermentation.

| Sample preparation
Dried cloves were purchased from online markets and ground into a fine powder using an HR1372 grinder (Philips, Huizhou, China).
Commercial kimchi paste was purchased from a local market in Gwangju, Korea; the composition (w/w) is shown in Table 1. The paste was divided into five groups. The first was left untreated to examine baseline quality characteristics, and clove powder (0.5%, 1%, 1.5%, and 2%) was added to the remaining treatment groups. Each group of kimchi paste was placed on a plastic tray (130 × 95 × 60 mm) and sealed with plastic film using an MS2 sealing machine (Packsis, Yangju, Korea), and stored at 10°C for 20 days.

| Determination of moisture content and salinity
The moisture content of samples was determined using an MB45 moisture analyzer (Ohaus, Greifensee, Switzerland). About 3 g of sample were placed in the sample holder and dried until a constant weight was obtained. Salinity was measured by Mohr's titration (Chen, Hsieh, Weng, & Chiou, 2005). Samples (1 g) were homogenized in 100 ml distilled water and then filtered 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 the mixture was titrated against 0.02 N AgNO 3 until the end point (a red-brown color) was reached.

| Scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical
The assay was carried out in a flat-bottomed 96-well plate as previously described (Chatatikun & Chiabchalard, 2013), with slight modifications. Clove powder (100 mg) and freeze-dried kimchi paste samples (100 mg) were extracted with 100 ml ethanol. Different concentrations of clove (0.125, 0.25, 0.5, and 1.0 mg/ml), kimchi paste sample (1 mg/ml), or standard ascorbic acid (100 μg/ml) were added to 100 μl of 0.2 mM methanolic DPPH solution in the 96-well plate. After mixing, the plate was incubated for 30 min at room temperature in the dark. The absorbance of each well was measured at 517 nm with a SPECTRO Star Nano spectrophotometer (BMG Labtech, Ortenburg, Germany). Percent inhibition was calculated with the following equation.

| Microbiological analysis
Samples (10 g) were mixed with 90 ml of sterile saline (0.85% NaCl, w/v) in a sterile stomacher bag and then homogenized using

| Extraction and analysis of sugars and organic acids
Kimchi paste (5 g) was homogenized in 20 ml of distilled water and then passed through an 8μm cellulose filter (Whatman) followed by a 0.45μm polyvinylidene difluoride syringe filter (Agilent, Santa Clara, CA, USA). The filtrate was diluted and injected into a Dionex 3000 high-performance liquid chromatography system (Dionex, New York, NY, USA). To analyze sugar content, water was used as the solvent at a flow rate of 0.5 ml/min; a Shodex RI-101 (Kawasaki, Japan) detector was used. Sucrose, glucose, fructose, and mannitol contents were calculated using external standards.
The extract used in the sugar analysis was also used for organic acid analysis. Acids were separated using an Aminex 87H column (300 × 10 mm; Bio-Rad, Hercules, CA, USA), and 0.01 N H 2 SO 4 was used as an eluent at a flow rate 0.5 ml/min. Acids were detected with an ultraviolet detector operating at 210 nm. External standards were used to calculate lactic, acetic, citric, and malic acid contents.

| Determination of pH and titratable acidity
Sample pH was measured using pH meter (Accumet AB 15, Thermo Fisher Scientific, Pittsburgh, PA, USA). The titratable acidity of the kimchi paste was determined according to a method described by the Association of Official Analytical Chemists (2005). Briefly, after homogenizing 10 g of kimchi paste in 90 ml distilled water, the sample was titrated with 0.1 N NaOH solution until the end point (pH 8.3) was reached. Total acidity was calculated as a percentage of lactic acid.

| Determination of reducing sugar content
Reducing sugar content was determined according to the 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959). Each sample (2 ml) was diluted with distilled water and mixed with 2 ml DNS.
After boiling at 100°C for 5 min and cooling in ice water, the absorbance at 550 nm was measured using a SPECTRO Star Nano spectrophotometer. The reducing sugar content (mg/g) is expressed as glucose equivalents.

| Statistical analysis
Data are expressed as mean ± SD of triplicate samples. Each experiment was performed at least three times. Data were analyzed using SPSS v.19.0 software (SPSS Inc., Chicago, IL, USA) by one-way analysis of variance. Duncan's multiple range test was used to compare the effects of clove powder addition or fermentation period.
Differences were considered statistically significant at p < 0.05.

| Initial moisture content and salinity
There were no differences in moisture content of kimchi paste at the early stage of storage between untreated control sample (72.23%) and samples treated with clove powder (71.56%-72.33%) ( Figure 1a). The initial salinity of kimchi pastes was 2.84% for the control and 2.67%-2.77% for treatment groups (Figure 1b).

| DPPH radical scavenging activity
The major constituent of clove oil and extract is eugenol, a phenolic compound with antioxidant activity (Chaieb et al., 2007;Nassar et al., 2007) that is exerted via mechanisms such as radical scavenging and metal ion chelation. Eugenol also participates in photochemical reactions (Mihara & Shibamoto, 1982). Here, we investigated the DPPH radical scavenging activity of various concentrations of clove powder (0.125-1 μg/ml) added to kimchi paste relative to ascorbic acid (100 μg/ml). Clove powder showed DPPH-scavenging activity

| Microbiological changes during storage
Changes were observed in the microbial composition of kimchi pastes containing various concentrations of clove powder during 20 days of storage at 10°C. The different uppercase letters in the row mean statistically significant (p < 0.05) differences according to fermentation days. The different lowercase letters in the column mean statistically significant (p < 0.05) differences according to concentration of clove powder ( Table 2). The initial amount of total aerobic bacteria in the pastes was 6.68 log CFU/g for the control and 6.66-6.75 log CFU/g for treatment groups. After 5 and 15 days of storage, total aerobic bacteria in the paste reached 7.50 and 7.57 log CFU/g, respectively, in the control and 6.67-6.81 and 6.66-6.89 log CFU/g, respectively, in samples with 0.5%-2% clove powder. This is consistent with previous studies reporting that clove oil reduced total aerobic bacteria content in cake (Ibrahium et al., 2013) and buffalo meat (Naveena, Muthukumar, Sen, Babji, & Murthy, 2006). The antibacterial activity is mainly attributable to eugenol; the partially hydrophobic nature of phenolic compounds can induce cell wall degradation, disrupt the cytoplasmic membrane, cause damage to membrane proteins, and interfere with membrane-integrated enzymes, eventually leading to cell death (Shan, Cai, Brooks, & Corke, 2007).
The initial amount of lactic acid bacteria in kimchi pastes was 5.60 log CFU/g for the untreated control and 5.14, 4.40, 4.19, and 3.42 log CFU/g for pastes containing 0.5%, 1%, 1.5%, and 2% clove powder, respectively. Thus, the amount of lactic acid bacteria was inversely related to clove powder concentration. After 10 and 20 days of storage, the population of lactic acid bacteria in the paste was 6.79 and 6.73 log CFU/g, respectively, for the control and 3.46-5.50 and 1.39-3.33 log CFU/g, respectively, for samples with 0.5%-2% clove powder. A key event in the fermentation of many foods is the conversion of sugars to lactic acid by lactic acid bacteria (Deegan, Cottera, Hilla, & Ross, 2006); inhibiting their growth could, therefore, delay the fermentation process. The initial population of yeast and molds in kimchi pastes was 5.45 log CFU/g for the untreated control and 5.18-5.43 log CFU/g for treatment groups with 0.5%-2% clove powder; these values did not change significantly during 20 days of storage. Excessive growth of yeast and mold during kimchi paste fermentation can impede sensory satisfaction during consumption (In & Chae, 2004;Kim, Hwang, Choi, & Kim, 1992); however, there was no visible yeast and mold growth in any of our samples during fermentation.
The initial population of coliforms in kimchi pastes was 3.59 log CFU/g for the untreated control and 3.39-4.21 log CFU/g for samples treated with 0.5%-2% clove powder. In agreement with these results, clove oil has been shown to inhibit the growth of coliform bacteria in buffalo meat (Naveena et al., 2006), and a pre-

| Changes in gas composition in packaged headspace during storage
We analyzed the gas composition in stored kimchi pastes treated with clove powder (Figure 3). The initial O 2 concentration in the pastes was 20.73%-20.90%, with no differences observed among samples. From day 5, the O 2 content of the container headspace increased with clove powder concentration. On day 10, the O 2 contents for samples treated with 0.5% and 2% clove powder were 10.23% and 17.67%, respectively.
The initial CO 2 composition in the kimchi paste container was 1.20% for the control and 1.13%-1.14% for samples treated with clove powder, with no differences among samples. From day 5, the CO 2 content of the container headspace decreased as a function of clove powder concentration. On day 10, the CO 2 content was 29.73% and 4.60% for samples containing 0.5% and 2% clove  A-E Means sharing the same uppercase letters in the same row differ significantly (p < 0.05). a-e Means sharing the same lowercase letters in the same column differ significantly (p < 0.05). and CO 2 concentration inside the kimchi paste packaging by inhibiting the growth of lactic acid bacteria.

| Sugar content
The sugar content of kimchi pastes with various concentrations of clove powder changed during 20 days of storage at 10°C. The different uppercase letters in the row mean statistically significant (p < 0.05) differences according to concentration of clove powder. The different lowercase letters in the column mean statistically significant (p < 0.05) differences according to fermentation days ( Mannitol content in kimchi pastes ranged from 1,230.45 to 1,307.78 mg/kg on day 0, with no differences among samples.
The mannitol content increased as fermentation proceeded.

| Organic acid content
Changes in the concentrations of organic acids such as lactic, acetic, citric, and malic acid in kimchi pastes upon addition of clove powder were measured. The different uppercase letters in the row mean statistically significant (p < 0.05) differences according to concentration of clove powder. The different lowercase letters in the column mean statistically significant (p < 0.05) differences according to fermentation days (

| Changes in pH, titratable acidity, and reducing sugar content during storage
Changes in pH, titratable acidity, and reducing sugar content of kimchi pastes containing various concentrations of clove powder during 20 days of storage at 10°C were analyzed (Figure 4). The pH and acidity are important factors determining the maturity and quality of kimchi and kimchi pastes; an important step in their fermentation is the transformation of sugar to lactic and other organic acids by the lactic acid bacteria, which reduces the pH and increases acidity (Jeong et al., 2011;Kang et al., 2015). Before storage and immediately after addition of clove powder, the pH of kimchi pastes was 5.10-5.29, with no differences among samples. The pH of the untreated control sample decreased from 5.29 on day 0 to 4.32 on day 20, whereas samples containing 2% clove powder showed a decrease in pH from 5.10 to 4.84 at these time points. This indicated that adding clove powder delayed the decrease in pH. Accordingly, acidity increased from 1.38% on day 0 to 1.57% on day 20 in control samples and from 0.92% to 1.05% in samples containing 2% clove powder. Clove extracts have been used as natural preservative in raw pork (Shan et al., 2009) since they contain high levels of phenolic compounds that not only extend shelf life, but also maintain meat color via antioxidant activities.
The reducing sugar content of kimchi paste increased until day 10 before decreasing, demonstrating that additional clove powder produces smaller changes; these are related to the decrease in pH and increase in acidity, and probably result from the decomposition and utilization of reducing sugars as a carbon source for microbial growth during fermentation (Afoakwa, Kongor, Takrama, & Budu, 2013;Kang et al., 2015).

| CON CLUS IONS
This study investigated the possibility of using clove powder as a natural additive in fermentation to improve the longevity of kimchi paste. The presence of clove powder significantly reduced the population of total aerobic and lactic acid bacteria in fermented kimchi paste, delaying changes in O 2 and CO 2 concentration, pH, titratable acidity, and reducing sugar content during storage. These results suggest that adding clove powder to kimchi paste can extend its shelf life without compromising other attributes.

ACK N OWLED G EM ENTS
This research was supported by grants from the World Institute of Kimchi (KE1602-3-1 and KE1801-4), funded by the Ministry of Science and ICT, Republic of Korea.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L R E V I E W
This study does not involve any human or animal testing.