Effect of long term storage of kimchi cabbage on kimchi fermentation and quantitative detection of white colony forming yeast using real-time PCR

다국어 초록 (Multilingual Abstract)

Kimchi cabbage (KC), the main ingredient used in kimchi, has a great influence on the quality characteristics of kimchi. In this study, the long-term storage method of KC, a quality evaluation of kimchi made with long-term stored KC, and a method to quickly detect undesirable white colony-forming yeast (WCFY) on the kimchi during fermentation were investigated.
To store KC for a long time, once harvested, they should be quickly maintained at a low temperature to suppress post-harvest physiology; however, the sudden temperature change may lead to chilling injury in KC. Consequently, the optimal cooling rate is key for the long-term storage of KC. To determine the optimal cooling rate, KCs were cooled at different cooling rates (1, 2, 4, 6, 8, and 24°C/d), and then stored at 1°C for 90 days. Thereafter, the changes in weight, trimming, total losses, pH, free sugar content, and total bacterial count were analyzed during storage. KC stored at a cooling rate of 6°C/d presented lower total loss and better quality properties than those stored at other cooling rates; thus, 6°C/d is an appropriate cooling rate for long-term storage of KC.
Kimchi made using stored KC exhibited different fermentation characteristics than kimchi made using fresh kimchi cabbage. In this study, viable microbial communities and metabolites of kimchi were monitored and sensory evaluation was performed. The storage of KC affected the microbial community structure of kimchi, which caused differences in metabolites. In the early and middle stages of kimchi fermentation, fructose and mannitol contents were higher in control kimchi than in kimchi made using stored KC. Additionally, control kimchi had higher levels of sweetness and texture than kimchi made using stored KC.
Long-term fermentation of kimchi, especially kimchi made using stored KC, resulted in WCFY appearing on the surface of kimchi. Real-time polymerase chain reaction (PCR) assays to rapidly detect Kazachstania servazzii and Candia sake known as WCFY in kimchi were developed. The K. servazzii- and C. sake-specific real-time PCR amplified the target yeast species only, without any cross-reactivity with non-target yeast species. The limit of detection of each assay was 1. ×102 and 3.1×102 CFU/mL for K. servazzii and C. sake respectively. The quantitative real-time PCR assays were validated through quantitative analysis of yeast content inoculated into kimchi. Therefore, these methods were applied to analyze the change in yeast content during kimchi fermentation. The real-time PCR assays developed in this study have the potential to be used for the quantitative detection of K. servazzii and C. sake present in kimchi.
This study can be used as basic data for kimchi manufacturers that use stored KC for the supply and demand of kimchi.

목차 (Table of Contents)

• BACKGROUND 1
• Purpose of study 3
• 1. PART 1 5
• 1.1. Abstract 6
• 1.2. Introduction 7
• 1.3. Materials and Methods 9
• 1.3.1. Kimchi cabbage samples 9
• 1.3.2. Storage method 9
• 1.3.3. Temperature, humidity, and gas concentration in the storage house 11
• 1.3.4. Weight, trimming, and total losses of kimchi cabbage 12
• 1.3.5. pH and free sugar content of kimchi cabbage 13
• 1.3.6. Total bacterial count of kimchi cabbage 14
• 1.3.7. Statistical analysis 15
• 1.4. Results and discussion 16
• 1.4.1. Temperature change in the storage house 16
• 1.4.2. Relative humidity change in the storage house 18
• 1.4.3. Change in oxygen and carbon dioxide concentrations in the storage house 19
• 1.4.4. Weight, trimming, and total losses of kimchi cabbage 21
• 1.4.5. Changes in the pH, total microbes, and total free sugar of kimchi cabbage during storage 25
• 1.5. Conclusions 28
• 1.6. Appendix 29
• 1.6.1. Part 1 publication in the journal 29
• 1.6.2. Do not need copyright for published paper in regards to Part 1 29
• 2. PART 2 30
• 2.1. Abstract 31
• 2.2. Introduction 32
• 2.3. Materials and Methods 34
• 2.3.1. Kimchi cabbage storage 34
• 2.3.2. Preparation of kimchi 35
• 2.3.3. Analysis of moisture content and pH 36
• 2.3.4. Viable cell count of total microbes, LAB, and yeast 37
• 2.3.5. Microbial community analysis 38
• 2.3.6. Metabolite analysis 39
• 2.3.7. Sensory evaluation 40
• 2.3.8. Statistical analysis and principal component analysis (PCA) 41
• 2.4. Results and Discussion 42
• 2.4.1. Physiological change of kimchi cabbage during long term storage 42
• 2.4.2. Changes in pH, acidity, viable cell count during kimchi fermentation 46
• 2.4.3. The microbial diversity and communities during kimchi fermentation 48
• 2.4.4. Metabolic changes during kimchi fermentation 51
• 2.4.5. Sensory evaluation during kimchi fermentation 55
• 2.5. Conclusions 57
• 2.6. Appendix 58
• 2.6.1. Part 2 publication in journal 58
• 3. PART 3 59
• 3.1. Abstract 60
• 3.2. Introduction 61
• 3.3. Materials and Methods 63
• 3.3.1. Yeast strain and culture conditions 63
• 3.3.2. Kimchi preparation 65
• 3.3.3. DNA extraction 66
• 3.3.4. Primer and probe design 67
• 3.3.5. Quantitative real-time PCR 69
• 3.3.5.1. Quantitative real-time PCR condition 69
• 3.3.5.2. Construction of standard curve and data analysis 70
• 3.3.6. Validation and application of quantitative real-time PCR 71
• 3.4. Results and Discussion 72
• 3.4.1. Specificity and sensitivity of real-time PCR for K. servazzii and C. sake 72
• 3.4.2. Quantitation of for K. servazzii and C. sake using quantitative real-time PCR 76
• 3.4.2.1. Standard curve and linearity 76
• 3.4.2.2. Validation of quantitative real-time PCR 77
• 3.4.3. Application of quantitative real-time PCR 79
• 3.5. Conclusions 83
• 3.6. Appendix 84
• 3.6.1. Part 3 publication in journal 84
• 3.6.2. Do not need copyright for published paper in regards to Part 3 84
• CONCLUSIONS 85
• REFERENCES 87