Novel Biopreservative Approach to Retard Tuna Red Meat Discoloration by Lactic Acid Bacteria, Leuconostoc citreum M8

Abstract

Seafood is an excellent protein, fats, vitamins, and minerals source, making it highly nutritious. However, the shelf-life of seafood is significantly limited due to various nutrients, a neutral pH, and high moisture content. Rapid microbial and biochemical reactions occurring immediately after catching lead to sensory and nutritional changes, thereby shortening the shelf-life. These characteristics of seafood have prompted extensive study into various methods to maintain seafood quality during distribution and storage. Tuna is one of the most consumed seafood products, with its global consumption rising. It is mainly distributed in raw form, such as sashimi and sushi. Consequently, the color of tuna red muscle meat (TRM) is considered a critical factor influencing consumer preferences. The redness of TRM varies based on the forms and compositions of different types of myoglobin (Mb). Changes in the color of TRM are primarily the result of the accumulation of met-Mb due to auto-oxidation, lipid oxidation, and protein oxidation. Numerous methods have been studied to delay the oxidation process, such as cold storage, antioxidants, modified atmosphere packaging, and active packaging. However, these previous studies have mainly focused on physical storage conditions or chemical reactions with additives. There have been few studies into preventing TRM discoloration and quality maintenance using microbiological approaches, including using microorganisms and their metabolic by-products (biopreservation). Therefore, this study aimed to evaluate the potential of using lactic acid bacteria (LAB) cell-free supernatant (LCFS) to prevent tuna meat discoloration and maintain its quality during storage. LAB strains were isolated and identified from traditional fermented seafood, Jeotgal. LAB strains were selected based on their superior antioxidant and met-Mb conversion effects. The selected LCFS strains were mixed with crude myoglobin extract (CME) from TRM to verify their ability to maintain the red color. This approach was applied to TRM, and the study included the assessment of color, the stability of Mb, and any quality changes. Additionally, the analysis of significant biomarkers affecting the changes of TRM during storage was performed based on metabolomics, and their correlation with antioxidant enzymes was analyzed. A total of 36 LAB strains (M1~M36) were isolated from six different types of Jeotgal. The antioxidant activities (DPPH, ABTS, FRAP, and ORAC assays), met-Mb converting effect, and microbiological safety of each isolated LAB strain were analyzed. Among the LAB strains, Lactococcus lactis M1 and Leuconostoc citreum M8 have superior overall antioxidant activity, excellent met-Mb converting activity, and confirmed microbiological safety. To evaluate the effectiveness of LCFS in preventing discoloration, M1 and M8 were cultivated in mMRS with adjusted glucose levels (mMRS-M1 and mMRS-M8). These LCFS were mixed with TRM's CME to determine their discoloration-prevention effects. A 1:9 mixture of mMRS-M8 and CME showed the inhibition of met-Mb production during storage. Furthermore, the effect of the LCFS on TRM was evaluated during storage at 4°C. The results showed that the redness (a* value) of TRM treated with LCFS was maintained after 48 h. This tendency was also identified in the relative quantification of Mb derivative forms. TRM treated with M8 CFS maintained over 60% oxy-Mb content up to 60 h. The M8 CFS-treated TRM also showed both microbiological and physicochemical quality maintenance effects. Metabolomics analysis was conducted using gas chromatography-mass spectrometry (GC-MS) to analyze changes in tuna metabolites during storage. The analysis involved principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), and orthogonal partial least squares-discriminant analysis (OPLS-DA), which identified significant biomarkers in the M8 CFS-treated TRM group. It was found that M8 CFS treatment could impact the amino acid metabolism, potentially affecting the preservation of tuna quality. Through the OPLS-DA model, we analyzed differential metabolites at each storage time, identifying 12 in the control group and 23 in the M8 treatment group. Subsequent KEGG pathway analysis utilizing these differential metabolites revealed the phenylalanine, tyrosine, and tryptophan biosynthesis pathway as a significant metabolic pathway in both control and M8 treatment groups. Furthermore, correlation analysis between differential metabolites and antioxidant enzymes, including SOD, CAT, and POD showed tyrosine has opposite relation in the control and M8-treated groups. Tyrosine's involvement in antioxidant-related enzymes was further elucidated through GO analysis, particularly in the ROS detoxification pathway, indicating a significant impact on metabolic processes associated with antioxidant effects. This suggests a meaningful association between M8 treatment and tyrosine in biological processes related to antioxidant activity. These findings indicate that LCFS treatment can alleviate oxidative stress and contribute to the overall quality of stored TRM. Based on these results, further study into the enzymatic pathways and the impact of oxidative stress on enzyme activity and regulation is necessary. Such results present the potential for LCFS treatment as a bio-preservation strategy in the food industry, extending shelf-life and providing a novel approach to maintaining seafood quality.