1. Effect of Lactic Acid Bacteria on D- and L-Lactic Acid Contents of Kimchi D-form of lactic acid is often found in fermented foods and excessive dietary intake of D-lactic acid may cause a metabolic stress in infants and patients. To determine the prevailing microorganisms involved in D-lactic acid accumulation in kimchi, D-/L-lactic acid synthesis profiles of the major lactic acid bacteria (LAB) in kimchi were analyzed and the relationship between D-/L- lactic acid contents and microbial populations was investigated during the fermentation. When cultured in a medium containing glucose, Leuconostoc mesenteroides and Leuc. citreum mainly synthesized D-lactate (75.28 mM and 85.00 mM, respectively) with small amount of L-form. Leuc. gelidum and Leuc. inhae showed a similar pattern with it. Lactobacillus plantarum and Lb. brevis converted glucose into a balanced mixture of D-/L-lactic acid (114.13/81.74 and 115.47/84.77 mM, respectively), while Lb. casei synthesized mainly L-lactic acid (185.36 mM) and a very little amount of D-lactic acid (8.20 mM). When kimchi was incubated at 8 ℃ or 22 ℃, the D-lactic acid (11-14 mM) was over-produced than L-form (5-8 mM), and leuconostocs was the leading genus during the period of D-lactic acid accumulation. Accordingly, it can be concluded that the main strains for D-lactic acid production in kimchi are leuconostocs and some of lactobacilli: leuconostoc is the major producer between the initial to mid-phase of fermentation and Lb. plantarum or Lb. brevis may boost D-lactic acid content at the late stage of acid accumulation. ABSTRACT Qing Jin 1, Hyang- Sik Yoon 2, Jin Soo Han 3, Junsoo Lee 1 and Nam Soo Han 1 * 1 Department of Food Science and Technology, Research Center for Bioresource and Health(RCBH), Chungbuk National University, Cheongju 361-763, Korea 2 Team for Food Research & Development, Chungcheongbuk-do Agricultural Research and Extension Services, Ochang 363-883, Korea 3 College of Veterinary Medicine, Konkuk University, Seoul 143-701, Korea References 1. FAO/WHO. Toxicological evaluation of certain food additives with a review of general principles and of specifications. Seventeenth report of the Joint FAO-WHO Expert Committee on Food Additives. WHO Tech. Rep. Ser. 539: 1-40 (1974) 2. Yoon HS, Son YJ, Han JS, Lee JS, Han NS. Comparison of D- and L-lactic acid contents in commercial kimchi and sauerkraut. Food Sci. Biotechnol. 14: 64-67 (2005) 3. Choi HJ, Shin YJ, Yu JH, Yoon SS. A new selective medium for the isolation and the detection of leuconostocs in foodstuffs. Korean Journal of Food Science and Technology. 28: 279-284 (1996) 4. Okubo S, Mashige F, Omori M, Hashimoto Y, Nakahara K, Kanazawa H, Matsushima Y. Enantiometric determination L- and D-lactic acid in human cerebrospinal fluid by chiral ligand exchange high-performance liquid chromatography. Biomed. Chromatogr. 14: 474-477 (2000) 5. Lee CW, Ko CY, Ha DM. Microfloral changes of the lactic acid bacteria during kimchi fermentation and identification of the isolates. Korean Journal of Applied Microbiology and Biotechnology. 20: 102– 109 (1992) 6. Garvie, EI. The production of L(+)- and D(-)-lactic acid in cultures of some lactic acid bacteria, with a special study of Lactobacillus acidophilus NCDO 2. J. Dairy Res. 34: 31-38 (1967) D-/L-lactic acid profiles of LAB in kimchi FIGURE 1. Chromatograms of D-/L-lactic acid in LAB culture broth. 1. Standards (5 mM of acetic acid, D-/L- lactic acids) 2-4. Culture broths of Leuc. mesenteroides, Lb. plantarum and Ent. faecalis in MRS medium were analyzed after 24 hrs incubation at 30 ℃. D- and L-lactic acids are optical isomers, which are mainly derived from pyruvate by the action of D- and L-LDH respectively. The presence of D-lactic acid in fermented foods and ingestion of lactic acid bacteria through foods do not pose an adverse effect for the vast majority of the adult population, but it can accumulate in the blood of patients suffering from short-bowel syndrome and intestinal failure, leading to a manifestation of D-lactic acidosis and encephalopathy. Furthermore, newborn infants may fail to completely metabolize D-lactic acid ingested or produced by intestinal microorganisms because of liver immaturity. Thus, food products containing these ingredients are not recommended for nutrition to infants and young children to the age of 3 years. Humans ingest D-lactic acid by consuming fermented vegetables or milk such as pickle, sauerkraut, kimchi, and yogurt, which contain both D- and L-lactic acids. D- and L-lactic acid contents were analyzed in those foods. Otherwise, it was suggested that D-lactic acid producing colonizing intestinal lactobacilli were the main factor in the pathogenesis. Kimchi is a Korean traditional fermented dish, which has a sour, hot, salty, and characteristically carbonic taste from the lactic acid fermentation of vegetables. Our previous work revealed that a significant amount of D-lactic acid exist in all types of kimchi tested, regardless of producers and raw materials. Considering the substantial amount of kimchi consumption (total 500 million USD in Korea in 2004), intake of D-lactic acid through kimchi can not be overlooked. In lactic acid bacteria (LAB), the presence of one type of LDH or another varies between species. For LAB in kimchi, understanding their roles in the accumulation of D- and L-lactic acid is necessary. Research Objectives Aanalyze D-/L-lactic acid synthesis profiles of major LAB in kimchi Investigate D-lactic acid production level during kimchi fermentation Determine the prevailing microorganisms involved in D-lactic acid accumulation in kimchi Strains: Leuconostoc mesenteroides KCTC 3719, Leuc. gelidum KCTC 3527, Leuc. inhae KCTC 3774, Pediococcus pentosaceus KCCM 11729 and Enterococcus faecalis KCCM 11902 are studied for type strains. Leuc. citreum, Lb. plantarum, Lb. brevis, Lb. sakei are isolated from kimchi and identified by 16s rRNA analysis in our research group. Growth condition: Cultures of LAB were inoculated in MRS fermentation medium (Difco, Detroit, MI, USA) at optimum temperature for 24 hrs under static condition. Gene sequences of lactic acid bacteria (LAB) in kimchi were obtained in the genomic database of GeneBank. The growth of LAB in kimchi was measured in colony forming units (CFU) per mL of liquid using the culture pouring method. For the viable cell count of leuconostocs, the NLS agar medium containing novobiocin and vancomycin was used and for total LAB cell counts Lactobacilli MRS medium (Difco) was used. Strains and Growth Conditions Introduction Fermentation of Dongchimi-Kimchi HPLC Quantitation ItemsCondition PumpYOUNG-LIN, M930, Korea DetectorUV(YOUNG-LIN, M720, Korea) at 254nm Column Shodex Orpac CRX853(8.0×50mm, Japan) Guard Column Shodex Orpac CRX-G(4.0×10mm, Japan) Injection Volume20ul Mobile Phase1.0mM CuSO 4 Flow Rate1.0mL/min L-Lactic acidLithium Salts(Sigma, U.S.A.) D-Lactic acidLithium Salts(Sigma, U.S.A.) Strains OD (550 nm) D- lactic acid (mM) L- lactic acid (mM) Total lactic acid (D+L) (mM) Ratio (D:L) Leuconostoc mesenteroides 2.33675.2819.6894.9680:20 Leuc. Citreum2.37285.0016.87101.8783:17 Leuc. gelidum2.56491.0017.29108.2984:16 Leuc. inhae1.01657.9119.0376.9475:25 Lactobacillus plantarum 4.784114.1381.74195.8758:42 Lb. brevis4.688115.4784.77200.2458:42 Lb. fermentum2.85648.5972.07120.6640:60 Lb. casei6.3768.20185.36193.564:96 Pediococcus pentosaceus 4.93843.30114.22157.5228:72 Enterococcus faecalis 0.716Nd 1) 30.78 0:100 TABLE1. Analytical conditions of HPLC TABLE 2. Analytical conditions of HPLC 1)Nd means not detected. D-/L-lactic acid Contents during kimchi fermentation Figure 2. Changes in lactic acid concentration during kimchi fermentation at 8 ℃. Figure 3. Changes in lactic acid concentration during kimchi fermentation at 22 ℃. Conclusions Lb. plantarum, Lb. brevis and Lb. fermentum made comparable amounts of D-/L-forms but Lb. casei made simply L-form. The total amounts of lactic acids (D + L) fermented by lactobacilli were between 130-200 mM. Ped. pentosaceus and Ent. faecalis also synthesized D-lactic acids as 43mM vs 114 mM and L-lactic acids as 31mM vs 0 mM, respectively. The high producers of D-lactic acid of LAB in kimchi would be Leuconostoc spp., Lb.plantarum and Lb. brevis. All tested-leuconostocs synthesized D-lactic acid as a major form(58-91mM) compared to L-form(17-20mM) and the ratio of D-/ L-lactic acid was about 4:1. The main strains for D-lactic acid production in kimchi are leuconostocs and some of lactobacilli: leuconostoc is the major producer between the initial to mid-phase of fermentation and Lb. plantarum or Lb. brevis may boost D-lactic acid content at the late stage of acid accumulation. When dongchimi-kimchi was prepared and incubated at 8 ℃ and 22 ℃, the viable cell counts of total LAB and leuconostocs gradually increased to reach 1×10 9 and 1×10 8 level after 9 days and 3days of fermentation and decreased thereafter. Kimchi was incubated at 8 ℃ or 22 ℃, the D-lactic acid (11-14 mM) was over-produced than L-form (5-8 mM), the contents of D-lactic acid were higher at 22 ℃. Radish(80g) Cutted, washed and peeled Pretreatment Mixed with Salt(40g) Incubation Seasoning 20 ℃, 12hrs Crushed garlic (10g), ginger (3 g), chopped green onions (20 g), filled with 4 L of water Fermentation Dongchimi-Kimchi 8 ℃ and 22 ℃, 14 days