Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a glycolytic enzyme, is known as a moonlighting protein. Previous study showed that cell surface GAPDH of Lactiplantibacillus plantarum bound to ABO blood group antigens expressed in the intestinal mucosa. However, the binding site of GAPDH to the blood group antigens has remained unclear. Therefore, we searched for the A-type blood group antigen binding site based on the crystal structure information of the recombinant GAPDH (rGAPDH) from L. plantarum subsp. plantarum JCM 1149^T, which has already been determined. We found that there is a cavity (357 Ų) in which lysine residues and aspartic acid residues form clusters on the molecular surface between subunits. Furthermore, we predicted that the 175th lysine residue (Lys175), located in the center of the cavity, plays an important role in the binding of ABO blood group antigen. Lys175Ala mutant gap (Lys175Ala) gene was prepared by mutating Lys175 to uncharged alanine by site directed mutagenesis using the DpnI method. Using an enzyme, the intermolecular interaction with A-type blood group antigen was measured by the Quartz Crystal Microbalance (QCM) method. As a result, the mean value of ΔF of wild-type (－173 Hz) was 1.7 times higher than the mean value of ΔF of Lys175Ala mutant enzyme (－103 Hz). Thus, it was clarified that Lys175 of rGAPDH from L. plantarum plays an important role in the recognition of A-type blood group antigen. Moreover, molecular docking simulation using AutoDock Vina was performed to determine the detailed binding structure of L. plantarum rGAPDH and A-type blood group antigen. The binding energy between these two was －6.3 kcal/mol. As for the binding to the A-type blood group antigen, NH in the main chain of Gln312 was hydrogen bonded to N-acetylgalactosamine (GalNAc), and the side chain of Lys175 was hydrogen bonded to galactose (Gal). In addition, the side chain of Asn311 was hydrogen bonded to Gal, and the side chain of Asp255 was hydrogen bonded to fucose (Fuc).

 Cyclic dipeptides, 2,5-diketopiperazines (DKPs), are present in various fermented and/or heated foods and are well-known taste and bioactive components. However, there are few studies on DKPs in cheese, which is a typical fermented food. Herein, we determined the amount of DKPs in various cheeses and investigated the relationship between the DKP content and production process of cheese. We observed that ripened cheeses (semi-hard, hard, white mold, blue veined, hybrid of white and blue mold, and washed-rind type) had higher DKP contents than unripened cheese (Mozzarella cheese), based on the analyses of 42 types of DKPs. The ripened cheese also contained various types of DKPs, while Mozzarella cheese contained only cyclo(-Arg-Pro). Moreover, in Gouda cheeses with different ripening periods, both the total content and types of DKPs increased as the ripening period increased. The amino group content, which was used as an index for the degree of protein degradation in cheese ripening, also increased during the ripening process. Therefore, we concluded that the amount and types of DKPs in cheese were affected by the ripening process. The amino acid sequences of four types of bovine casein, α_s1, α_s2, β, and κ were compared with the sequences of DKPs detected in cheese. The results showed that 23 out of 25 types of DKPs corresponded to the amino acid sequences of four caseins, indicating that most of the DKPs in cheese are derived from casein.

 The effect of Lactobacillus rhamnosus GG (GG) fermented milk on postprandial blood glucose level was investigated by study on mice and humans. ICR mice were administered GG fermented milk (n=6) or control water (n=7) with starch solution. At 30 min after administration of starch solution, the GG fermented milk group showed significantly lower blood glucose level than the control group (p<0.05). Subjects (n=20, female, aged 21-35 years) skipped breakfast and received GG fermented milk or control beverage with 114 g bread and 10 g butter. Blood was collected at 0 (before), 30, 60, and 120 min after meal ingestion for analysis of blood glucose and insulin levels. At 30 min after meal ingestion, ingesting GG fermented milk led to a significantly lower blood glucose level than ingesting control beverage (p<0.01). These results show that GG fermented milk is effective in the prevention of postprandial hyperglycemia.