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In this review, we focus on the cholesterol‐lowering activity of Lactobacillus gasseri, the immunomodulatory function of casein phosphopeptides (CPPs) and lactic acid bacteria as starters for fermented milk, and the probability and difficulty of creating immunomodulatory fermented milk as a ‘food for specified health use’. Among the starter bacteria used for the preparation of fermented milk, L. gasseriSBT0270 could exert a hypocholesterolemic effect in rats fed a diet high in cholesterol through deconjugation of bile salts. On the other hand, CPPs, having a tripeptide sequence consisting of two phosphoserine residues at N‐ and C‐terminal positions, stimulated lymphocyte proliferation and IgA formation via the increase of interleukin‐5 and interleukin‐6 produced by type 2 helper T cells. The oral ingestion of CPPs enhanced the level of antigen‐specific and total intestinal IgA. Moreover, one strain of bifidobacteria (Bifidobacterium breveYIT4064) and one strain of Lactobacilli (Lactobacillus caseistrain Shirota), used as a starter for fermented milk, activated the humoral and cellular immune system, respectively, and prevented various diseases. It is important to elucidate what components modulate and what mechanisms are responsible for the physiological function of fermented milk in order for it to be accepted as a ‘food for specified health use’ claiming immunomodulatory activity.

ABSTRACTThe antimicrobial activity of the intraurethrally administered probiotic Lactobacillus caseistrain Shirota againstEscherichia coliin a murine urinary tract infection (UTI) model was examined. UTI was induced by intraurethral administration ofEscherichia colistrain HU-1 (a clinical isolate from a UTI patient, positive for type 1 and P fimbriae), at a dose of 1 × 106to 2 × 106CFU in 20 μl of saline, into a C3H/HeN mouse bladder which had been traumatized with 0.1 N HCl followed immediately by neutralization with 0.1 N NaOH 24 h before the challenge infection. Chronic infection with the pathogen at 106CFU in the urinary tract (bladder and kidneys) was maintained for more than 3 weeks after the challenge, and the number of polymorphonuclear leukocytes and myeloperoxidase activity in the urine were markedly elevated during the infection period. A single administration of L. caseiShirota at a dose of 108CFU 24 h before the challenge infection dramatically inhibited E. coligrowth and inflammatory responses in the urinary tract. Multiple daily treatments with L. caseiShirota during the postinfection period also showed antimicrobial activity in this UTI model. A heat-killed preparation ofL. caseiShirota exerted significant antimicrobial effects not only with a single pretreatment (100 μg/mouse) but also with multiple daily treatments during the postinfection period. The otherLactobacillusstrains tested, i.e., L. fermentumATCC 14931T, L. jenseniiATCC 25258T, L. plantarumATCC 14917T, and L. reuteriJCM 1112T, had no significant antimicrobial activity. Taken together, these results suggest that the probiotic L. caseistrain Shirota is a potent therapeutic agent for UTI.

ABSTRACTWe examined colonization patterns of Shiga toxin-producingEscherichia coli(STEC), concentrations of Shiga toxins (Stxs) and specific immunoglobulin A (lgA) against Stxs and STEC bacterial cell surface antigen in various portions of the gastrointestinal tract in an infant rabbit infection model. After inoculation of 3-day-old infant rabbits with STEC strain 89020087 at low doses (∼103CFU/body), numbers of colonizing STEC bacteria and concentrations of Stxs in the intestine increased dramatically and the animals developed diarrhea within a couple of days after infection. Daily administration ofLactobacillus caseifrom the day of birth dramatically decreased the severity of diarrhea and lowered STEC colonization levels in the gastrointestinal tract 100-fold day 7 after infection. Both Stx1 and Stx2 concentrations in the intestines and histological damage to the intestinal mucus induced by STEC infection were decreased by the administration of L. casei. Examination of the concentrations of volatile fatty acids and pH of the intestinal contents revealed that the protective effect of L. caseiadministration against STEC infection was not due to fermented products such as lactic acid in the gastrointestinal tract. Administration ofL. caseiincreased levels of lgAs against Stx1, Stx2, and formalin-killed STEC cells in the colon approximately two-, four-, and threefold, respectively, compared with those of the untreated controls by day 7 after infection. These results suggest that administration ofL. caseistrain Shirota enhances the local immune responses to STEC cells and Stxs and leads to elimination of STEC and thus decreases Stx concentrations in the intestines.

Rats fed a diet containing β1-4 linked galactooligosaccharides (GOS) (5 g/100 g of diet) absorbed calcium and magnesium more efficiently than those fed the control diet. However, the increment obtained through GOS-feeding was reduced by neomycin sulfate 0.67 g/100 g of diet). Since the decrease in cecal pH in rats fed GOS was suppressed by neomycin-feeding, bacterial action in the digestive tract was considered to be reduced by neomycin-feeding. Our findings suggest that the action of intestinal bacteria is necessary for the effects of GOS.

The effect of fermented skim milk (FSM) by Lactobacillus caseistrain Shirota on plasma lipids in hamsters was examined. Hamsters fed on cholesterol-free and -enriched diets containing 30% FSM had lower levels of plasma triglyceride than those fed on the control diet. In the experiment with the cholesterol-enriched diet-fed hamsters, the plasma triglyceride level was suppressed by FSM at concentrations of 10% to 30%. Unfermented milk tended to lower the level of triglyceride, but not significantly. The plasma cholesterol concentration was not affected by an FSM and unfermented skim milk supplement to the diet. L. caseistrain Shirota grew well in the presence of mixed lipid micelles containing bile acid, but did not have the ability to remove cholesterol from the culture broth. These results indicate that FSM lowered the plasma triglyceride level in hamsters.

We evaluated the properties of recombinant staphyloki-nase in comparison with those of tissue-type plasminogen activator (t-PA) and streptokinase (SK). The presence of fibrin(ogen) fragment FCB-2 in the reaction mixture increased plasminogen activation by staphylokinase more than 20-fold. Such characteristics are similar to those of t-PA. On the other hand, SK was not affected by the presence of FCB-2. The thrombolytic properties of staphy-lokinase were studied in a system consisting of a radioactive human plasma clot (125l-fibrinogen-labeled) suspended in the circulating citrated plasma. Significant thrombolysis (50% in 3 hours) was obtained with 2 μg/mL of staphyloki-nase and 4.45 μg/mL t-PA, as compared with 12  μ g/ml. for SK. The relative molar potency of staphylokinase, calculated from the molecular weight, was about two times more effective than that of SK, but about half of that of t-PA. Systemic fibrinolytic activation and fibrinogen break-down was not observed with staphylokinase or t-PA, but was observed with SK. The thrombolytic efficiency of staphylokinase, which was calculated as the ratio of the degree of thrombolysis/the degree of fibrinogenolysis, was about five times greater than that of SK, and about half of that of t-PA. These findings suggest that staphylokinase has higher specific thrombolytic properties and lesser fibrinogenolytic properties than those of SK.

The rate of side-chain cleavage of cholesterol by mitochondria derived from mouse adrenal tumour cells was measured. Incubation of the cells in the presence of adrenocorticotrophin (ACTH) for periods of up to 1 h was without effect on the subsequent side-chain cleavage by mitochondria. However, if the cells were incubated in the presence of aminoglutethimide phosphate (0·76 mmol/l), addition of ACTH (final concentration 86 u./l) to the medium containing the cells increased the subsequent rate of side-chain cleavage by the isolated mitochondria. This response reached a maximum after incubation of cells with ACTH for 2 h and decayed when the isolated mitochondria were left at 0 °C, although a significant difference was still apparent after 120 min. Similar stimulation of mitochondrial side-chain cleavage by ACTH was observed when the reaction was inhibited by anaerobiosis instead of aminoglutethimide phosphate. Addition of dibutyryl cyclic AMP, at final concentrations greater than 10−5mol/l, to cells during incubation with aminoglutethimide phosphate (0·76 mmol/l) also stimulated the conversion of cholesterol to pregnenolone by the mitochondria.Provided the adrenal tumour cells were incubated with aminoglutethimide, or anaerobically, the mean cholesterol content of the inner mitochondrial membrane was significantly higher (P< 0·01) when ACTH was included in the incubation medium than when it was not.It is concluded that ACTH increases the movement of cholesterol to the mitochondrial membrane which contains the side-chain cleavage enzyme system and that part of this cholesterol is used for the enhanced conversion of cholesterol to pregnenolone brought about by ACTH.