Kouichirou Shin

Twenty-five years of research on bovine lactoferrin applications.

Lactoferrin (LF) was identified as a milk protein in 1960. Large-scale manufacturing of bovine LF (bLF) was established more than 20 years ago. Using this commercially available material, research for bLF applications has advanced from basic studies to clinical studies, and bLF has been applied to commercial food products for the last 25 years. During this period, it was found that LF is digested by gastric pepsin to generate a multi-potent peptide, lactoferricin. It was also demonstrated that oral administration of bLF augments host protection against infections via antimicrobial action and immunomodulation of the host. In addition, researchers have demonstrated that oral administration of bLF prevents cancer development. In this review, we look back on 25 years of bLF research and development.

Oral Lactoferrin Prevents Body Weight Loss and Increases Cytokine Responses during Herpes Simplex Virus Type 1 Infection of Mice

Lactoferrin (LF), a multifunctional milk protein, is known to inhibit in vitro infection by viruses such as herpes simplex virus type 1 (HSV-1). We evaluated the influence of LF feeding on the HSV-1 cutaneous infection of mice. Bovine LF was administered to mice and, after 10 d, the mice were infected with HSV-1. LF administration did not affect the viral clearance in the skin, but inhibited the appearance of skin lesions. LF prevented body weight loss and the decrease of splenocyte number associated with HSV-1 infection. LF increased the serum interleukin (IL)-18 level and splenocyte production of interferon-γ and IL-12. These results suggest that LF feeding was not effective for eradication of the virus, but may contribute to the maintenance of homeostasis and the concomitant increases of cytokine responses during HSV-1 infection.

Inhibition of Escherichia coli respiratory enzymes by the lactoperoxidase-hydrogen peroxide-thiocyanate antimicrobial system.

Aims: The lactoperoxidase‐hydrogen peroxide‐thiocyanate antimicrobial system (LPAS) is known to inhibit bacterial respiration. In the present study, several respiratory enzymes of Escherichia coli were compared in terms of their susceptibility to the LPAS.

Polymerase Chain Reaction–Restriction Fragment Length Polymorphism Analysis of Clarithromycin‐Resistant Helicobacter pylori Infection in Children Using Stool Sample

Background.  To analyze clarithromycin‐resistant Helicobacter pylori infection in children, we developed a method of polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis using stool samples.

Susceptibility of Helicobacter pylori and its urease activity to the peroxidase-hydrogen peroxide-thiocyanate antimicrobial system

The susceptibility of Helicobacter pylori to the antimicrobial system involving lactoperoxidase, hydrogen peroxide and thiocyanate was investigated. The inhibitory effect of the system on the urease activity of H. pylori, which plays a role in its colonisation of the stomach, was also investigated. Twelve H. pylori strains examined, including 10 clinical isolates, were all inhibited by the peroxidase system in brain-heart infusion broth supplemented with fetal calf serum, but to different extents. The killing effect was observed within 3 h. Although bacterial viability recovered afterwards, there was still a clear difference between cultures incubated in the presence of the complete system and control cultures incubated in the absence of lactoperoxidase, after incubation for 24 h. The urease activity and viability of H. pylori were both inactivated by this system in phosphate buffer. These effects were dependent on the concentrations of both lactoperoxidase and hydrogen peroxide and were abolished by the addition of cysteine. Furthermore, these effects were observed when bovine lactoperoxidase was replaced by recombinant human lactoperoxidase or native or recombinant human myeloperoxidase. The peroxidase system found in saliva and milk may contribute to the host defence against H. pylori infection and inhibition of transmission via the oral route.

The Mechanism of in Vivo Bacteriostasis of Bovine Lactoferrin

Recently we have reported that orally administered bovine Lf(bLf) exerts bacteriostatic effects against bacterial overgrowth in the intestine of specific-pathogen-free (SPF) mice fed milk. In this animal model, the in vivo bacteriostatic effect of bLf against the proliferation of intestinal Enterobacteriaceae, the bacteria most sensitive to bLf, was independent of the iron-chelating ability of bLf. In addition various proteolytic hydrolysates of bLf (with differing antibacterial activities in vitro) showed the same bacteriostatic effect as undigested bLf. These results suggest that the mechanism of in vivo bacteriostasis of Lf differs from the in vitro mechanism reported. In SPF mice fed milk differing in concentrations of lactose, glucose and galactose, the proliferation of intestinal Enterobacteriaceae was dependent on the carbohydrate concentration in the diet. The addition of 2% bLf to the diets significantly suppressed this carbohydrate-dependent proliferation of bacteria except in the case of diets containing excess carbohydrate. In germ-free mice fed sterile milk, the addition of 2% bLf to milk resulted in a significant decrease in concentrations of lactose, glucose and galactose in the cecal contents. In an in vitro assay system using everted sacs of the small intestine of SPF mice, both bLf and its pepsin hydrolysate apparently stimulated glucose absorption. Based on these findings, we propose that the in vivo mechanism of action of ingested bLf involves the stimulation of carbohydrate absorption resulting in a bacteriostatic effect against Enterobacteriaceae in the intestine of mice fed milk.

In vitro and in vivo effects of a composition containing lactoperoxidase on oral bacteria and breath odor

The antimicrobial activity of a composition containing bovine lactoperoxidase (LPO), glucose oxidase, glucose and buffer salts was tested against oral bacteria in vitro. A preliminary in vivo study was conducted to test the effect on breath odor of the tablets containing this composition. Suspension of the composition in filter-sterilized saliva or phosphate-buffered saline containing sodium thiocyanate (NaSCN) at a physiological concentration showed bactericidal activity against Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Although hydrogen peroxide (H(2)O(2)) was not detected in the suspension, hypothiocyanite (OSCN(-)) was detected at an average concentration of 0.161 mM. Three tablets made with the composition were continuously sucked by three subjects, and the levels of volatile sulfur compounds (VSCs) in their oral air samples were monitored over a 2 h period by compact gas chromatography. Ingestion of the tablets reduced the average levels of VSCs in the oral air, whereas non-treatment or ingestion of the control tablets without enzymes did not. These results suggest that the composition shows bactericidal activity through the formation of OSCN(-) in saliva and is effective for reducing breath odor, although further clinical studies are needed.

Antibacterial activity of the lactoperoxidase system combined with edible Laminaria hot-water extract as a source of halide ions.

Hot-water extracts prepared from nine out of 12 samples of dried edible Laminaria reduced the viable numbers of Aggregatibacter actinomycetemcomitans, Staphylococcus aureus, and Esherichia coli below the detection limit after incubation for 5 min when combined with lactoperoxidase, glucose oxidase, and glucose. Some extracts showed higher bactericidal activity and a higher OI− concentration in the assay mixture after ultrafiltration.

Effects of orally administered bovine lactoperoxidase on dextran sulfate sodium-induced colitis in mice

The effect of lactoperoxidase (LPO) on dextran sulfate sodium-induced colitis was examined in mice. After 9 d of colitis induction, weight loss, colon shortening, and the histological score were significantly suppressed in mice orally administered LPO (62.5 mg/body/d) as compared to a group administered bovine serum albumin. These results suggest that LPO exhibits anti-inflammatory effects in the gastrointestinal tract.

Orally Administered Lactoperoxidase Increases Expression of the FK506 Binding Protein 5 Gene in Epithelial Cells of the Small Intestine of Mice: A DNA Microarray Study

Lactoperoxidase (LPO) is a component of milk and other external secretions. To study the influence of ingested LPO on the digestive tract, we performed DNA microarray analysis of the small intestine of mice administered LPO. LPO administration upregulated 78 genes, including genes involved in metabolism, immunity, apoptosis, and the cell cycle, and downregulated nine genes, including immunity-related genes. The most upregulated gene was FK506 binding protein 5 (FKBP5), a glucocorticoid regulating immunophilin. The upregulation of this gene was confirmed by quantitative RT-PCR in other samples. In situ hybridization revealed that expression of the FKBP5 gene in the crypt epithelial cells of the small intestine was enhanced by LPO. These results suggest that ingested LPO modulates gene expression in the small intestine and especially increases FKBP5 gene expression in the epithelial cells of the intestine.