Lactoferrin research, technology and applications

ARTICLE IN PRESS

International Dairy Journal 16 (2006) 1241–1251 www.elsevier.com/locate/idairyj

Review

Lactoferrin research, technology and applications Hiroyuki Wakabayashi, Koji Yamauchi, Mitsunori Takase Nutritional Science Laboratory, Morinaga Milk Industry Co., Ltd., 5-1-83 Higashihara, Zama, Kanagawa 228-8583, Japan Received 6 September 2005; accepted 29 May 2006

Abstract Lactoferrin is an iron-binding glycoprotein present in milk as well as other exocrine secretions and neutrophil granules in mammals. Lactoferrin is considered to be an important host defense molecule and has a diverse range of physiological functions such as antimicrobial/antiviral activities, immunomodulatory activity, and antioxidant activity. During the past decade, it has become evident that oral administration of lactoferrin exerts several beneficial effects on the health of humans and animals, including anti-infective, anticancer, and anti-inflammatory effects. This has enlarged the application potential of lactoferrin as a food additive. The technology of producing bovine lactoferrin on a factory scale was established over 20 years ago. Bovine lactoferrin is purified by cation-exchange chromatography from bovine skim milk or whey, and is commercially available from several suppliers. Recombinant human lactoferrin is produced by Aspergillus niger, transgenic cows, and rice, and its efficacy is being evaluated. In this article, we review basic research and technological aspects of the application of lactoferrin. r 2006 Elsevier Ltd. All rights reserved. Keywords: Lactoferrin; Host defense; Antiinfective; Cation-exchange; Food additive

Contents 1. 2.

3.

4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Health benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Animal studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Human clinical studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Mechanism of action. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technology and chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Purification from milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Recombinant production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Heat treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

Corresponding author. Tel.: +81 46 252 3045; fax: +81 46 252 3049.

E-mail address: [email protected] (H. Wakabayashi). 0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2006.06.013

Lactoferrin (LF) is an 80-kDa iron-binding glycoprotein of the transferrin family, which was first fractionated as an unknown ‘‘red fraction’’ from cows’ milk by Sørensen and

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Sørensen (1939), and the red protein from both human and bovine milk was defined as a transferrin-like glycoprotein (Johansson, 1960; Groves, 1960). LF is a major component of milk and is present in neutrophil granules or other exocrine secretions such as tears, saliva, and the cervical mucus. LF is thought to play a role in innate defense and exhibits a diverse range of biological activities, including antimicrobial activities, antiviral activities, antioxidant activities, immunomodulation, modulation of cell growth, and binding and inhibition of several bioactive compounds, such as lipopolysaccharide and glycosaminoglycan (Baveye, Elass, Mazurier, Spik, & Legrand, 1999; Chierici, 2001). The in vitro activity of LF also includes transcriptional activation of several genes (Oh, Pyo, Kim, & Choi, 2004). We found that pepsin-hydrolysate of LF (LFhyd) has more potent antimicrobial activity than the native protein and we purified the active peptide from LFhyd (Tomita et al., 1991). The antimicrobial peptide derived from LFhyd was named lactoferricin (Lfcin; Bellamy et al., 1992). Interestingly, LFcin and its derivatives exhibit various biological activities, like LF. Therefore, the LFcin-region seems likely to be an important functional domain of LF (Wakabayashi, Takase, & Tomita, 2003).

Recently it has been recognized that oral administration of LF exerts various health beneficial effects such as antiinfective activities not only in infants, but also in adult animals and humans (Tomita, Wakabayashi, Yamauchi, Teraguchi, & Hayasawa, 2002; Teraguchi, Wakabayashi, Kuwata, Yamauchi, & Tamura, 2004). In this review, we summarize these effects and the safety observed in animal studies and human clinical studies. We also discuss the mechanism of action, especially focusing on immunomodulation. LF content in milk varies depending on the species. The amount of LF is lower in cows’ milk (i.e., 0.1–0.4 mg mL
1) than in human milk (i.e., 1–3 mg mL
1). However, a factory scale technology to produce large amounts of bovine LF (bLF) at high purity from cows’ milk was established over 20 years ago (Law & Reiter, 1977). We describe several technological aspects of the production and heat treatment of bLF. We also introduce technologies for the production of recombinant human LF (rhLF). 2. Health benefits Investigations have been done to evaluate the effect of orally administered LF in healthy or diseased human

Table 1 Effectiveness of orally administered LF-related compounds on bacterial flora and infections in animals Model or disease

Efficacy

Animal

Administered agent and dose

Reference

Bacterial flora Human flora inoculated as intestinal flora Overgrowth of intestinal bacteria Translocation of intestinal bacteria

Increase of Bifidobacterium and other bacteria Decrease of E. coli, Streptococcus, and Clostridium Prevention of translocation

Mouse

Hentges et al. (1992)

Mouse

bLF 2 mg mL
1 in infant formula diet bLF, bLFhyd 0.5–5% in diet

Mouse

bLF, bLFhyd 2% in diet

Teraguchi et al. (1995)

Infection (digestive tract) Peroral systemic infection with E. coli Gastric infection with Helicobacter pylori

Decrease of pathogen in blood and liver Decrease of pathogen

Neonatal rat

rhLF 0.35 g kg
1

Edde et al. (2001)

Mouse

bLF 0.01 g body
1 bLF 0.4 g kg
1

Decrease of infection rate

Mouse

rhLF 0.1 g kg
1

Decrease of pathogen, promotion of cure Increase of survival rate

Mouse

bLF 0.5, 2.5 g kg
1

Wada et al. (1999) Wang, Hirmo, Wille´n, and Wadstro¨m (2001) Dial, Romero, Headon, and Lichtenberger (2000) Takakura et al. (2003)

Mouse

LFcin B 0.005 g body
1

Isamida et al. (1998)

Mouse

bLF 2% in water

Mouse

Bhimani, Vendrov, and Furmanski (1999) Ha˚versen et al. (2000)

Gastric infection with Helicobacter felis Oral candidiasis Peroral systemic infection with Toxoplasma gondii Infection (other than digestive tract) Systemic infection with Staphylococcus aureus Urinary tract infection with E. coli Tinea corporis, tinea pedis Cutaneous infection with herpes simplex virus-1 Pneumonia due to influenza virus infection

Decrease of infection in kidney and pathogen Decrease of pathogen Decrease of pathogen, promotion of cure Prevention of body weight loss

Guinea pig

bLF, hLF, LFcin H-pep 0.5 mg body
1 bLF 0.25, 2.5 g kg
1

Mouse

bLF 1.5% in water

Prevention of pneumonia

Mouse

bLF 0.06 g body
1

Teraguchi et al. (1994)

Wakabayashi, Uchida, et al. (2000) Wakabayashi, Kurokawa, et al. (2004) Shin et al. (2005)

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Table 2 Effectiveness of orally administered LF-related compounds on growth/nutritional status and inflammation/drug-induced injury in animals Model or disease Growth/nutritional status Iron-deficient anemia Excess-iron feeding Normal Normal Normal Normal Inflammation/drug-induced injury Intractable stomatitis Endotoxin shock Trinitrobenzenesulfonic acidor dextran sulfate-induced colitis Dextran sulfate-induced colitis Adjuvant-induced arthritis NSAID-induced intestinal injury Methotrexate-induced small intestinal damage

Efficacy

Animal

Administered agent and dose

Reference

Increase of red blood cells, hamatocrit, hemoglobin Decrease of hepatic iron Increase of hepatic protein synthesis Decrease of triacylglycerol and cholesterol Enhancement of VEGF-Amediated angiogenesis Increase of weight gain, gainto-feed ratio

Rat

Fe-bLF 0.035 g body
1

Kawakami et al. (1988)

Weaning mouse Newborn pig

Fe-bLF 18% in diet bLF 1 mg mL
1 in formula

Mouse

bLF 1% in diet

Hagiwara et al. (1997) Burrin, Wang, Heath, and Dudley (1996) Takeuchi et al. (2004)

Rat

hLF 0.01 g body
1

Norrby (2004)

Preweaning calf

bLF 1 g body
1

Robblee et al. (2003)

Cat

bLF 0.04 g kg
1

Sato et al. (1996)

Germfree piglet Rat

bLF 2 g body
1 bLF 0.2 g kg
1

Lee et al. (1998) Togawa et al. (2002a, b)

Mouse

Ha˚versen et al. (2003)

Rat

hLF, LFcin H-pep 0.002 g body
1 bLF 0.1 g kg
1

Rat

rhLF 0.2 g kg
1

Dial et al. (2005)

Rat

bLF 1 g kg
1

Van’t Land et al. (2004)

Improvement of inflammatory lesions Decrease of mortality Prevention of inflammatory lesion Prevention of inflammatory parameters Inhibition of inflammation and pain Prevention of intestinal bleeding and inflammation Inhibition of damage, epithelial cell proliferation, and permeability

Hayashida et al. (2004)

Table 3 Effectiveness of orally administered LF-related compounds on cancers in animals Cancer model

Efficacy

Animal

Administered agent and dose

Reference

Carcinogen-induced tumor in colon, esophagus, lung, tongue, bladder, liver

Inhibition of tumor development

Rat

bLF 0.2, 2% in diet

Spontaneously developed intestinal polyposis Tumor cell injection

Inhibition of polyp development Inhibition of lung metastasis Inhibition of tumor development

ApcMin mouse

bLF 2% in diet

Sekine, Ushida et al. (1997), Sekine, Watanabe et al. (1997), Ushida et al. (1999), Tanaka et al. (2000), Masuda et al. (2000), Fujita, Ohnishi, Sekine, Iigo, and Tsuda (2002) Ushida et al. (1998)

Mouse

bLF, bLFhyd 0.3 g kg
1

Iigo et al. (1999)

Mouse

rhLF 1 g kg
1

Varadhachary et al. (2004)

Tumor cell injection

beings/animals (Tomita et al., 2002; Teraguchi et al., 2004). To date, it has become evident that the oral administration of LF exerts various beneficial effects against diseases and is safe for health. This enlarged the application potential of LF as a food additive for humans and animals. We summarize the results obtained so far from animal studies and human clinical studies, and also discuss the mechanisms of action.

2.1. Animal studies A wide range of effects of orally administered LF and related compounds have been found in studies in mammals, as shown in Tables 1–3. Table 1 shows the effects on bacterial flora and infections. Table 2 shows the effects on growth/nutritional status and inflammation/drug-induced injuries. Table 3 shows the effects on cancers. Although the

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investigations were mainly done using commercially available bLF, other LF-related compounds, including ironsaturated bLF (Fe-bLF), pepsin-hydrolysate of bLF (bLFhyd), bovine LFcin (LFcin B), human LFcin-derivative peptides (LFcin H-pep), human LF purified from human milk (hLF), and rhLF, have also been used for some of the studies. Earlier studies investigated the effect on intestinal bacterial flora and digestive tract-related infections (Table 1). Feeding cows’ milk to mice resulted in a great increase in the numbers of Enterobacteriaceae in the faeces and the gastrointestinal tract. When mice were fed cows’ milk containing 2% bLF or bLFhyd, the overgrowth of Enterobacteriaceae and Streptococcus was significantly suppressed (Teraguchi et al., 1994). On the other hand, LF did not suppress the intestinal Bifidobacterium. Additionally, ingested bLF or bLFhyd inhibited the translocation of aerobic bacterial species from the gut to the mesenteric lymph nodes (Teraguchi et al., 1995). Another study showed that oral bLF increases the intestinal Bifidobacterium (Hentges, Marsh, Petschow, Thal, & Carter, 1992). Orally administered LF or its peptide also suppressed gastric infection with Helicobacter pylori (Wada et al., 1999), oral infection with a pathogenic yeast Candida albicans (oral candidiasis) (Takakura et al., 2003), and peroral systemic infection with a parasitic protozoon Toxoplasma gondii (Isamida et al., 1998). Interestingly, orally administered LF also exhibits beneficial effects on infections in sites other than the digestive tract (Table 1). When Staphylococcus aureus was injected intravascularly or Esherichia coli was inoculated into the urinary bladder, mice given oral LF showed

reduced numbers of bacteria in the infected organs (Bhimani et al., 1999; Ha˚versen et al., 2000). A guinea pig model of dermatophytosis caused by the fungus Trichophyton mentagrophytes was examined (Wakabayashi et al., 2000). The results indicated that bLF given orally reduced the fungal burden in the skin of the back (tinea corporis) and foot (tinea pedis), and promoted the cure of tinea corporis. Oral bLF prevented body weight loss caused by infection with herpes simplex virus-1 on the skin (Wakabayashi et al., 2004). In a murine pneumonia model of influenza virus infection, oral bLF attenuated the inflammatory symptoms (Shin et al., 2005). Orally administered LF affects growth and nutritional status in animals (Table 2). Regarding iron metabolism, Fe-bLF administration increased hemoglobin and red blood cells in iron-deficient anemic rats (Kawakami, Hiratsuka, & Dosako, 1988), whereas administration of Fe-bLF at high dose decreased hepatic iron content in weaning mice fed excess iron (Hagiwara et al., 1997). It is reported that oral LF reduces fat content in the serum and liver in mice (Takeuchi, Shimizu, Ando, & Harada, 2004), vascular endothelial growth factor A (VEGF-A)-mediated angiogenesis in rats (Norrby, 2004), and weight gain in preweaning calves (Robblee et al., 2003). Oral LF shows beneficial effects against inflammation and drug-induced injuries (Table 2). Intractable stomatitis in cats (Sato, Inanami, Tanaka, Takase, & Naito, 1996), endotoxin shock in germfree piglets (Lee, Farmer, Hilty, & Kim, 1998), drug-induced colitis in rats and mice (Togawa et al., 2002a, b; Ha˚versen, Baltzer, Dolphin, Hanson, & Mattsby-Baltzer, 2003), and adjuvant-induced arthritis in rats (Hayashida et al., 2004) have been shown to be

Table 4 Effectiveness of orally administered LF in humans Subject or disease

Efficacy

Administered agent and dose

Reference

Bacterial flora Fecal flora in low birth weight infants Fecal flora in infants

Increase of Bifidobacterium, decrease of Clostridium Increase of Bifidobacterium

bLF 1 mg mL
1 in infant formula

Kawaguchi et al. (1989)

bLF 1 mg mL
1 in infant formula

Roberts et al. (1992)

Infection (digestive tract) Gastric infection with Helicobacter pylori

Increase of eradication rate by triple therapy

bLF 0.2 g body
1

Di Mario et al. (2003)

hLF 0.8 g body
1

Tru¨mpler et al. (1989)

bLF 1.8, 3.6 g body
1

Tinea pedis

Decrease of incidence of bacteremia and severity of infection Decrease of ALT and HCV RNA in serum Promotion of cure

bLF 0.6, 2 g body
1

Tanaka et al. (1999); Iwasa et al. (2002) Yamauchi, Hiruma et al. (2000)

Growth/nutritional status Infants

Increase of ferritin

bLF 1 mg mL
1 in infant formula

Chierici et al. (1992)

Prevention of small intestinal permeability

rhLF 5  3 g body
1

Troost et al. (2003)

Infection (other than digestive tract) Neutropenic patients Chronic hepatitis C

Drug-induced injury NSAID-induced enteropathy

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improved by oral administration of LF. In addition, nonsteroidal anti-inflammatory drug (NSAID)-induced intestinal injury (Dial, Dohrman, Romero, & Lichtenberger, 2005) and methotrexate-induced small intestinal damage (Van’t Land, Van Beek, Van Den Berg, & M’rabet, 2004) were prevented by LF administration. Several animal studies have suggested that LF can inhibit the development and progression of tumors (Table 3). The first such report showed that the development of azoxymethane-initiated colonic tumors in rats was inhibited by oral bLF (Sekine, Ushida et al., 1997; Sekine, Watanabe et al., 1997). Similarly, a chemopreventive effect of LF on cancers induced by carcinogen in the esophagus, lung, tongue, bladder, and liver has been reported. Moreover, an inhibitory effect of bLF on spontaneous intestinal polyposis in ApcMin mice, a model for both familial adenomatous polyposis and sporadic colon cancers, was reported (Ushida et al., 1998). In other studies, oral bLF showed significant inhibition of lung metastasis in mice with subcutaneously implanted tumors (Iigo et al., 1999). The antimetastatic effect of bLFhyd was comparable to that of native bLF. An anti-tumor effect was also shown for rhLF (Varadhachary et al., 2004). 2.2. Human clinical studies Human clinical studies have been done in infants as well as in adults, mainly using bLF (Table 4). In infants, increases of Bifidobacterium in the fecal flora and the serum ferritin level were obtained by supplementing infant formula with bLF at 1 mg mL
1 (Roberts et al., 1992; Chierici, Sawatzki, Tamisari, Volpato, & Vigi, 1992). After feeding of the bLF-enriched formula for 2 weeks, the ratio of Bifidobacterium in the fecal flora of low-birth weight infants was increased, while the ratios of Enterobacteriaceae, Streptococcus and Clostridium showed a tendency to decrease (Kawaguchi et al., 1989). These results suggest that feeding of bLF-enriched formula leads to the establishment of a Bifidobacterium-predominant flora in infants. In adults, it was reported that 7-day bLF treatment increased the eradication rates of the one-week triple therapy with rabeprazole, clarithromycin, and tinidazole for H. pylori gastric infection (Di Mario et al., 2003). A significant difference was found between LF group (100%) and control group (78.9%). A pilot study showed that the ingestion of bLF for 8 weeks can decrease serum hepatitis C virus (HCV)- RNA levels and serum alanine aminotransaminase (ALT) levels in chronic hepatitis C patients with low viral load (Tanaka et al., 1999). In another study, 25 patients with chronic hepatitis C genotype 1b received a 6-month course of bLF (Iwasa et al., 2002). The serum level of HCV RNA significantly decreased in patients given bLF during the 6 months of treatment. The effectiveness of LF was investigated in the treatment of tinea pedis in a placebo-controlled doubleblind study (Yamauchi, Hiruma et al., 2000). Either 0.6 or

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2 g of bLF, or a placebo, was orally administered daily for 8 weeks to 37 adults who were judged to have mild or moderate tinea pedis. In the analysis limited to subjects with moderate vesicular or interdigital tinea pedis, dermatological symptom scores in the bLF-treated groups were significantly decreased compared with those in the placebo group. In a study using rhLF, a preventive effect on NSAID-induced enteropathy was observed (Troost, Saris, & Brummer, 2003). 2.3. Mechanism of action A study demonstrated that over than 60% of administered bLF survives passage through the adult human stomach and enters the small intestine in an intact form (Troost, Steijns, Saris, & Brummer, 2001). On the other hand, analysis of the gastric contents revealed that LFcin B was formed at a molar concentration corresponding to 4.5% of ingested bLF (Kuwata, Yip, Tomita, & Hutchens, 1998). When the animals were given free access to milk containing bLF at 40 mg mL
1 (482 mmol L
1), the levels of bLF fragments containing the LFcin B region in the contents of the stomach, upper small intestine, and lower small intestine were approximately 200, 20, and 1 mmol L
1, respectively (Kuwata, Yip, Yamauchi, et al., 1998; Kuwata et al., 2001). Some parts of ingested LF are likely to be not fully digested and to be present in the lower gastrointestinal tract. These intact bLF and partially digested bLF peptides, which retain biological activities, may exert various physiological effects in the digestive tract. Orally administered LF exhibits several beneficial effects at sites other than the digestive tract. In the case of infants or adults with injury in the intestine, it is possible that ingested LF enters the blood circulation and acts systemically. However, neither bLF nor functional bLF fragments (LFcin B-containing peptides and anti-bLF antibodybinding peptides) were at a level higher than the detection limit (1 ng mL
1) detected in the portal blood of normal adult rats orally given bLF at a maximal dose of 5 g kg
1 (Wakabayashi, Kuwata, Yamauchi, Teraguchi, & Tamura, 2004). Therefore, LF-related molecules are not likely to be transported from the intestine into the circulation. Based on the large body of accumulated evidence, it is rational to consider that oral bLF or its digested products acts initially on the intestinal immune system and then augments the protective immunity systemically (Teraguchi et al., 2004). Orally administered bLF enhances interleukin (IL)-18 production in the intestinal epithelial cells, and increases the numbers of CD4+ cells, CD8+ cells, and natural killer (NK) cells in the intestinal mucosa (Kuhara et al., 2000; Wang et al., 2000). In the systemic immune system, oral bLF increases the numbers of cells in lymph nodes and the spleen, enhances the activity of peritoneal macrophages and splenic NK cells, and enhances the production of Th1 type cytokines (IL-12 and interferon (IFN)-g) (Sekine, Ushida et al., 1997; Sekine, Watanabe et al., 1997;

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Takakura et al., 2004; Wakabayashi, Kurokawa, et al., 2004; Wakabayashi, Takakura, Teraguchi, & Tamura, 2003). The circulation of humoral factors such as IL-18 and the recruitment of immune cells may be signals bridging the intestinal immune system and the systemic immune system in the action of oral LF. However, the detailed molecular mechanisms must be elucidated in future studies. 2.4. Safety bLF as a food ingredient is thought to be safe, because there is a long dietary history of its use. People who live on dairy products must have ingested bLF for a long time, because raw milk and natural cheese contain 0.1– 0.4 mg mL
1 and about 3 mg g
1 of bLF, respectively. Reverse mutation tests and oral dose toxicity tests were carried out in order to check the safety of highly purified bLF. bLF purified from cheese-whey did not show any reverse mutation in the Ames test using five types of bacteria such as Salmonella typhimurium, and showed negative results in all cases of the Ame’s test, chromosome aberration test and micronucleus test (Yamauchi, Toida, Kawai et al., 2000). bLF did not show any toxicity upon single-dose oral ingestion or 4-week or 13-week oral repeated-dose ingestion at a maximum dose of 2 mg kg
1 day
1 in rats (Yamauchi, Toida, Nishimura et al., 2000). A human clinical study in chronic hepatitis C patients showed that high oral doses, up to 7.2 g body
1 day
1 were well tolerated (Okada et al., 2002). Therefore, the safety of bLF as a dietary component must be very high, although the antigenicity should be considered, as is true for other proteins. Use of bLF as a nutritional supplement is considered to be Generally Recognized As Safe (GRAS) by the US Food and Drug Administration. 3. Technology and chemistry 3.1. Purification from milk Since LF is denatured by heat treatment depending on the conditions, pasteurized milk is not suitable as a source for bLF purification. Therefore, skim milk and cheese whey that have not undergone rigorous heating can be sources of bLF. Because LF has a cationic nature according to its amino acid composition, it can be purified by cation-exchange chromatography such as carboxymethyl (CM)-Sephadex (Law & Reiter, 1977; Yoshida, Wei, Shinmura, & Fukunaga, 2000) and this purification method is the most popular procedure for bLF purification in bLF-supplying companies. For example, skim milk (pH 6.7) or cheese-whey (pH 6.4) is filtered and applied to a cation-exchange chromatography column without pH adjustment. The column is washed with a low-concentration (1.6%) NaCl solution, by which lactoperoxidase is eluted. Then bLF is eluted with a high-concentration (5%) NaCl solution. The bLF is concentrated by ultrafiltration

and is separated from NaCl by diafiltration. After low heat treatment, bLF is freeze-dried to make a powder. Alternatively, bLF is spray-dried after sterile filtration. The amount of bLF produced in the world was estimated by our company to be about 79 tonnes in 2003. On a laboratory scale, several other chromatographic methods have been evaluated for purification of LF. Since LF can bind transition metals and anionic compounds such as heparin and DNA, these materials have been used to purify LF. Metal (copper)-chelate affinity chromatography followed by gel-filtration was reported for purification of hLF from human whey (Lo¨nnerdal, Carlsson, & Porath, 1977). Guinea pig LF was isolated by one-step copperchelate affinity chromatography with pH-gradient elution from neutrophil granules (Torres, Peterson, Evans, Mage, & Wilson, 1979). Use of heparin-Sepharose affinity chromatography for purification of hLF from human whey was first described by Bla¨ckberg and Hernell (1980). Heparin-Sepharose affinity chromatography was also used for purification of LF from human skim milk, human pancreatic juice, and bovine skim milk (Wang, Chan, & Kloer, 1984). Heparin-agarose affinity chromatography was used to purify bLF from the secretions of the involuting mammary gland (Rejman, Hegarty, & Hurley, 1989). Single-stranded DNA-agarose affinity chromatography was used to purify hLF from human whey (Hutchens, Magnuson, & Yip, 1989). 3.2. Recombinant production Production of hLF by purification from human milk has potential problems of safety such as contamination by human pathogens and a limitation of the amount of the supply. Therefore, rhLF for use in animal studies and human clinical studies has been produced in Aspergillus niger var. awamori by Agennix Inc., in transgenic cows by Pharming Technologies B.V., and in rice by Ventria Bioscience. Production of rhLF by fermentation of Aspergillus oryzae and A. niger var. awamori was reported in 1992 (Ward et al. 1992) and 1995 (Ward et al., 1995), respectively. rhLF was produced at 5 g L
1 of fermentation medium of A. niger var. awamori and purified by cationexchange chromatography. It was evaluated for the physicochemical characteristics, biological activities, and safety (Headon, 2000). Aside from a difference in oligosaccharides, A. niger-produced rhLF has similar properties with native hLF. Transgenic cows harboring the genomic hLF gene under the regulatory control of the bovine as1-casein promoter produced rhLF at 0.4–3.0 g L
1 of milk (van Berkel et al., 2002). rhLF and bLF in the transgenic cows’ milk were separated by Mono S chromatography. Although rhLF and native hLF underwent differential N-linked glycosylation, they were equally effective in in vivo infection models. The amount of rhLF expressed in rice reached 25% of total soluble protein and 0.5% of grain weight (Bethell &

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Table 5 Influence of heat treatment on bLF activities Activities of bLF treated under the indicated conditionsa 90 1C b

Iron binding capacity (%) Antigenicity (%) Antibacterial activity (OD)d

100 1C

Control

pH 2

pH 3

pH 4

pH 5

pH 6

pH 2

pH 3

pH 4

pH 5

pH 6

100 100 0.7

95 95 0.6

95 95 0.6

95 95 0.7

92 95 0.7

75 60 1.2

62 75 0.3

75 72 0.1

90 75 1.0

70 55 1.3

NDc 0 2.4

a Five percent bLF in distilled water was adjusted to the indicated pH and treated at the indicated temperature for 5 min. After adjustment to near neutral pH, samples were assayed for the indicated activities. b Control is bLF not treated by heating. c ND: Not determined because of appearance of turbidity. d Antibacterial activity was tested by incubation of E. coli for 20 h in the presence of the test sample at 500 mg mL
1 and the bacterial growth (OD660) was determined.

Huang, 2004). Apart from rhLF produced in rice having plant pattern glycosylation, the physicochemical and biochemical properties of rhLF and native hLF were similar. 3.3. Heat treatment The question of heat stability is important when LF is used as a bioactive component of foods. In order to develop a practical method for pasteurization of LF, the heat stability has been studied. To study the influence of pH on the heat stability of bLF, solutions of 5% bLF at pH 2–11 in distilled water were heated at 80–120 1C for 5 min (Abe et al., 1991). The heated solutions of bLF gelled at neutral and alkaline pH. At acidic pH, bLF in the heated samples remained soluble and the solutions remained clear. bLF exhibited comparative stability to heating at pH 4 and 90–100 1C regarding iron-binding capacity and antigenic activity, as shown in Table 5. When 1% bLF at pH 4 in distilled water was preheated at 70 1C for 3 min followed by UHT at 130 1C for 2 s, there was only a 3% loss of residual iron-binding capacity compared with that of the unheated sample. These results indicate that bLF is stable to heating at pH 4 and this heating condition is suitable as a practical method for pasteurization of bLF. Rigorous heat treatment of bLF at pH 2–3 increases the antibacterial activity (Table 5). bLF heat-treated at pH 2 and 120 1C for 15 min had no iron-binding capacity and significantly decreased antigenicity, but its antibacterial activity was increased (Saito, Miyakawa, Tamura, Shimamura, & Tomita, 1991). Reverse-phase high performance liquid chromatography (HPLC) fractionation of the heatteated bLF revealed the generation of several peptide fractions having strong antibacterial activity. These antibacterial peptide fractions contained the N-terminal region of bLF overlapping LFcin B. The heat stability of bLF also depends on the matrix. The heat-sensitivity of apo-bLF and Fe-bLF was higher in milk than in phosphate buffer, in which apo-bLF was

denatured faster than Fe-bLF (Sanchez et al., 1992). The heat stability of LF is affected by environmental conditions such as pH, salts, and whey proteins (Kussendrager, 1994). Therefore, the parameters of the heat-induced denaturation of LF have to be examined under the conditions of the application of interest. 3.4. Analysis method The concentration of LF in the supplemented foods must be measured to monitor the stability of LF during the processing and storage. Although sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (Ronayne de Ferrer, Baroni, Sambucetti, Lo´pez, & Ceriani Cernadas, 2000), HPLC (Hutchens, Henry, & Yip, 1991), and mass spectrometry (Natale et al., 2004) can quantify LF in food samples, these analytical methods can not discriminate between intact LF and denatured LF. Because immunological methods can discriminate the tertiary structures of proteins, these methods would be suitable for monitoring intact LF in food samples. A single radial immunodiffusion assay (Masson & Heremans, 1971), the Rocket assay (Nagasawa, Kiyosawa, & Kuwahara, 1972), and enzymelinked immunosorbent assay (ELISA) (So¨derquist, Sundqvist, Jones, Holmberg, & Vikerfors, 1995) have been used to measure bLF in supplemented products. ELISA kits for bLF and hLF are commercially available from several suppliers such as Bethyl Laboratories (Montgomery, USA) and Merck/EMD Biosciences (San Diego, USA), and are generally used. Recently, a new immunoassay method to quantify bLF was reported (Yamauchi et al., 2004). An automated latex assay was developed using F(ab0 )2 fragments of anti-bLF rabbit IgG-coated polystyrene latex beads and an automated multi-purpose analyzer. The latex assay employs agglutination of the antibody-coated latex particles in the presence of the antigen. This method enabled the quantification of bLF in bLF-supplemented products such as infant formula in a simple, rapid, highly sensitive, and

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Table 6 Examples of bLF application available in Japan Category

Product

Brand name

Expected effect

Food

Infant formula

Hagukumi, Chilmil Ayumi, Non-Lact, EAkachan, GP-P, New-NA-20 (Morinaga)

Antiinfection, Improvement of oro-gastrointestinal microflora, Immunomodulation, Antiinflammation, Antioxidation

Supplemental tablet

Lactoferrin Plus, Lactoferrin Original Type (Livewell), Actio Lactoferrin (Asahi), Lactoferrin (DHC) Lactoferrin 200 Yogurt, Onakani-Haitatsu Yogurt, Ikiikigenki-Nomu Yogurt (Morinaga), Bifiene M (Yakult) Ca Lactoferrin Skim Milk (Morinaga), Tetsu Lactoferrin Skim (Snow Brand) Lactoferrin Plus (Morinaga) Lactoferrin 200, Lactonin (Morinyu Sunworld)

Yoghurt

Skim milk Drink Pet food Skin care (cosmetics)

Lotion, cream, face wash

Milk Protein (DHC), Miss Yoko essential lotion/white cream/essence (Yoko)

Hygiene, Moistening, Antioxidation

Oral care

Mouth wash, mouth gel, toothpaste chewing gum

Biotene Oral Balance/mouse wash/tooth paste (Laclede) Hamigaki Gum (Kanebo)

Hygiene, Moistening

precise manner. A reagent for the automated latex assay of bLF and hLF is commercially available as a kit (Cosmo Bio, Tokyo, Japan). 4. Applications The first application of bLF in a commercial product was its supplementation in the infant formula ‘‘BF-L’’ by Morinaga Milk Industry in 1986. bLF is currently used for the supplementation of various foods and skin/oral care products. Examples of bLF application available in Japan are listed in Table 6. bLF is used to supplement foods such as infant formula, supplemental tablets, yoghurt, skim milk, drinks, and pet foods. The expected effects of these products include antiinfection, improvement of oro-gastrointestinal microflora, immunomodulation, antiinflammation, and antioxidation. Infant formula supplemented with bLF is available not only in Japan but also in Korea and Indonesia. Other fields of application are skin care and oral care products. Skin care products (cosmetics) containing bLF include lotions, creams, and face washes, and these products are expected to contribute to hygiene, moistening, and antioxidation in the skin. Oral care products containing bLF include mouth washes, mouth gels, toothpaste, and chewing gum, and these products are expected to contribute to hygiene and moistening in the mouth. bLFcontaining oral care products are sometimes combined with other mucosal defense proteins such as lactoperoxidase and lysozyme. rhLF has not been applied as a commercial product yet. A major target of rhLF application may be the pharmaceutical field, because genetically modified food is not entirely accepted by consumers at present.

5. Conclusion Technology for the production of bLF and rhLF has already been established. The pasteurizing conditions during processing of LF-supplemented products have also been assessed. It is now possible to supply a larger amount of LF than the current supply. Using these products, various beneficial effects of LF as a food additive have been demonstrated and this enabled us to use LF in a large number of fields. Because LF has multifunctional properties, new effects of LF consumption should be discovered in future studies. In addition, the use of LF in combination with other milk components or drugs will be an increasing consideration.

References Abe, H., Saito, H., Miyakawa, H., Tamura, Y., Shimamura, S., Nagao, E., et al. (1991). Heat stability of bovine lactoferrin at acidic pH. Journal of Dairy Science, 74, 65–71. Baveye, S., Elass, E., Mazurier, J., Spik, G., & Legrand, D. (1999). Lactoferrin: A multifunctional glycoprotein involved in the modulation of the inflammatory process. Clinical Chemistry and Laboratory Medicine, 37, 281–286. Bellamy, W., Takase, M., Yamauchi, K., Wakabayashi, H., Kawase, K., & Tomita, M. (1992). Identification of the bactericidal domain of lactoferrin. Biochimica et Biophysica Acta, 1121, 130–136. Bethell, D. R., & Huang, J. (2004). Recombinant human lactoferrin treatment for global health issues: Iron deficiency and acute diarrhea. Biometals, 17, 337–342. Bhimani, R. S., Vendrov, Y., & Furmanski, P. (1999). Influence of lactoferrin feeding and injection against systemic staphylococcal infections in mice. Journal of Applied Microbiology, 86, 135–144. Bla¨ckberg, L., & Hernell, O. (1980). Isolation of lactoferrin from human whey by a single chromatographic step. FEBS Letters, 109, 180–183.

ARTICLE IN PRESS H. Wakabayashi et al. / International Dairy Journal 16 (2006) 1241–1251 Burrin, D. G., Wang, H., Heath, J., & Dudley, M. A. (1996). Orally administered lactoferrin increases hepatic protein synthesis in formulafed newborn pigs. Pediatric Research, 40, 72–76. Chierici, R. (2001). Antimicrobial actions of lactoferrin. Advances in Nutritional Research, 10, 247–269. Chierici, R., Sawatzki, G., Tamisari, L., Volpato, S., & Vigi, V. (1992). Supplementation of an adapted formula with bovine lactoferrin: 2. Effect on serum iron, ferritin and zinc levels. Acta Paediatrica, 81, 475–479. Dial, E. J., Dohrman, A. J., Romero, J. J., & Lichtenberger, L. M. (2005). Recombinant human lactoferrin prevents NSAID-induced intestinal bleeding in rodents. Journal of Pharmacy and Pharmacology, 57, 93–99. Dial, E. J., Romero, J. J., Headon, D. R., & Lichtenberger, L. M. (2000). Recombinant human lactoferrin is effective in the treatment of Helicobacter felis-infected mice. Journal of Pharmacy and Pharmacology, 52, 1541–1546. Di Mario, F., Aragona, G., Dal Bo`, N., Cavestro, G. M., Cavallaro, L., Iori, V., et al. (2003). Use of bovine lactoferrin for Helicobacter pylori eradication. Digestive and Liver Disease, 35, 706–710. Edde, L., Hipolito, R. B., Hwang, F. A. Y., Headon, D. R., Shalwitz, R. A., & Sherman, M. P. (2001). Lactoferrin protects neonatal rats from gut-related systemic infection. American Journal of Physiology Gastroentestinal and Liver Physiology, 281, G1140–G1150. Fujita, K., Ohnishi, T., Sekine, K., Iigo, M., & Tsuda, H. (2002). Downregulation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced CYP1A2 expression is associated with bovine lactoferrin inhibition of MeIQx-induced liver and colon carcinogenesis in rats. Japanese Journal of Cancer Research, 93, 616–625. Groves, M. L. (1960). The isolation of a red protein from milk. Journal of the American Chemical Society, 82, 3345–3350. Hagiwara, T., Ozawa, K., Fukuwatari, Y., Hayasawa, H., Hirohata, Y., Adachi, A., et al. (1997). Effects of lactoferrin on iron absorption in immature mice. Nutrition Research, 17, 895–906. Ha˚versen, L. A., Baltzer, L., Dolphin, G., Hanson, L. A˚., & MattsbyBaltzer, I. (2003). Anti-inflammatory activities of human lactoferrin in acute dextran sulphate-induced colitis in mice. Scandinavian Journal of Immunology, 57, 2–10. Ha˚versen, L. A., Engberg, I., Baltzer, L., Dolphin, G., Hanson, L. A., & Mattsby-Baltzer, I. (2000). Human lactoferrin and peptides derived from a surface-exposed helical region reduce experimental Escherichia coli urinary tract infection in mice. Infection and Immunity, 68, 5816–5823. Hayashida, K., Kaneko, T., Takeuchi, T., Shimizu, H., Ando, K., & Harada, E. (2004). Oral administration of lactoferrin inhibits inflammation and nociception in rat adjuvant-induced arthritis. Journal of Veterinary Medical Science, 66, 149–154. Headon, D. R. (2000). Human lactoferrin: production at large scale, characterization and applications. In K. Shimazaki, et al. (Eds.), Lactoferrin: Structure, function and applications (pp. 415–427). Amsterdam: The Netherlands, Elsevier Science B.V. Hentges, D. J., Marsh, W. W., Petschow, B. W., Thal, W. R., & Carter, M. K. (1992). Influence of infant diets on the ecology of the intestinal tract of human flora-associated mice. Journal of Pediatric Gastroenterology and Nutrition, 14, 146–152. Hutchens, T. W., Henry, J. F., & Yip, T.-T. (1991). Structurally intact (78-kDa) forms of maternal lactoferrin purified from urine of preterm infants fed human milk: Identification of a trypsin-like proteolytic cleavage event in vivo that does not result in fragment dissociation. Proceedings of the National Academy of Sciences of the United States of America, 88, 2994–2998. Hutchens, T. W., Magnuson, J. S., & Yip, T.-T. (1989). Interaction of human lactoferrin with DNA: one-step purification by affinity chromatography on single-stranded DNA-agarose. Pediatric Research, 26, 618–622. Iigo, M., Kuhara, T., Ushida, Y., Sekine, K., Moore, M. A., & Tsuda, H. (1999). Inhibitory effects of bovine lactoferrin on colon carcinoma 26 lung metastasis in mice. Clinical and Experimental Metastasis, 17, 35–40.

1249

Isamida, T., Tanaka, T., Omata, Y., Yamauchi, K., Shimazaki, K., & Saito, A. (1998). Protective effects of lactoferricin against Toxoplasma gondii infection in mice. Journal of Veterinary Medical Science, 60, 241–244. Iwasa, M., Kaito, M., Ikoma, J., Takeo, M., Imoto, I., Adachi, Y., et al. (2002). Lactoferrin inhibits hepatitis C virus viremia in chronic hepatitis C patients with high viral loads and HGV genotype 1b. American Journal of Gastroenterology, 97, 766–767. Johansson, B. (1960). Isolation of an iron containing red protein from human milk. Acta Chemica Scandinavica, 14, 510–512. Kawaguchi, S., Hayashi, T., Masano, J., Okuyama, K., Suzuki, T., & Kawase, K. (1989). A study concerning the effect of lactoferrinenriched infant formula on low birth weight infants—with special reference to changes in intestinal flora. Perinatal Medicine, 19, 557–562 (in Japanese). Kawakami, H., Hiratsuka, M., & Dosako, S. (1988). Effects of ironsaturated lactoferrin on iron absorption. Agricultural and Biological Chemistry, 52, 903–908. Kuhara, T., Iigo, M., Itoh, T., Ushida, Y., Sekine, K., Terada, N., et al. (2000). Orally administered lactoferrin exerts an antimetastatic effect and enhances production of IL-18 in the intestinal epithelium. Nutrition and Cancer, 38, 192–199. Kussendrager, K. (1994). Effects of heat treatment on structure and ironbinding capacity of bovine lactoferrin. In IDF bulletin: Indigenous antimicrobial agents of milk: Recent developments (pp. 133–146). Uppsala, Sweden: IDF. Kuwata, H., Yamauchi, K., Teraguchi, S., Ushida, Y., Shimokawa, Y., Toida, T., et al. (2001). Functional fragments of ingested lactoferrin are resistant to proteolytic degradation in the gastrointestinal tract of adult rats. Journal of Nutrition, 131, 2121–2127. Kuwata, H., Yip, T. T., Tomita, M., & Hutchens, T. W. (1998). Direct evidence of the generation in human stomach of an antimicrobial peptide domain (lactoferricin) from ingested lactoferrin. Biochimica et Biophysica Acta, 1429, 129–141. Kuwata, H., Yip, T. T., Yamauchi, K., Teraguchi, S., Hayasawa, H., Tomita, M., et al. (1998). The survival of ingested lactoferrin in the gastrointestinal tract of adult mice. Biochemical Journal, 334, 321–323. Law, B. A., & Reiter, B. (1977). The isolation and bacteriostatic properties of lactoferrin from bovine milk whey. Journal of Dairy Research, 44, 595–599. Lee, W. J., Farmer, J. L., Hilty, M., & Kim, Y. B. (1998). The protective effects of lactoferrin feeding against endotoxin lethal shock in germfree piglets. Infection and Immunity, 66, 1421–1426. Lo¨nnerdal, B., Carlsson, J., & Porath, J. (1977). Isolation of lactoferrin from human milk by metal-chelate affinity chromatography. FEBS Letters, 75, 89–92. Masson, P. L., & Heremans, J. F. (1971). Lactoferrin in milk from different species. Comparative Biochemistry and Physiology, 39B, 119–129. Masuda, C., Wanibuchi, H., Sekine, K., Yano, Y., Otani, S., Kishimoto, T., et al. (2000). Chemopreventive effects of bovine lactoferrin on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced rat bladder carcinogenesis. Japanese Journal of Cancer Research, 91, 582–588. Nagasawa, T., Kiyosawa, I., & Kuwahara, K. (1972). Amounts of lactoferrin in human colostrums and milk. Journal of Dairy Science, 55, 1651–1659. Natale, M., Bisson, C., Monti, G., Peltran, A., Garoffo, L. P., Valentini, S., et al. (2004). Cow’s milk allergens identification by two-dimensional immunoblotting and mass spectrometry. Molecular Nutrition and Food Research, 48, 363–369. Norrby, K. (2004). Human apo-lactoferrin enhances angiogenesis mediated by vascular endothelial growth factor A in vivo. Journal of Vascular Research, 41, 293–304. Oh, S. M., Pyo, C. W., Kim, Y., & Choi, S. Y. (2004). Neutrophil lactoferrin upregulates the human p53 gene through induction of NF-kB activation cascade. Oncogene, 23, 8282–8291.

ARTICLE IN PRESS 1250

H. Wakabayashi et al. / International Dairy Journal 16 (2006) 1241–1251

Okada, S., Tanaka, K., Sato, T., Ueno, H., Saito, S., Okusaka, T., et al. (2002). Dose-response trial of lactoferrin in patients with chronic hepatitis C. Japanese Journal of Cancer Research, 93, 1063–1069. Rejman, J. J., Hegarty, H. M., & Hurley, W. L. (1989). Purification and characterization of bovine lactoferrin from secretions of the involuting mammary gland: Identification of multiple molecular weight forms. Comparative Biochemistry and Physiology, 93B, 929–934. Robblee, E. D., Erickson, P. S., Whitehouse, N. L., McLaughlin, A. M., Schwab, C. G., Rejman, J. J., et al. (2003). Supplemental lactoferrin improves health and growth of Holstein calves during the preweaning phase. Journal of Dairy Science, 86, 1458–1464. Roberts, A. K., Chierici, R., Sawatzki, G., Hill, M. J., Volpato, S., & Vigi, V. (1992). Supplementation of an adapted formula with bovine lactoferrin: 1. Effect on the infant faecal flora. Acta Paediatrica, 81, 119–124. Ronayne de Ferrer, P. A., Baroni, A., Sambucetti, M. E., Lo´pez, N. E., & Ceriani Cernadas, J. M. (2000). Lactoferrin levels in term and preterm milk. Journal of the American College of Nutrition, 19, 370–373. Saito, H., Miyakawa, H., Tamura, Y., Shimamura, S., & Tomita, M. (1991). Potent bactericidal activity of bovine lactoferrin hydrolysate produced by heat treatment at acidic pH. Journal of Dairy Science, 74, 3724–3730. Sanchez, L., Peiro, J. M., Castillo, H., Perez, M. D., Ena, J. M., & Calvo, M. (1992). Kinetic parameters for denaturation of bovine milk lactoferrin. Journal of Food Science, 57, 873–879. Sato, R., Inanami, O., Tanaka, Y., Takase, M., & Naito, Y. (1996). Oral administration of bovine lactoferrin for treatment of intractable stomatitis in feline immunodeficiency virus (FIV)-positive and FIVnegative cats. American Journal of Veterinary Research, 57, 1443–1446. Sekine, K., Ushida, Y., Kuhara, T., Iigo, M., Baba-Toriyama, H., Moore, M. A., et al. (1997). Inhibition of initiation and early stage development of aberrant crypt foci and enhanced natural killer activity in male rats administered bovine lactoferrin concomitantly with azoxymethane. Cancer Letters, 121, 211–216. Sekine, K., Watanabe, E., Nakamura, J., Takasuka, N., Kim, D. J., Asamoto, M., et al. (1997). Inhibition of azoxymethane-initiated colon tumor by bovine lactoferrin administration in F344 rats. Japanese Journal of Cancer Research, 88, 523–526. Shin, K., Wakabayashi, H., Yamauchi, K., Teraguchi, S., Tamura, Y., Kurokawa, M., et al. (2005). Effects of orally administered bovine lactoferrin and lactoperoxidase on influenza virus infection in mice. Journal of Medical Microbiology, 54, 717–723. So¨derquist, B., Sundqvist, K.-G., Jones, I., Holmberg, H., & Vikerfors, T. (1995). Interleukin-6, C-reactive protein, lactoferrin and white blood cell count in patients with S. aureus septicemia. Scandinavian Journal of Infectious Diseases, 27, 375–380. Sørensen, M., & Sørensen, S. P. L. (1939). The proteins in whey. Compt. Rendus Laboratoire Carlsberg, 23, 55–99. Takakura, N., Wakabayashi, H., Ishibashi, H., Teraguchi, S., Tamura, Y., Yamaguchi, H., et al. (2003). Oral lactoferrin treatment of experimental oral candidiasis in mice. Antimicrobial Agents and Chemotherapy, 47, 2619–2623. Takakura, N., Wakabayashi, H., Ishibashi, H., Yamauchi, K., Teraguchi, S., Tamura, Y., et al. (2004). Effect of orally administered bovine lactoferrin on the immune response in the oral candidiasis murine model. Journal of Medical Microbiology, 54, 495–500. Takeuchi, T., Shimizu, H., Ando, K., & Harada, E. (2004). Bovine lactoferrin reduces plasma triacylglycerol and NEFA accompanied by decreased hepatic cholesterol and triacylglycerol contents in rodents. British Journal of Nutrition, 91, 533–538. Tanaka, K., Ikeda, M., Nozaki, A., Kato, N., Tsuda, H., Saito, S., et al. (1999). Lactoferrin inhibits hepatitis C virus viremia in patients with chronic hepatitis C: A pilot study. Japanese Journal of Cancer Research, 90, 367–371. Tanaka, T., Kawabata, K., Kohno, H., Honjo, S., Murakami, M., Ota, T., et al. (2000). Chemopreventive effect of bovine lactoferrin on 4-nitroquinoline 1-oxide-induced tongue carcinogenesis in male F344 rats. Japanese Journal of Cancer Research, 91, 25–33.

Teraguchi, S., Ozawa, K., Yasuda, S., Shin, K., Fukuwatari, Y., & Shimamura, S. (1994). The bacteriostatic effects of orally administered bovine lactoferrin on intestinal Enterobacteriaceae of SPF mice fed bovine milk. Bioscience, Biotechnology, and Biochemistry, 58, 482–487. Teraguchi, S., Shin, K., Ogata, T., Kingaku, M., Kaino, A., Miyauchi, H., et al. (1995). Orally administered bovine lactoferrin inhibits bacterial translocation in mice fed bovine milk. Applied and Environmental Microbiology, 61, 4131–4134. Teraguchi, S., Wakabayashi, H., Kuwata, H., Yamauchi, K., & Tamura, Y. (2004). Protection against infections by oral lactoferrin: Evaluation in animal models. Biometals, 17, 231–234. Togawa, J., Nagase, H., Tanaka, K., Inamori, M., Nakajima, A., Ueno, N., et al. (2002a). Oral administration of lactoferrin reduces colitis in rats via modulation of the immune system and correction of cytokine imbalance. Journal of Gastroenterology and Hepatology, 17, 1291–1298. Togawa, J., Nagase, H., Tanaka, K., Inamori, M., Umezawa, T., Nakajima, A., et al. (2002b). Lactoferrin reduces colitis in rats via modulation of the immune system and correction of cytokine imbalance. American Journal of Physiology Gastrointestinal and Liver Physiology, 283, G187–G195. Tomita, M., Bellamy, W., Takase, M., Yamauchi, K., Wakabayashi, H., & Kawase, K. (1991). Potent antibacterial peptides generated by pepsin digestion of bovine lactoferrin. Journal of Dairy Science, 74, 4137–4142. Tomita, M., Wakabayashi, H., Yamauchi, K., Teraguchi, S., & Hayasawa, H. (2002). Bovine lactoferrin and lactoferricin derived from milk: Production and applications. Biochemistry and Cell Biology, 80, 109–112. Torres, A. R., Peterson, E. A., Evans, W. H., Mage, M. G., & Wilson, S. M. (1979). Fractionation of granule proteins of granulocytes by copper chelate chromatography. Biochimica et Biophysica Acta, 576, 385–392. Troost, F. J., Saris, W. H. M., & Brummer, R.-J. M. (2003). Recombinant human lactoferrin ingestion attenuates indomethacin-induced enteropathy in vivo in healthy volunteers. European Journal of Clinical Nutrition, 57, 1579–1585. Troost, F. J., Steijns, J., Saris, W. H. M., & Brummer, R.-J. M. (2001). Gastric digestion of bovine lactoferrin in vivo in adults. Journal of Nutrition, 131, 2101–2104. Tru¨mpler, U., Straub, P. W., & Rosenmund, A. (1989). Antibacterial prophylaxis with lactoferrin in neutropenic patients. European Journal of Clinical Microbiology and Infectious Diseases, 8, 310–313. Ushida, Y., Sekine, K., Kuhara, T., Takasuka, N., Iigo, M., Maeda, M., et al. (1999). Possible chemopreventive effects of bovine lactoferrin on esophagus and lung carcinogenesis in tha rat. Japanese Journal of Cancer Research, 90, 262–267. Ushida, Y., Sekine, K., Kuhara, T., Takasuka, N., Iigo, M., & Tsuda, H. (1998). Inhibitory effects of bovine lactoferrin on intestinal polyposis in the ApcMin mouse. Cancer Letters, 134, 141–145. van Berkel, P. H. C., Welling, M. M., Geerts, M., van Veen, H. A., Ravensbergen, B., Salaheddine, M., et al. (2002). Large scale production of recombinant human lactoferrin in the milk of transgenic cows. Nature Biotechnology, 20, 484–487. Van’t Land, B., Van Beek, N. M. A., Van Den Berg, J. J. M., & M’rabet, L. (2004). Lactoferrin reduces methotrexate-induced small intestinal damage, possibly through inhibition of GLP-2-mediated epithelial cell proliferation. Digestive Diseases and Sciences, 49, 425–433. Varadhachary, A., Wolf, J. S., Petrak, K., O’Malley, B. W., Jr., Spadaro, M., Curcio, C., et al. (2004). Oral lactoferrin inhibits growth of established tumors and potentiates conventional chemotherapy. International Journal of Cancer, 111, 398–403. Wada, T., Aiba, Y., Shimizu, K., Takagi, A., Miwa, T., & Koga, Y. (1999). The therapeutic effect of bovine lactoferrin in the host infected with Helicobacter pylori. Scandinavian Journal of Gastroenterology, 34, 238–243. Wakabayashi, H., Kurokawa, M., Shin, K., Teraguchi, S., Tamura, Y., & Shiraki, K. (2004). Oral lactoferrin prevents body weight loss and increases cytokine responses during herpes simplex virus type 1

ARTICLE IN PRESS H. Wakabayashi et al. / International Dairy Journal 16 (2006) 1241–1251 infection of mice. Bioscience, Biotechnology, and Biochemistry, 68, 537–544. Wakabayashi, H., Kuwata, H., Yamauchi, K., Teraguchi, S., & Tamura, Y. (2004). No detectable transfer of dietary lactoferrin or its functional fragments to portal blood in healthy adult rats. Bioscience, Biotechnology, and Biochemistry, 68, 853–860. Wakabayashi, H., Takakura, N., Teraguchi, S., & Tamura, Y. (2003). Lactoferrin feeding augments peritoneal macrophage activities in mice intraperitoneally injected with inactivated Candida albicans. Microbiology and Immunology, 47, 37–43. Wakabayashi, H., Takase, M., & Tomita, M. (2003). Lactoferricin derived from milk protein lactoferrin. Current Pharmaceutical Design, 9, 1277–1287. Wakabayashi, H., Uchida, K., Yamauchi, K., Teraguchi, S., Hayasawa, H., & Yamaguchi, H. (2000). Lactoferrin given in food facilitates dermatophytosis cure in guinea pig models. Journal of Antimicrobial Chemotherapy, 46, 595–601. Wang, C., Chan, W., & Kloer, H. U. (1984). Comparative studies on the chemical and immunochemical properties of human milk, human pancreatic juice and bovine milk lactoferrin. Comparative Biochemistry and Physiology, 78B, 575–580. Wang, W. P., Iigo, M., Sato, J., Sekine, K., Adachi, I., & Tsuda, H. (2000). Activation of intestinal mucosal immunity in tumor-bearing mice by lactoferrin. Japanese Journal of Cancer Research, 91, 1022–1027. Wang, X., Hirmo, S., Wille´n, R., & Wadstro¨m, T. (2001). Inhibition of Helicobacter pylori infection by bovine milk glycoconjugates in a

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BALB/cA mouse model. Journal of Medical Microbiology, 50, 430–435. Ward, P. P., Lo, J.-Y., Duke, M., May, G. S., Headon, D. R., & Conneely, O. M. (1992). Production of biologically active recombinant human lactoferrin in Aspergillus oryzae. Biotechnology, 10, 784–789. Ward, P. P., Piddington, C. S., Cunningham, G. A., Zhou, X., Wyatt, R. D., & Conneely, O. M. (1995). A system for production of commercial quantities of human lactoferrin: A broad spectrum natural antibiotic. Biotechnology, 13, 498–503. Yamauchi, K., Hiruma, M., Yamazaki, N., Wakabayashi, H., Kuwata, H., Teraguchi, S., et al. (2000). Oral administration of bovine lactoferrin for treatment of tinea pedis. A placebo-controlled, double-blind study. Mycoses, 43, 197–202. Yamauchi, K., Soejima, T., Ohara, Y., Kuga, M., Nagao, E., Kagi, K., et al. (2004). Rapid determination of bovine lactoferrin in dairy products by an automated quantitative agglutination assay based on latex beads coated with F(ab’)2 fragments. Biometals, 17, 349–352. Yamauchi, K., Toida, T., Kawai, A., Nishimura, S., Teraguchi, S., & Hayasawa, H. (2000). Mutagenicity of bovine lactoferrin in reverse mutation test. Journal of Toxicological Science, 24, 63–66. Yamauchi, K., Toida, T., Nishimura, S., Nagano, E., Kusuoka, O., Teraguchi, S., et al. (2000). 13-week oral repeated administration toxicity study of bovine lactoferrin in rats. Food and Chemical Toxicology, 38, 503–512. Yoshida, S., Wei, Z., Shinmura, Y., & Fukunaga, N. (2000). Separation of lactoferrin-a and -b from bovine colostrums. Journal of Dairy Science, 83, 2211–2215.