ANTI-MICROBIAL ACTION

Several milk proteins have antimicrobial activity:

Lactoferrin

Lactoperoxidase

Lactoferrin

Lactoferrin is a molecule of 76,000 molecular weight. It is similar to the transferrin found in the blood stream where it functions as a carrier of iron. It has been hypothesized that its role in milk may also involve iron binding. It may bind the iron so tightly that is no longer available for microbial growth. Human lactoferrin is a major component of human whey.

There have been reports that lactoferrin is an effective inhibitor of microbial growth and it is produced for commercial use in toothpaste in Belgium. Recent reports suggest that peptide of lactoferrin is overtly bactericidal. Reports from Texas A& M suggest the hydrolysis of the lactoferrin in whey and greatly enhances its ability to inhibit the growth of certain pathogenic organisms. It has been reported that hydrolysis of whole whey with pepsin results in the formation of significant quantities of such a peptide. Richter and others have suggested that the hydrolyzed whey might be useful for inclusion in meat products to inhibit the growth of E coli. As the inclusion of whey in such products makes it possible to decrease their lipid content, a hamburger product that could be labeled as low fat and that is safer from a microbiological standpoint may be possible.

The economic viability of such a process will depend upon the degree of hydrolysis necessary to obtain bactericidal activity. If the requisite hydrolysis renders the remaining whey non functional, the cost may be too high. If on the other had, the proteins are still functional, there may be a future for this procedure. The peptides have been reported to bind to microbial membranes and to alter their permeability resulting in cell death. There is some work underway to isolate these peptides by an ion exchange process. Others are working in this area and it is one that I feel holds considerable potential for eventual commercialization.

Companies that I am aware of that are commercially producing lactoferrin are:

Swedish Diaries Association (SMR) - they use their own patented process

Tatua Co-operative Dairy Company in New Zealand

Lacto Bretagne Associes' in Belgium

Milei in Germany under a license from Morinaga

DOMO Food Ingredients a subsidiary of Friesland Dairy Foods in the Netherlands

DMV is reported to have a process ready to go some time this year. Some of the original procedures use raw whey as a feed stock, but all will probably convert to whey and more likely, WPC. The use of WPC reduces the volume of liquid and the amount of salts initially loaded on the ion exchange columns. These sites produce from 2,000 to 17,0000 pounds of lactoferrin annually.

Biotechnology may change this whole market. For example, Agennix, Inc. was issued patent WO 9614413 in May of this year. It describes the production of lactoferrin and selected fragments of lactoferrin in yeast cells. I expect that it wont be too long before transgenic goats or cows are capable of similar production.. This may eliminate traditional whey as a source of lactoferrin.

. More recently lactoferrin has been alleged to have a number of other physiological and biological functions ( Brock, 1995; Kussendrager, 1993; Lonnerdal and Iyer, 1995; Adamik and Wlaszczyk, 1996).

Although human and bovine lactoferrin differ, there is increasing evidence that both serve similar biological functions (Adamik and Wlaszczyk, 1996; Shidonda, et. al, 1996)

The suggested biological activities of lactoferrin, in addition to its antimicrobial activity, include:

-ion transport (Nagasako, et al., 1993)

-antiviral activity (Ellison and Giehl, 1991; Shimizu, etal. , 1996; Marchetti, etal, 1996.)

-enhanced antimicrobial activity of peptides derived from lactoferrin ( lactoferricin)

-toxin binding properties ( Kawakasi, etal., 1992:Giugliano, et al., 1995; Mattsby-Baltzer, et al.,

1996)

-promotion of growth of some animal cells ( Nicols, and McKee, 1990;Burrin, et. al, 1996; Mita, et

al. , 1996)

-platelet binding (Quian, et al, 1995)

-immunomodulating effects ( Brock, 1995; Konig, et al. , 1995; Shinoda, et. al., 1996)

-participation in local secretory immunity in synergism with immunoglobulins and other protective

factors (Ellison and Giehl, 1991;)

-wound and wound healing(Bockman and Guidon, 1996;

-anti-inflammatory (Mattsby-Baltzer, etal., 1996)

Receptors for lactoferrin have been detected and isolated on activated T and B cells, monocytes, intestinal brush border cells, platelets and neoplastic cells (Adamik and Wlaszczwk, 1996). Very low physiologic serum levels of lactoferrin increase upon infection and in some rheumatoid patients.

Antiviral Activity:

Lactoferrin has been shown to be effective in protecting against a number of different viruses (Marchetti, etal.,

1996; Shimizu, etal., 1996 )

Marchetti, etal. (1996) reported that both human and bovine lactoferrin were effective against the herpes simplex virus type 1 (HSV-1) by inhibiting adsorption of the virus. This activity was independent of the iron with-holding, since both iron saturated and apolactoferrin were equally effective.

Lactoperoxidase

Lactoperoxidase is a minor protein that has been reported to have antimicrobial activity. It is produced commercially in Belgium under the French dairy group CLE, a subsidiary of Lacto Bretagne Associes by an ion exchange process and may also be produced in New Zealand. It is reportedly used as an anti-caries agent in tooth paste.

Lactoperoxidase is a glycoprotein consisting of a single peptide chain with a molecular weight of 78, 431 Dal. It has 15 half-systemic residues and a much higher isoelectric point (pH 9.2) than most of the other whey proteins. The carbohydrate content is about 10%, structured into four or five potential binding sites. Partial loss of some of the glycosidic components during isolation has been attributed to its electrophoretic heterogeneity. The enzyme contains a haeme structure, with 1 iron molecule per mole of lactoperoxidase. The conformation of the protein is stabilized by a strongly chelated calcium ion.

Applications of lactoperoxidase include:

Product	LPO system	Mode of action	Results
raw milk	natural	preserving	4 days at 4 C
raw milk	SCN/hydrogen peroxide	shelf life	3 days at 10 C
past. Milk	SCN/hydrogen peroxide	shelf life	21 days at 10 C
cheese milk	SNC/hydrogen peroxide	shelf life	8 days at 4-7 C
yogurt	LPO	acidity control	14 days at 20 C
Emulsions	LPO/KI/GO	preserving	14 days at 20 C
Cosmetics	LPO/KI/SCN/GO	preserving	2-4 months
Dentifrice	LPO/SCN/LYS/GO	healing	daily
Ophthalmic	LP/KI/SCN/GO	protectant	1 week
Anti-tumor	LPO/GO/Antibodies	healing	periodical

GO = glucose oxidase; LYS = lysozyme; KI = potassium iodide

Applications are being found in addition to use as antibacterial agents and the use of the LPs in cosmetics, ophthalmic solutions, dental and wound treatment, and as anti-tumor and anti viral agents are of particular interest.

Godfrey, et.al. (1990) found that there was a critical combination of LPO, glucose, glucose oxidase (GO), iodide and thiocyanate to be effective in cosmetics. The treatment was effective against a range of yeasts, fungi and viruses, as well as bacteria for periods of up to 4 months.

Poulson (1986) patented a process for using a lactoperoxidase system for dental and wound treatment. Hoogedoorn ( 1985) used LPs in toothpaste or a mouthrinse to reduced acid formation by oral microoganisms. Clinical studies have supported the possibility that plaque accumulation, gingivitis and early onset carries may be reduced by appropriate LPs preparations.

LPO, together with glucose oxidase and monoclonal antibodies, have been applied in tumor therapy (Stanilawski, etlal., 1989;Lefkowitz, et.al., 1990). Such treatments may also possibly delay or eliminate the human virus (HSV I) (Courtois, et.al., 1990) -- or possibly decrease the transcription of human immunodeficiency virus (HIV)-coded protein (Pourtois, et.al. , 1990)

There has been a suggestion the lactoperoxidase acts synergistically with lactoferrin (Reiter, 1985); secretory IgA (Tenovuo, 1985) and lysozyme (Roger, etal., 1994)