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    LYSOZYME

    First draft prepared by
    Dr G.J.A. Speijers and Mrs M.E. van Apeldoorn
    National Institute of Public Health and Environmental Protection
    Laboratory for Toxicology
    Bilthoven, The Netherlands

    1.  EXPLANATION

         Lysozyme has not been previously evaluated by the Joint FAO/WHO
    Expert Committee on Food Additives.

         Lysozyme is an enzyme that consists of 129 amino acids
    cross-linked by 4 disulfide bridges. Lysozyme is found in animal
    tissues, organs and serum as well as in tears, milk and cervical
    mucus. The major commercial source is hen egg white. Lysozyme
    concentration in egg albumen is about 0.5% (Proctor & Cunningham,
    1988; Solchem Italiana, 1991).

         Lysozyme (as the hydrochloride) is used in cheese production to
    prevent "late blowing". This phenomenon is caused by the growth of
     Clostridium tyrobutyricum, a contaminant present in milk used for
    cheese production.  C. tyrobutyricum ferments the lactate resulting
    from fermentation of lactose, to produce carbon dioxide, hydrogen,
    butyric acid and acetic acid. In many cheeses, especially those that
    are pressed, accumulation of gases during the later stages of curing
    causes the cheese to "blow", literally to explode.

         No general oral toxicity studies with lysozyme were available.
    Therefore its biological properties were reviewed and the few
    studies in which data on toxicity of lysozyme were given are
    summarized under Section 2.1.

    2.  BIOLOGICAL DATA

    2.1  Biological properties

         Lysozyme can attack cell wall polysaccharides of different
    bacterial species, especially Gram positive bacteria, leading to
    rupture of the cell wall and killing of the microorganism (Proctor &
    Cunningham, 1988; Solchem Italiana, 1991).

         Lysozyme is known as an effective immunological agent. It is
    used in human therapy for the treatment of viral and bacterial
    infections. Lysozyme has analgesic properties (Bianchi, 1981;
    Bianchi, 1983; Bruzzese  et al., 1989; Proctor & Cunningham, 1988;
    Verhamme, 1985) and has been used as a potentiating agent in
    antibiotic therapy. Lysozyme is used in the prophylaxis and therapy
    of leukopenia induced by antiblastic and ionizing radiation.
    EDTA-tris-lysozyme solutions are effective in the treatment of
    coliform infections of the bladder in humans (Proctor & Cunningham,
    1988; Verhamme, 1985).

         The anti-inflammatory action of lysozyme consists in the
    neutralization of acid substances released in the inflammatory
    process. Stipulation of phagocytosis by lysozyme favours wound
    healing and regression of degenerative and necrotic processes.
    Lysozyme-mediated decrease of mast cell degranulation leads to
    reduction of histamine release and a subsequent anti-oedema effect
    (Verhamme, 1985; Verhamme  et al., 1988).

         It has been suggested that polysaccharides, glycopropteins and
    glycolipids of the cell membrane can be bound to lysozyme in a
    substrate-specific way. This has led to the hypothesis that lysozyme
    has a regulatory function in membrane-dependent cellular processes
    and in protection against membrane abnormalities associated with
    neoplastic transformation (Bregant  et al., 1990; Sava  et al.,
    1989; Verhamme  et al., 1988).

         Due to its protein nature, lysozyme has immunogenic properties
    and can provoke anaphylactic reactions (Le Moli  et al., 1986;
    Verhamme  et al., 1988). However, its potency seems to be of
    moderate degree and considerably lower than that of other proteins
    such as albumen and ovalbumin (Bianchi, 1982). Immunogenic
    properties of lysozyme have been well studied in various animal
    models. In such studies immunization in adjuvant was generally
    performed in order to reveal the immunogenicity (Bianchi, 1982;
    Colizzi  et al., 1985; Strossberg & Kanarek, 1970; Semma  et al.,
    1981a,b; Verhamme  et al., 1988).

         In man immunogenicity of hen egg lysozyme seems to be a minor
    problem. Treatment of cancer patients with a daily intravenous
    administration of lysozyme for a few weeks was without apparent
    adverse consequences (as cited in Bianchi, 1982). Exposure through
    the oral route generally results in tolerance to the compound and
    does not cause adverse effects (LodinovŠ & Jouja, 1977). Patients
    who suffer from adverse reactions after consumption of eggs most
    frequently show antibody response to one of the many protein
    components of egg white, but very rarely to egg-white lysozyme
    (Langeland & Aas, 1987).

    2.2  Toxicological studies

    2.2.1  Acute toxicity

         Lysozyme was tolerated orally at up to 4000 mg/kg bw in mice
    and rats. Intravenously administered lysozyme was tolerated at up to
    1000 mg/kg bw in mice and up to 2000 mg/kg bw in rats and rabbits
    (Bianchi, 1982).

    2.2.2  Short-term studies

    2.2.2.1  Rabbits

         Two groups of 10 male New Zealand rabbits were treated
    intravenously with 500 mg/kg bw lysozyme hydrochloride or 200 mg/kg
    bw albumen (both in 2 ml/kg bw of normal saline) 5 days/week for 4
    weeks. Four weeks after the last injection a second, similar, 4-week
    treatment period was started. Symptoms of anaphylactic reactions
    were scored. Twenty-four hours after the last injection haematology
    and blood biochemistry were carried out. All animals were examined
    macroscopically. During the first treatment period one rabbit of the
    albumen and one of the lysozyme groups died. During the 4-week
    interval between the two treatment periods, 2 more rabbits in the
    albumen group but and none in the lysozyme group died During the
    second treatment period rabbits in the lysozyme group showed a
    moderately increased depth and frequency of respiration, but they
    recovered quickly and survived the 4 weeks until the end of
    treatment. Rabbits in the albumen group showed severe anaphylactic
    reactions and some died immediately after injection. After injection
    of albumen all animals were profoundly prostrated, manifested
    irregular respiration and cyanosis of the snout and ears, refused to
    eat and drink and were unable to stand on their hind legs. All died
    within the second treatment period. The animals in the lysozyme
    group showed a normal behaviour and remained in good health. No
    effects on food or water consumption, defecation, haematology, blood
    biochemistry or macroscopy were observed (Bianchi, 1982).

    2.2.3  Special studies on immune responses

    2.2.3.1  Guinea-pigs

         Male guinea-pigs were immunized against lysozyme, hen egg white
    or ovalbumin by means of intraperitoneal injections on the 2nd, 4th
    and 6th day of 2 consecutive weeks with 6 ml/animal of solutions of
    the compounds (10 mg/ml); 30-35 days later the animals were
    challenged by intracardiac puncture with 1 ml/animal of 10 mg/ml of
    the antigen solutions. During the immunization period a number of
    animals in the egg white and ovalbumin groups died. Following the
    intracardiac challenge such severe anaphylactic reactions were seen
    in the egg white and ovalbumin groups that 40-75% of the animals
    died. Lysozyme did not cause any mortality and only in a limited
    number of the immunized animals were anaphylactic reactions seen. An
    anaphylactic shock was provoked by lysozyme in albumen- or
    ovalbumin-immunized guinea-pigs and  vice versa (Bianchi, 1982).

    2.2.3.2  Rabbits

         Groups of 10 rabbits were immunized intradermally against
    lysozyme, albumen and ovalbumin in Freund's complete adjuvant and
    challenged intravenously 30-35 days later with 1 ml of the 10 mg/ml
    solutions of the antigens. During the immunization period 2 animals
    in the lysozyme group and 4 animals in each of the albumen and
    ovalbumin groups died. After challenge in each group 4 animals died.
    A marked fall in platelet counts was observed, the percentage
    reduction being similar before and after challenge. Lysozyme was
    less toxic than albumen or ovalbumin, but the difference was not
    significant (Bianchi, 1982).

    2.3  Observations in humans

         Fifteen full-term and 18 premature infants were given egg-white
    lysozyme in the milk formula (10 mg/100 ml) from the 1st to the 8th
    week of age as substitute for the lysozyme in breast milk (2 mg/ml)
    to stimulate the production of immunoglobulins. Thirteen full-term
    and 13 premature artificially-fed infants, as well as 20 breast-fed
    infants, were followed as controls. Assuming that the infants
    consumed daily 600-900 ml of milk formula, daily intake of lysozyme
    in this study was 60-90 mg. The infants did not show ill-effects. No
    difference in the production of serum immunoglobulins between the
    lysozyme group and control group was seen. Secretory IgA was found
    in stool filtrates of full-term lysozyme-fed infants as well as in
    breast-fed controls. In other groups (full-term controls fed
    artificially without lysozyme, premature controls fed artificially
    without lysozyme and premature controls fed artificially with
    lysozyme) only traces of secretory IgA were detected in stool
    filtrates. Lysozyme feeding partly substituted for passive transfer
    of secretory IgA from maternal milk. No antibodies were found in
    serum of lysozyme-fed children (LodinovŠ & Jouja, 1977).

    3.  COMMENTS AND EVALUATION

         In studies related to allergenic effects, the reactions
    produced by egg-white lysozyme in animals and humans were less than
    those seen with other proteins, e.g., ovalbumin and albumin, which
    have a long history of use as food components.

         On the basis of the available data, the Committee concluded
    that the low additional intake of lysozyme via cheese was not a
    hazard to consumer health, provided that the enzyme complied with
    the specifications.

         Lysozyme is obtained from edible animal tissue commonly used as
    food and can thus be designated as a class I enzyme (Annex 1,
    reference 76, Annex III). It was therefore considered acceptable for
    use in food processing when used in accordance with good
    manufacturing practice.

    4.  REFERENCES

    BIANCHI, C. (1981) Is Fleming's lysozyme an analgesic agent? An
    experimental reappraisal of clinical data.  Eur. J. Pharmacol., 71:
    211-221.

    BIANCHI, C. (1982) Antigenic properties of hen egg white lysozyme
    (Fleming's lysozyme) and notes on its acute/sub-acute toxicity.
     Curr. Therap. Res., 31: 494-505.

    BIANCHI, C. (1983) Is Fleming's lysozyme an analgesic agent?
    Experiments on mice.  Clin. Exp. Pharmacol. Physiol., 10: 45-52.

    BREGANT, F., CESCHIA, V., PACOR, S. & SAVA, G. (1990) Reduction of
    MCa mammary carcinoma in mice fed with egg-white lysozyme.
     Pharmacol. Res., 22: 95-96.

    BRUZZESE, T., CEDRO, A., FANCIANO, C.A., BASILICO, L., ANGELI, A.,
    FERRARI, F., PATRINI, G. & GIAGNONI, G. (1989) Antinociceptive
    properties of lysozyme fragments.  Boll. Chim. Farmaceutico, 128:
    33-37.

    COLLIZI, V., PALMIERI, G., SETTE, A., APPELLA, E., DORIA, G. &
    ADORINI, L. (1985) Synthetic peptides in the analysis and regulation
    of delayed-type hypersensitivity to lysozyme.  Folia Biologica, 31:
    396-401.

    LANGELAND, T. & AAS, K. (1987) Allergy to hen's egg white; Clinical
    and immunological aspects. In: Brostoff, J. & Challacombe, S.J.
    (eds.) Food Allergy and Intolerance, London, BailliŤre Tindall, pp.
    367-374. (ISBN 0-7020-1156-8)

    LE MOLI, S., PAGANELLI, R., SIRIANNI, M.C., NISINI, R., QUINTI, I.,
    SEMINARA, R., D'AMELIO, R. & AIUTI, F. (1986) Gli effetti  in vitro
    del lisozima su alcune fuzioni specifiche e non specifiche del
    sistema immunitario.  Boll. Ist. Sieroterap. Milan, 65: 283-289.

    LODOVINú, R. & JOUJA, V. (1977) Influence of oral lysozyme
    administration on serum immunoglobulin and intestinal secretory IgA
    levels in infants.  Acta Pediatr. Scand., 66: 709-712.

    PROCTOR, V.A. & CUNNINGHAM, F.E. (1988) The chemistry of lysozyme
    and its use as a food preservative and a pharmaceutical.  CRC Crit.
     Rev. Food Sci. Nutr., 26: 359-395.

    SAVA, G., BENETTI, A., CESCHIA, V. & PACOR, S. (1989) Lysozyme and
    cancer: role of exogenous lysozyme as anticancer agent (review).
     Anticancer Res., 9: 583-592.

    SEMMA, M., AMANO, T., FUJIO, H. & SAKATO, N. (1981a) Suppression of
    hen egg-white lysozyme (HEL)-specific delayed-type hypersensitivity
    responses in mice by suppressor T cells after neonatal
    administration of anti-idiotypic antibodies.  Microbiol. Immunol.,
    25: 1303-1315.

    SEMMA, M., SAKATO, N., FUJIO, H. & AMANO, T. (1981b) Regulation of
    delayed-type hypersensitivity (DHT) response to hen egg-white
    lysozyme (HEL). Induction of suppressor T-cells with soluble HEL
    derivative peptide in A/J mice.  Immunol. Letters, 3: 57-61.

    SOLCHEM ITALIANA (1991) Toxicological evaluation of lysozyme.
    Dossier provided by Solchem Italiana S.p.a. Italy.

    STROSSBERG, A. & KANAREK, L. (1970) Immunochemical studies on hen's
    egg-white lysozyme. The role of the lysine, the histidine and the
    methionine residues.  Eur. J. Biochem., 14: 161-168.

    VERHAMME, I. (1985) Klinisch en farmaceutisch belang van lysozyme.
     Farm. Tijdschr. Belg., 62: 339-346.

    VERHAMME, I., RACCHELLI, L. & LAUWERS, A. (1988) Lysozyme
    (N-acetylmuramyl-Ŗ(1 -> 4)glycanohydrolase EC 3.21.17).  Int.
     Pharm. J., 2: 129-132.


    See Also:
       Toxicological Abbreviations