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 (Lodinová & 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 (Lodinová & 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
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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, Baillière 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.
LODOVINœ, 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-ß(1 -> 4)glycanohydrolase EC 3.21.17). Int.
Pharm. J., 2: 129-132.
See Also:
Toxicological Abbreviations