FR2656311A1 - INHIBITORS OF LIPOLYTIC ENZYMES. - Google Patents

Abstract

Enzyme inhibitor comprising as an active ingredient at least one basic protein, one basic polypeptide or their salts. The inhibitor is useful as a dietary agent for the prevention of obesity and lipemia as well as a food additive.

Description

INHIBITORS OF LIPOLYTIC ENZYMES

  The subject of the invention is enzyme inhibitors which participate in lipolysis. More particularly, the subject of the invention is lipolytic enzyme inhibitors which comprise base proteins and / or

basic polypeptides.

  Many publications have mentioned that proteins such as serum albumin, β-lactoglobulin and certain soy proteins inhibit certain types of lipases (see, for example, Journal of Lipid Research, vol., 1984, pages 1214-1221). bile acids, these proteins lose their lipase inhibiting activity and do not exert in vivo their

role of lipase inhibitor.

  Continuous tests carried out on protein lipase inhibitors have shown that the basic proteins, the base peptides and their salts inhibit or suppress the activity of lipolytic enzymes in the presence of

bile.

  Thus, the invention provides a lipolytic enzyme inhibitor which comprises as an active ingredient at least

  a base protein, a base peptide or their salts.

  The expression "lipolytic enzyme inhibitor" used hereinafter refers to an agent whose function is to inhibit or suppress the activity of lipolytic enzymes such as lipases, thereby inhibiting or suppressing the hydrolysis of lipids. which results in inhibiting or suppressing the absorption of lipids by the intestine. The lipolytic enzyme inhibitors according to the present invention are effective under the conditions where the lipids are emulsified in the presence of bile acids.

  and so they are effective in vivo.

  The basic proteins, basic polypeptides and their salts which can be used according to the invention include the purothionines contained in wheat; purothionin-like products, contained in cereals other than wheat (including barley and rye) such as purothionine-like polypeptides widely distributed in barley as described in the Japanese patent published under NO 57840/1986 and the purothionin-like polypeptides found in rye as reported by J Agric Food Chem Vol. 26, No. 4, pages 794-796 (1978); protamine; histone;

  polylysine; lapolyarginine and their salts.

  Three types of purothionines, purothionin a 1, a 2 and B have some differences in the amino acid sequence each containing the following amino acid sequences.

  purothionines according to the present invention.

I 5 10 15 20 25

  Purothionine Ni H'N-Lys-Ser-Cys-Cys-Arg-Ser-Thr-Leu-Gly-Arg-Asn-Cys-TyrAsn-Leu-Cys-Arg-Ala-Arg-Gly-Ala-G In-Lys Leu-Cys

  Purothionine "2 11 {N-Lys-Ser-Cys-Cys-Arg-Thr-Thr-Leu-Gly-Arg-Asn-Cys-Tyr-Asn-Leu-Cys-Arg-Ser-Arg-Gly-Ala-Gln-Lys Leu-Cys

  Purothionine H 2 N-Lys-Ser-Cys-Cys-Lys-Ser-Thr-Leu-Gly-Arg-Asn-Cys-TyrAsn-Leu-Cys-Arg-Aia-Arg-Gly-Ala-Gln-Lys- Leu-Cys

35 40 45

  -Ala-Gly-Val-Cys-Arg-Cys-Lys the-Ser-Ser-Gly-Leu-Ser-Cys-Pro-Lys-G Iy-PhePro-Lys-COOH -Ser-Thr-Va I-Cys- Arg-Cys-Lys-Leu-Thr-Ser-Gly-Leu-Ser-CysPro-Lys-Gly-Phe-Pro-Lys-COOH-Ala-Asn-Vα-Cys-Arg-Cys-Lys-Leu-Thr- Ser-GlyLeu-Ser-Cys-Pro-Lys-Asp-Phe-Pro-Lys-COOH Three types of protamines including mono-, di- and tri-protamines and five types of histones including Hi, H 2 A, are known. H 2 B, H 3, H 4 and H 5 All of these types can

  be used in the present invention.

  Polylysine is distinguished by the location of the peptide bond and consists of the e-polylysine represented by the formula (I)

{NH- (CH 2) 4-CH (I)

  NH 2 N is the degree of polymerization of lysine and apolylysine represented by formula (II)

4 NH-CH-COI

(CH 2) 4 (II)

  The polylysines and their salts can be used according to the invention. E-polylysine and its salts are preferred because they can maintain in vivo the function which suppresses or inhibits lipolytic enzymes. over a long period of time, thus allowing a lower absorption of total lipids compared to α-polylysine and other basic proteins, base polypeptides and their salts. The polylysines of formulas (I) and (II) denote 4 or more, more particularly 5 or more, are more effective due to their higher activity of inhibiting the lipolytic enzyme. The e-polylysines o N has a lower value at 9 have weak antimicrobial activity Thus, o-polylysines where N is 5 to 9 will have an effect on the inhibition of lipid absorption

  without damage to the intestinal flora.

  The basic proteins constituting the amino acids and the peptides include two types of optical isomers, the L and D forms. The basic proteins and the basic peptides derived from natural products are known as usually consisting of L amino acids. protein and the basic polypeptides and their salts used according to the invention may consist of amino acids either L or D. The lipolytic enzyme inhibitors according to the present invention may contain a basic protein,

  a base peptide and their salts alone or in combination.

  The lipolytic enzyme inhibitors according to the invention can be administered to humans and to various animals including domestic animals and poultry, such as cattle, horses, chickens.

  as well as house animals such as dogs and cats.

  The effective doses vary according to the type, age and physical conditions etc. of the subjects to whom they are administered. Preferably, they may be administered to any

  appropriate dose according to the individual subjects.

  The lipolytic enzyme inhibitors according to the invention are formulated into a preparation for oral administration They can be administered either alone or in admixture with a vehicle usually used in the pharmaceutical industry or in combination with other medicaments. can use them in all forms of preparation such as tablets,

  granules, capsules, powders and similar products.

  In addition, the lipolytic enzyme inhibitors according to the invention can also be administered as food additives. Thus, the inhibitors according to the invention are useful as food or feed additives. Administration of the lipolytic enzyme inhibitors according to the invention to humans and animals can inhibit lipolytic enzymes to suppress or inhibit lipid hydrolysis so that rapid intestinal absorption of ingested lipids can be inhibited. it is possible to control the absorption of total fats to keep them at a low level thus making it possible to obtain results such as the prevention of lipemia and obesity. Thus, the inhibitors according to the invention are useful as dietetic agents for

  prevention of obesity and lipemia.

  Among the lipolytic enzyme inhibitors of the invention, the epolylysines more particularly maintain their action which inhibits the absorption of lipids in the living being for a long period of time, which results in a strong reduction of

  absorption of total fats in vitro.

  The invention will be better understood by means of

non-limiting examples below.

EXAMPLE 1

  (Preparation of purothionine) A mixture of crude purothionine containing the purothionines al, a 2 and B, is obtained from the wheat flour by the method described in Agr Biol Chem, Vol. 34, NO. 7, pp. 1089-1094 (1970) The resulting crude mixture is purified to isolate the 2-purothionin and the

purothionine B, respectively.

  (Preparation of emulsion of olive oil).

  250 mg of olive oil, 21.5 mg of sodium cholate as a bile acid and 30 mg of phosphatidylcholine are added to 5 ml of a 300 mM potassium phosphate buffer solution at p H 6.8 (called

  hereinafter potassium phosphate buffer).

  The mixture is sonicated to prepare

an emulsion of olive oil.

  (Preparation of a solution containing a protein (polypeptide) One ml of potassium phosphate buffer is added to each 4 mg of a mixture of crude purothionine, purified purothionin 2, purified purothionin B, bovine serum albumin ( hereinafter referred to as "BSA", A 7030, Sigma Co., Ltd., USA), B-lactoglubulin (L 0130, Sigma Co, Ltd) and egg albumin (A 5503, Sigma Co Ltd) for preparing six types of solutions containing a protein (polypeptide) (Preparation of a lipase solution) Porcine pancreatic lipase (manufactured by Sigma Co., Ltd) is added to the potassium phosphate buffer solution to prepare a lipase solution containing

  per ml 100 units of porcine pancreatic lipase.

  (Test of the activity of proteins inhibiting lipase) The control and six samples to be tested are

prepared in the following manner.

  Al of an emulsion of prepared olive oil

  as indicated above are used as support.

  of potassium phosphate buffer are added to the support to prepare a control emulsion sample. Each of the 50 μl of protein-containing solutions (polypeptide) prepared above is added to 100 μl of support to prepare six sample emulsions. test All emulsions are incubated for 5 minutes To each of these emulsions is added 50 ml of a lipase solution prepared as indicated above The resulting mixture is incubated at 370 C for 1 hour and then they are added 3 ml of a extraction solvent (chloroform:

methanol: n-heptane = 49: 1: 49).

  After stirring for 5 minutes, the mixture is centrifuged at 3000 rpm for 5 minutes. The upper layer is removed by means of a vacuum cleaner and 1 ml of a copper reagent (0.45 M triethanolamine) is added to the remaining solution. 0.05 N acetic acid, 3.4% copper sulfate pentahydrate and 20% sodium chloride). The mixture is stirred for 5 minutes and centrifuged at 3000 rpm for 5 minutes. 0.5 ml of solution is taken from the top layer and 0.5 ml of a color developing agent (a solution) is added to this solution. 0.1% bathocupron and 0.05% butyl

  hydroxyanisole in the above extraction solvent).

  The absorbance at 480 nm (Abs 480) is measured on the control solutions and the test solutions, using a photometer to determine the amount of free acids formed, ie the activity of the lipase.

Pancreatic porcine.

  The results are shown in Table 1 where the activity is expressed as a percentage of Abs 480 in the case where the control sample is hydrolyzed by

pancreatic lipase.

Table 1

  Protein (polypeptide) Activity of the lipase contained in the porcine pancreatic isoelectrococcal sample (%) Control 100 crude purothionins approx. 10 6.0 purothionine at 2 approx. 10 O purothionin B env 10 O egg albumin ca 4-5 107 It can be seen from the results in Table 1 that lipase activity is strongly inhibited when the inhibitor according to the invention comprises crude purothionines or a purified purothionin a 2 or a purified purothionin B is added in the presence of an acid

  of bile, while egg albumin, B-

  lactoglobulin and BSA, which do not belong to the basic proteins do not have lipase inhibiting activity in the

  conditions specified in this example.

EXAMPLE 2

  The activity of porcine pancreatic lipase was monitored in the same manner as in Example 1 except that the solution containing crude purothionin or purothionin B was diluted to a protein concentration of 0.2 mg / ml L The lipase activity of the sample containing crude purothionin was 97.5%, indicating that crude purothionin in such a concentration exhibits a low activity of inhibiting porcine pancreatic lipase. On the contrary, the lipase activity of the sample containing β-purothionine was 0% thus showing that β-purothionine is able to strongly inhibit the activity of porcine pancreatic lipase even at such a concentration. In addition, in a separate test implemented a

  similar to that indicated above, the B-

  purothionine was able to reduce the activity of porcine pancreatic lipase to 5.2% even at a concentration of 0.04 mg / ml showing its high

porcine pancreatic lipase.

EXAMPLE 3

  (Preparation of a cholesterol oleate emulsion) 32.5 mg of cholesterol oleate and 25 mg of phosphatidylcholine and 21.5 mg of sodium cholate are added to 5 ml of potassium phosphate buffer The solution is subject ultrasound to prepare a

  cholesterol oleate emulsion.

  (Preparation of a solution containing the protein) According to the method indicated in Example 1, four solutions containing protein are prepared, each of these solutions containing the crude purothionines, BSA,

  B-lactoglobulin or egg albumin.

  (Preparation of cholesterol esterase solution) A cholesterol esterase isolated from the porcine pancreas is added to 300 mM potassium phosphate buffer solution to prepare an enzyme solution

  containing 40 μg / ml of cholesterol esterase.

  (Control of Cholesterol Esterase Inhibitory Activity of the Protein) One control and four test samples are prepared as follows. Al of an emulsion of prepared olive oil

  as above are used as support.

  M1 of a potassium phosphate buffer solution are added to the support to prepare a control emulsion. Each 50 μl solution containing the protein (polypeptide), prepared as indicated above is added to 100 μl of support to prepare four sample emulsions. to test All emulsions are incubated for 5 minutes Each of these emulsions is

  add 50 41 of lipase solution prepared as above.

  The resulting mixture is incubated at 370 ° C. for 1 hour and then 3 ml of extraction solvent used in Example 1 are added. After stirring for 5 minutes, the mixture is centrifuged at 3000 rpm for 5 minutes. The upper layer is removed with an aspirator and 1 ml of copper reagent used in Example 1 is added to the remaining solution. The mixture is stirred for 5 minutes.

  minutes and centrifuged at 3000 rpm for 5 minutes.

  0.5 ml of a solution is taken from the top layer and 0.5 ml of the developing agent is added to this solution.

  the color used in Example 1.

  The resulting solutions used as a control and test to be tested are measured for absorbance at 480 nm (Abs 480) using a photometer to determine the amount of free fatty acid formed, i.e.

of cholesterol esterase.

  The results are shown in Table 2 where the activity is expressed as a percentage of Abs 480 when the control sample was hydrolyzed by

cholesterol esterase.

  Table 2 Protein (polypeptide) contained in the sample Cholesterol esterase activity (%) Control 100 crude purothionins 9.8 egg albumin 93.6 B-lactoglobulin 89.9

BSA 120.0

  The results shown in Table 2 show that cholesterol esterase activity is strongly inhibited when the inhibitor according to the invention contains crude purothionines, that is to say when the base protein has been added in the presence of bile acid, whereas egg albumin, B-lactoglobulin and BAS not belonging to the basic proteins have no or low esterase inhibiting activity of

cholesterol, under these conditions.

EXAMPLE 4

  (Preparation of an olive oil emulsion) 250 mg of olive oil, 21.5 mg of sodium cholate as acid component of the bile and 30 mg of phosphatidylcholine are added to 5 ml of a Tris buffer solution of 200 M at a pH of 6.8 (hereinafter referred to as "Tris buffer"). The mixture is sonicated to prepare

an emulsion of olive oil.

  (Preparation of a solution containing the protein or peptide) 10 ml of Tris buffer are added to 1 mg of each of the following compounds: Purified purothionine used in Example 1, protamine (P 4005, Sigma Co, Ltd, US A) , histone H 2 A (H 6881, Sigma Co., Ltd), histone H 3 (H 4380, Sigma Co, Ltd), α-poly-L-lysine (3075, Peptide Research Co., Ltd.) to prepare six types of solutions

  containing a protein or a peptide.

  (Preparation of lipase solution) Porcine pancreatic lipase (manufactured by Sigma Co., Ltd) is added to Tris buffer to prepare an enzyme solution containing 100 units of lipase

Pancreatic swine per ml.

  (Control of lipase inhibiting protein activity) The control and the six samples to be tested are

prepared in the following manner.

  100 Ml of olive oil emulsion prepared as

  indicated above are used as support.

  1 μl of Tris buffer are added to the support to prepare a control emulsion 50 ml portions of solution containing the protein (polypeptide) prepared as above are added to 100 μl of support to prepare six test emulsions All emulsions are incubated For 5 minutes each of these emulsions is added 50 to 4 l of a lipase solution prepared as above. The resulting mixture is incubated at 370 ° C. for 1 hour and then 3 ml of extraction solvent used in the suspension are added. EXAMPLE 1 After stirring for 5 minutes, the mixture is centrifuged at 3000 rpm for 5 minutes. The top layer is removed with an aspirator and to the resulting solution is added 1 ml of the copper reagent used in Example 1. The mixture The mixture is stirred for 5 minutes and centrifuged at 3000 rpm for 5 minutes. 0.5 ml of solution is taken from the top layer and 0.5 ml of agent developing the color is added to this solution. eur

used in Example 1.

  The control solutions and test samples thus obtained are monitored to measure their absorbance at 480 nm (Abs 480) using a photometer to determine the amount of free fatty acids formed, i.e.

  the activity of porcine pancreatic lipase.

  The results are shown in Table 1 where the activity is expressed as a percentage of Abs 480 when the control sample is hydrolyzed by

porcine pancreatic lipase.

Table 3

  Protein (polypeptide) Lipase activity contained in the porcine porcine pancreatic isoelectrical sample (%) Control 100 B-purothionin env 10 1,3 protamine env 10 1,3 histone H 2 A env 10 3,7 histone H 3 env The results of Table 3 show that lipase activity is strongly inhibited when the inhibitor according to

  the present invention comprises the following compounds:

  purified purothionine, protamine, histone, α-poly-L-lysine or poly-Larginine, a kind of basic protein or peptin added under the conditions specified therein

example.

EXAMPLE 5

  Two groups of 10 male SD rats (9 months old,

  weighing about 200 g) were prepared.

  g of corn oil, 6 g of egg yolk lecithin and 12.5 g of glycerin are added to the distilled water to make up to 100 ml. The mixture is

  sonicated to prepare an emulsion.

  The rats of the first group are given orally, using a gastric tube, 2 ml of emulsion and 100 mg of e-poly-L-lysine manufactured by Chisso Corp (containing dextrin and e-poly-L). -lysine in a weight ratio of 1: 1 and having the formula (I) where N is about 30) Blood is removed from the tail vein at predetermined intervals, and the serum concentration is measured as triglycerides, using the kit "Kyowa Medics Enzyme Kit TG" and the average value (mg / dl) per animal, calculated on the basis of the serum triglyceride concentration before

  administration (taken as 0 mg / dl) (according to the invention).

  The rats of the second group are fed orally with 2 ml of emulsion and 50 mg of dextrin. The serum triglyceride concentration is measured at predetermined intervals and the calculated average value is determined in the same way as above, for example

witness.

  The results are shown in Table 4 below.

Table 4

  Sancuine concentration in neutral fat (ml / dl) Time elapsed since administration <hi According to the invention Control O (before administration) OO 1 3,3 2,, to 176,8 16,3 2 32,4 4 ,, a 124.1 25.2 3 16.7 3.2 a 79.6 11.0 4 11.1 3, Oa 62.0 10.5 7 4.2 1, Oa 17.3 3.0 a : Significantly different at P <0.01 As shown by the results in Table 4, in the case of rats administered a sample containing e-poly-L-lysine (a base peptide according to the invention), Intestinal lipid absorption is inhibited and the serum triglyceride concentration can be kept low for a period of time from onset to 7 hours after administration, resulting in very low total fat absorption. These results indicate that e-polylysine exerts an in vivo inhibition activity of

  the lipolytic enzyme for a long period of time.

EXAMPLE 6

  (Preparation of peptide-containing solutions) Two solutions containing peptide are prepared by adding 1 mg of distilled water to each 1 mg of α-poly-L-lysine hydrochloride (3075, Peptide Research Co., Ltd) and hydrochloride. a-poly-D-lysine (P 7886, Sigma

Co., Ltd).

  (Polylysine assay for its lipase inhibiting activity) One control and two test samples were

prepared in the following manner.

  M1 of an olive oil emulsion prepared in the same manner as in Example 4 was used as a carrier. 50 μl of Tris buffer was added to the support to prepare a control emulsion. To each 100 μl of support was added 50 μl of solution containing the polypeptide prepared as above to obtain two emulsions to be tested. After incubation of the emulsions for 5 minutes 50 μl of enzyme solution prepared as in Example 4 were added. The resulting mixtures were incubated at 370 ° C. for one hour. Then, the incubated mixtures were treated and their activity was measured on porcine pancreatic lipase.

  in the same way as in Example 4.

  The results are shown in Table 5 below.

  Table 5 Polylysine contained in Activity on pancreatic lipase porcine samples (%) Control 100 a-poly-L-lysine hydrochloride 1, 6 a-poly-D-lysine hydrobromide 1,1 The following figures can be seen: above that polylysines both L and D can strongly inhibit the activity of lipase under the conditions specified in

this example.

EXAMPLE 7

  (Preparation of peptide-containing solutions) Lysine oligomers having a degree of polymerization of 2 to 5 were prepared as shown in Table 6, using a peptide synthesizer (Biolynx 4170, LKB-Pharmacia Co., Ltd.) Each of the oligomers was purified by high performance liquid chromatography To each of the oligomers was added distilled water to make up to 1 pmol / ml. Seven solutions containing

  the lysine oligomer were prepared.

  (Test of lysine oligomer lipase inhibition activity) The control and the six samples to be tested were

prepared in the following manner.

  Al olive oil emulsion prepared in the same manner as in Example 4 were used as

support.

  1 g of Tris buffer were added to the support to prepare a control emulsion. Each 50 μl of solution containing the lysine oligomer prepared as indicated above was added with 100 μl of support to prepare six test emulsions. After incubation of the emulsions for 5 minutes. 50 ml of lipase solution containing 100 units of porcine pancreatic lipase per ml prepared in the same manner as in Example 4 was added. The resulting mixture was incubated at 370 ° C.

during one hour.

  Then, the incubated mixtures were processed to measure their activity on the porcine pancreatic lipase of

  the same way as in example 4.

  The results are shown in Table 6.

Table 6

  Oligomer contained lysine Pancreatic lipase activity in porcine samples (%) Control 100 a- (L-lysine) 2 103.6 a- (L-lysine) 4 103, 8 a- (L-lysine) 5 2.1 E - (L-lysine) 3 89.0 e- (L-lysine) 4 53.8 e- (L-lysine) 5 3, 1 The above figures show that oligomers

  lysine having a degree of polymerization of 5 or more

  above 5 strongly inhibit the activity of the lipase, whereas oligomers with a degree of polymerization of 4 or less do not have a lipase inhibiting activity under the conditions specified in the Example In addition, the oligomers of E-type lysine with a degree of polymerization of 4 has a lipase inhibiting activity but this activity is not sufficient while the oligomer with a degree of polymerization of 5 has a high lipase inhibiting activity. That the lysine oligomer having a higher degree of polymerization has a higher lipase inhibiting activity than lysine having a lower degree of polymerization. It has been reported that oligomers of e-lysine with a degree of polymerization of about 10 or higher possess high antimicrobial activity. As a result, as well as the results shown in Table 6, it is concluded that the e-lysine oligomers having a degree of polymerization of 5 to 9 and which do not exert an adverse effect on the intestinal flora can be used more effectively

  as a lipolytic enzyme inhibitor.

Claims (12)

  A lipolytic enzyme inhibitor comprising as active ingredient at least one basic protein, a base polypeptide or their salts.   Inhibitor according to claim 1, wherein the base protein is chosen from histones and protamines. An inhibitor according to claim 1 or 2 wherein the basic protein is selected from the group consisting of   histones H1, H2A, H2B, H3, H4, H5, the mono-, di- and tri-   protamine. An inhibitor according to claim 1, wherein the basic polypeptide is selected from the group consisting of purothionine, purothionin analogue, polylysine, polyarginine and mixtures thereof.   An inhibitor according to claim 1, wherein the basic polypeptide is selected from the purothionins al, a 2 and B. The inhibitor according to claim 1, wherein the base polypeptide is selected from a and B-polylysine and their mixtures.   An inhibitor according to claim 1, wherein the basic polypeptide is an α-lysine oligomer having a degree of polymerization of 5 or more, an ε-lysine oligomer having a degree of polymerization of 5 or more or mixtures thereof. Inhibitor according to claim 1, wherein the salt of the basic polypeptide is chosen from hydrochloride   a-poly-L-lysine and α-poly-D-lysine hydrobromide.   A dietary agent comprising a lipolytic enzyme inhibiting substance selected from the group consisting of a basic protein, a base polypeptide and their salts. Dietary agent according to claim 9, wherein the inhibiting substance is chosen from histones, protamines and their mixtures.   11. The dietetic agent according to claim 9, wherein the inhibiting substance is chosen from the histones Hi, H 2 A,   H 2 B, H 3, H 4, H 5 and mono-, di and tri-protamines.   12 dietary agent according to claim 9 o the inhibiting substance is selected from purothionine, purothionine analog, polylysine, polyarginine and their mixtures.   13 The dietetic agent according to claim 9, wherein the inhibiting substance is selected from the purothionines of 1, 2 and B. The dietary agent according to claim 9, wherein   inhibiting substance is selected from a and B-polylysines.   An inhibiting agent according to claim 9 wherein the inhibiting substance is an α-lysine oligomer having a   degree of polymerization of 5 or more, an oligomer of e-   lysine having a degree of polymerization of 5 or more their mixtures.   Dietetic agent according to claim 9, wherein the inhibiting substance is α-poly-L-lysine hydrochloride.   or α-poly-D-lysine hydrobromide.   A food additive or food comprising a lipolytic enzyme inhibiting substance selected from a basic protein, a base polypeptide and salts thereof. 18 Additive according to claim 17 o the inhibiting substance is chosen from histones and protamines. 19 Additive according to claim 17 o the inhibiting substance is chosen from the histones Hi, H 2 A,   H 2 B, H 3, H 4, H 5 and mono-, di and tri-protamines.   Additive according to claim 17, wherein the inhibiting substance is chosen from purothionine,   purothionine analogue, polylysine and polyarginine.   An additive according to claim 17, wherein the inhibiting substance is selected from the purothionines 1, 2 and B. The additive according to claim 17, wherein   inhibiting substance is α or β-polylysine.   Additive according to claim 17, where the   inhibiting substance is selected from the oligomers of   lysine having a degree of polymerization of 5 or more, the oligomers of elysine having a degree of polymerization of 5 or more and their mixtures.   24 Additive according to claim 17 o the inhibiting substance is a-poly-L-lysine hydrochloride   or α-poly-D-lysine hydrobromide.