AU606698B2

AU606698B2 - Agent for inactivating spores and process for prolonging the storage life of products, substances or produce which can be spoiled by bacterial spore infestation

Info

Publication number: AU606698B2
Application number: AU18604/88A
Authority: AU
Inventors: Christian Klug; Andreas Lotz; Gerhard Wohner
Original assignee: Hoechst AG
Current assignee: Hoechst AG
Priority date: 1987-07-03
Filing date: 1988-07-01
Publication date: 1991-02-14
Anticipated expiration: 2008-07-01
Also published as: DK367088D0; JPS6434906A; DE3722044A1; FI883140A; FI883140A0; ZA884728B; IL86944A0; EP0297598A3; EP0297598A2; NO882955D0; AU1860488A; PT87882A; DK367088A

Description

5) Signature of Applicant (S) or Seal of Company and S ignature: of Its Officera as prescribed by its Articles of Association.

DATED this.. aay HOECHST AKT.E.NG.E.SELLS CRAFT by -14 1 D. B. Mischlewski RegisteredPatent Attorney....

A

6 6 Form MONWVEALTH OF AUSTRALIAAf o06 PATENTS ACT 1952-69 COMPILETE 'SPEC IFICATION

(ORIGINAL)

Class I t. Class Application Number: Lodged: Pomplewe Specification Lodged: Accepted: Published: Priority Related Art: This document contains I.

ani~e.ndmeints made ttn. :r Section 49 and is curi-ect fcr cc ccc C cc Narie of Applicant: Aldress of Applicalit: Actual Inventor: Address for Seritice HOECHST AK72IENGESELLSC-HAFT 45 Bruningstrasse, D-6230 Frank furt /,Id in Federal Republic of Germany ANDREAS LOTZ, VCYERHARD WOHNER, CHRISTIAN KLUG EDWD. WATERS SONS, 50 QU;I0N STREET, MELBOURNE, AUSTRALIA, 3000.

Complete Spvcification for the Irivention entitled: AGENT FOR INACTIVATING SPORES AND PROCESS FOR PROLONGING 1HE STORAGE LIFE OF PRODUCTSj SUBSTANCES OR PRODUCE WHICH CAN BE SPOILED BY BACTERIAL SPORE INFESTATION The following statement is a fjull description of this invention, Including the best method of performing it known to To the Commissioner of Patents HOECHST AKTIENGESELLSCHAFT Prokurist Authorized SignatOry ppa. Isenbruck i.V. Lapice PAT 510

Y"I

ry 1.

1- HOECHST AKTIENGESELLSCHAFT HOE 87/F 191 Dr.KH/gm o o 000 co oo S0000 00 0000 0 00 0 0 o 0000 0 00 0 0 0 00 0 0 0 00 0 0 0 0 00 0 0 0 0 00 0 0 0 0 00 Description Agent for inactivating spores and process for proLonging the storage Life of products, substances or produce which can be spoiled by bacterial spore infestation The invention relates to an agent for inactivation of spores, the use of this agent and a process for prolonging the storage life of products, substances or produce which can be spoiled by bacterial spore in 'station. The agent contains, as active components, eithe, one or more 10 different amino acids or a) one or more synergists and b) one or more bacterialyzing enzyme products with Nacetylmuramidase activity.

In the preservation of foodstuffs by the action of tem- 15 perature (pasteurization and sterilization), the action of the heat destroys the vegetative microorganisms in the foodstuffs or damages them to the extent that they are no longer capable of development under the expected storage conditions. However, with preservation by heat treat- U0 mentthere is the danger that precisely the spores of various microorganisms which spoil foodstuffs are not destroyed if heating is inadequate and that these germinate at a later point in time and are tius indirectly responsible for the decay of the foodstuffs. On the other hand, in addition to complete destruction of spores, oo high a thermization often also leads to an impairment of the sensory and nutritional physiology properties (forexample a loss of vitamins) and in addition because of the high energy consumption is very costintensive.

Endospores, which are formed by so-called endosporeforming organisms, such as Bacillus species, in particular B. cereus, B. Licheniformis, B. subtilis, B. coagulans ;;i

I

0000 0000 Soooo 0 0 0 0 oo 0 00 0 A 6 0 0 00 0 00 V 00 2 or B. stearothermophilus or Clostridia species, in particular C. sporogenes, C. pasteurianum, C. botulinum or C.

perfringens, can be responsible, for example, for sweet curdling of milk (Bacillus cereus from A germ count of 10 7 /ml of milk upwards) or for decay of products containing meat (for example Clostridium sporogenes).

Bacterialyzing enzyme products with N-acetylmuramidase activity, such as, for example, Lysozyme, can be used for preserving foods-uffs, for example cheese, meat and fish products. Thr of lysozyme-like products from secretions of animals and plants for preventing late blowing in various types of cheese is described in French Patent 8,003,321.

Combination of non-enzymatic preservatives with bacterialyzing enzyme products which show N-acetylmuramidase activity, resulting in an action synergism which Leads to a prolonging of the storage life, has also already been proposed (German Patent Application P 3,704,004.9).

It has now been found, surprisingly, that both one or more amino acids and a combination of one or more synergists with one or more bacteriatyzing enzyme products with N-acetylmuramidase activity are excellent agents for inactivating spores.

The invention thus relates to: 1. An agent for inactivating spores, which ccntains, as active components, either a) one or more different amino acids or b) one or more synergists, together with one or more bacterialyzing enzyme products with N-acetyLmuramidase activity.

2. The use of the agent described under 1. as a preservative.

3. A process for prolonging the storage life of products, 00 0 0 0 9*4 St i i 1 i i i i i t

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i i i ii 3 substances or produce which can be spoiled by bctsrial spore infestation, which comprises adding to the products, substances or produce which can be spoiled by bacterial spore infestation an agent for inactivating spores as described under 1.

The invention, in particular in its preferred embodiments, is described in detail and defined in the patent claims below.

Active components are understood as the substances which are responsible for the actual action of the agent.

According to the invention, the agent or one or more constituents of the agent can be mixed, for example, with solvents, such as water, buffer solutions, such as carbonates, emulsifiers, such as mono- or diglycerides, 15 inactive carriers, such as hydrocolloids, for example starch or 9elatin, or flavor correctants with no specific action, such as glucono-delta-lactone.

Amino acids which can preferably be used are proteinogenic amino acids that is to say those which occur in proteins for example cysteine, alanine, tyrosine, in particular in their L-form, or glycine. Their physioloyically tolerated salts are also suitable.

In 'the context of the invention, synergists are understood as those substances which, apart from the amino acids mentioned below, do not themselves have a sufficiently preserving action, if any that is to say are not preservatives but which are capable of assisting enzyme products with N-acetylmuramidase activity if they are used together with them. Such synergists are, for example, complexing agents, such as citric acid or ethylenediaminetetraacetate (EDTA), and in addition also amino acids, in particular proteinogenic acids, such as cysteine, alanine, tyrosine and glycine, which can also be used as salts and in particular 'n thuir L-form, or r of 4 4 I 4 nucleosides and nucleotides, such as inosine or sine monophosphate, which are frequently used as flavor intensifiers, or phosphates, such as tetrasodium pyrophosphate (diphosphate), sodium tripolyphosphdte (triphosphate), poLyphospate (Graham's salt) or reddening agents, such as alkali-metal nitrites, in particular sodium nitrite, and alkali-metal nritrates, such as sodium nitrate or potassium nitrate.

I 10 The second component b) of the agent is at least one bacterialyzing enzyme product with N-acetylmuramidase a. activity. Preferred suitable enzymes are, for example, lysozymes produced by plants, animals or microbes, in particular from ovalbumin or from Streptomycetes, in particular from Streptomyces coelicolor DSM 3030 and its mutants or variants (called cellosyl below). The enzyme product from DSM 3030 is easily obtained by the process described in German Offenlegungsschrift 3,440,735. In this process, Streptomyces coelicolor DSM 3030 is cultured in a fermentation medium with the addition of S. sugarbeet molasses in an amount of 5 to 50 g, preferably to 20 g per liter of culture medium. A further increase in yield is achieved if calcium ions in the form of readily soluble non-toxic calcium salts, preferably in the form of the inexpensive calcium chloride, are added to the culture medium. A 0.05 to 1 molar calcium ion concentration is advantageous, and concentrations of 100 to 500 mmol, for example in the form of an addition of 0.2 to 0.5% by weight of calcium chloride dihydrate, are particularly preferred.

Inactivation of spores is understood as the ability of a substance, agent or process to prevent, or at least to delay, in particular Juring storage, germination of spores in products, substances or produce which can be spoited by bacterial spore infestation.

Spores in the context of the invention are understood as endospores and epospores (sic), in particular enospores (sic), that is to say those of the BaciLLus species and Clostridium species.

The active substances (either the amino acid(s) or synergist and enzyme) can be added at the same time or in succession to any products, substances or produce which can be spoiled by bacterial spore infestation, for example foodstuffs, such as, for example, meat and meat products, meat-containing products and ready-to-eat dishes, fish, crustacean, shellfish and mollusc products, edible gelatinous coating compositions for meat products, delicatessen products, liquid egg and liquid egg yolk, vegetable and fruit products, alcoholic and alcohol-free drinks, milk and milk products, fillings for pastries and bakery products, sweets and confectionery, and pharmaceutical and cosmetic products, feedstuffs, tobarco and tobacco products.

t' 20 The composition of the agent is chosen so that the concentration of the bacterialyzing enzyme product or prot ducts with N-acetylmuramidase activity based on the weight of the product to be preserved is at least 0.001% by weight, preferably 0.01% by weight and particu- Larly preferably 0.1% by weight, and not more than 3% by weight, preferably 1.5% by weight and particularly preferably 1% by weight. An enzyme content of 0.01% by weight approximately correspor's to 2,000 units/g of product.

The synergists used are preferably the substances Listed in the following Table 1. These can be used either individually or in any desired combinations. The concentration of these substances with preferred ranges with respect to the weight of the product to be preserved is given under the head "g "at least" and "not more than".

If the agent consists uf only one or more different amino I -i 6 acids, the concentration data given in Table 1 under the amino acids heading apply.

Table 1 at least not more than Synergist I II III IV VI C 3 ooo 0 0 0 0 o Qo 0 o 0 o o 0 0 0 0o0000 a ooa o S0 0 a O 0 0 o p S0 00 00 0 0 Amino acids: L-cysteine L-alanine L-tyrosine glycine Nucleosides or Nucleotides: 15 Inosine monophosphate Complexing agents:

EDTA

20 citrate Phosphates: diphosphate triphosphate polyphosphate Reddening agents sodium nitrite sodium nitrate potassium nitrate 100 50 50 100 5000 1500 1500 5000 1000 750 750 2500 500 150 150 500 ppm 11 "i 10 25 50 1000 500 100 10 50 100 5000 2500 5JO 0.001 0.001 0.01 0.01 0.01 1 1 1 0.005 0.005 0.05 0.05 0.05 5 5 5 0.01 0.01 0.1 0.1 0.1 10 10 1 10 5 5 5 1000 1000 1000 0.5 0.1 5 1 1 1 1 500 500 500 100 100 100 ppm ppm ppm

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II III V VI minimum concentration preferred minimum concentration particularly preferred minimum concentration maximum concentration preferred maximum concentration particularly preferred maximum concentration If expedient, the concentrations can also be below or in excess of these minimum and maximum conentrations.

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-1 ii r 0000 0 0 o0 o 0oo 0 0 0 00 0 00 o t o o t S0 0 1 0 06 z l/i 7 According to the invention, the agent can be composed either solely of one or more different amino acids or of in each case only one substance of the particular class synergist and bacterialyzing enzyme products with N-acetylmuramidase activity, and also of any desired combinations, for example one, two or more synergists and one, two or more bacterialyzing enzyme products with Nacetylmuramidase activity. The agent can likewise both be made up from the desired individual substances before 10 use or these can also be added separately from one another, i.e. first the synergist and then the enzyme or vice versa are added to the product in which the spores are to be deactivated.

The agent according to the invention can furthermore also be used in combinations with physical methods of preservation, in particular thermal preservation processes, such as pasteurization or sterilization. The agent can be added either before or after the heating process, but preferably at least the enzyme is added after the heating process.

The invention is illustrated in more detail in the following examples.

The following synergists were used in the embodiment examples: 1: 2: 3 4 6: L-Cysteine L-Alanine L-Tyrosine Glycine Inosine

EDTA

Diphosphate Polyphosphate Triphosphate Citrate Sodium nitrite -j *8 Example 1 In vitro sporolysis: In each case 9.9 ml. of an aqueous buffer solution (acetate or Tris Luffer, depending on the desired pH) which contained cellosyi and synergist at pH values of 6 and 7 were incubated with 0.1 ml of a spore suspension (B.cereus or B.licheniformis) which had a titer of 10 106 spores/mL of suspension. The concentration of the synergist per 10 in of solution was 1:100 ppm or 500 ppm; 2:150 ppm; 2 3: in each case 150 ppm; 4:500 ppm 5:100 ppm.

The concentration of the ceLlosyl was in all cases 0.1%, based on 10 mL of solution.

The reduction of the spores capable of germination was t in each case determined after an incubation time of 24 o and 48 hours by means of germ count determination.

In the samples with the pH values of 5 and 6, the spore reduction was in all cases about 1 (after 24 hours) and 2 (after 48 hours) log phases (1 Log phase a 90% reduetion; 2 log phases 99% reduction).

Example 2 In vitro long-term action Delay in spore germination 49.5 ml of meat extract caseine peptone broth which con- Stained cellosyl and the synergists 6-11 at pH values af 6 and 7 (acetate or Tris buffer) were inoculated with 0.5 ml of a spore suspension (B.cereus, B.licheniformis, titer 105 106 spores/ml of suspension). The cellosyl concentration based on 50 ml of solution was 0.1%.

The synergists were present in the solution in the -9following concentrations: 6: 0.1% 9: 7: 0.5% 10: 1% 8: 0.5% 11: 0.005% After 7 days the evaluation was carried out by means of germ count determination of the vegetative cells and by means of spore titer determination.

Result: CeLLosyl in combination with the synergists 6-11 at pH 5.0 and in part at pH 6.0 effects a prevention of the spore germination and thus a prolonging of the storage life. Spore germination is prevented for in excess of at li^ast 20 days by the combination of 0.1% cel osyl with ppm sodium nitrite, whilst the addition of sodium nitrite alone prevents gemination for only 5 days. Without any addition of substance, the spores germinate even after incubation for one day.

Example 3 Foodstuffs experiment sporolysis 10 mi of sterile milk were inoculated with B.cereus S.spores (titer about 10 spores/mt of milk). After addition of synergists 1-3 500 ppm; 2: 150 or 500 ppm; i 2+3: in each case 150 ppm), in each case in combination with 0.1% of cellosyl, the milk was kept at refrigeration temperature (7°C to 10 0 C) and then pasteurized (75 0

C,

minutes). The reduction of endospores was determined by germ count determinations. With the combined use of cellosyL with L-alanie (150 or 500 ppm), L-cysteine (500 ppm) and L-aLanine to ;-eter with L-tyrosine (in each case 150 ppm), the proportion of endospores in the final product is reduced by 90% to 99%, a prolonging in storage life of 1-2 days being achieved.

ExamPLe 4 Foods tuff s experiment deLa/ in Spore germination, A mea t-conta ining bread spread was pre;parpd in accrda' e with the foLLowing recipe., margarine 1.

vegetabLe f at (par tLy hardened) D sod ium case inate .7 l.

0 10 pork, cooked 25.2 o) pepper G I 0 0 The product was prepared in a3 process L ine.

Working cycke: -boiL meat melt fat and margarine- 0 0 a pre-warm cottLoid miLL wi tl hot wa ter 0 11 pre-emuLs if y hot water, caseine and fat -add comminuted meat, seasoning and saLt (processing temperature about 40-50 0

C)

-adjust pH to 5.0 to 6.0 with citric acid homogenize by means of the coltoid niU -sterilize at 300 to 116 0

C~

The bread spread thus prepared was then contaninated with B.cereus, B.Licheniformis and C.sporogenes spores Ctiter 2 /g of bread spread). Af ter addition of 0.05% ceLLosyL together with a) 50 ppm of sodium nitrite and b) 0.1'9 of poLyphosphate (the data reLate to the weight of the bread spread used), the products were kept at +10 0 C. The evaLuatin,,n war. carried out with the aid of germ count and spore titer determinations. With combineHi use of ceLLosyL with sodium. nitrite or cetlosyL with polyphosphate, germinatior of the endlospores couid be prevented for in excess of 14 days, whilst in control batches spore germination and an increase in the vegetative germ counts were aLready found after a few hours.

Claims

1. An agent .for irnact 4 'vating spores comprising as active components: t:osine and/or alanine, or one or more synergists as herein defined, both t;ogether with one or more bacterialyzing enzyme products with N-acetylmaramidase activity.

2. An agent as claimed in cl,,im 1, in which -the synergist or synergists are chosen from the group comprising: amin,- acids, sale;s thereof, complexing agents, phosphates, flavor intensifiers and reddening agents.

3. An agent as claimed in claim 1, in which -the synergist or synergists are chosen from the group Comprisiaq: alanine, tyrosine, inosine, 5'-1,nosine monophosphate, EDTA, citrate, diphosphat, tripho,.ph, te, p)olyphosphate and sodium nitrite.

4. An agent as claimed in claim 1, in which the bacterialyzing enzyme product or products with. N-acetylN inurridase activity are chosen from the group comprising ly~iozymes produced by plan animals or microbes.

An agent as claimed in claim 1, in which the bacterialyzing enzyme product or prodticts, with N-acetyl- muramidase activity are chosen from the group comprising: '1bacterialyzing enzyme products with N-acety'Imuramidase activity from Streptomycetes.

6. An a-gent as claimed in claim 4, In which the bacterialyzing enzyme product with N-acetylmuramidase Meib Disk 9/1.3 t4G R RA 4 Ui f (Ot11 mI l -12- activity originates from Streptomyces coelicolor DSM 3030 and/or mutants and/or variants thereof (cellosyl).

7. The use of an agent as claimed in claim 1 as a preservative in products, substances or produce whic i can be spoiled by bacterial spore infestation.

8. A process for prolonging the storage life of products, substances or produce which can be spoiled by bacterial spore infestation, which comprises adding to the products, substances or produce which can be spoiled by bacterial spore infestation an agent for inactivating spores S° as claimed in claim 1. o o Sn o 0 *o

9. The process as claimed in claim 8, in which the agent for inactivating spores as claimed in ,:laim 1 is added 0 to the productr, substances or produce !,hich can be spoiled by bacterial spore infestation in a concentration such that the amino azid(s) are present in a concentration of 10 to 5000 ppm or the bacterialyzing enzyme products with 00 N-acetylmuramidase activity are present in a concentration of 0.01 to 1.5% by weight, in each case based on the weight of the product to be preserved.

10. A process for the preparation of the agent as j claimed in claim 1, which comprises bringing either the amino acid(s) or the synergist or synergists and the bacterialyzing enzyme product or products with N-acetyl- muramidase activity into a suitable application form. DATED this 25th day of October, 1990. HOECKST AKTIENGESELLSCHAFT WATERMARK PATENT ATTORNEYS 2ND FLOOR "THE ATRIUM", 290 BURWOOD ROAD, HAWTHORN, VIC. 3122. Melb Disk 9/1 3 MG T- I j