AU602949B2

AU602949B2 - Bacteria-lysing enzyme product form streptomycetes, its preparation and its use for preserving cheese

Info

Publication number: AU602949B2
Application number: AU10710/88A
Authority: AU
Inventors: Christian Klug; Andreas Lotz; Erich Luck; Gerhard Wohner
Original assignee: Hoechst AG
Current assignee: Hoechst AG
Priority date: 1987-01-23
Filing date: 1988-01-22
Publication date: 1990-11-01
Anticipated expiration: 2008-01-22
Also published as: EP0280032A2; PT86606B; EP0280032A3; NZ223254A; PT86606A; IL85160A0; FI880262A; NO880279D0; DE3701835A1; IE880163L; DK31188A; AU1071088A; DK31188D0; FI880262A0; ZA88445B; JPS63192386A; NO880279L

Description

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COMMONWEALTH OF AUSTRALIA6 0 2 1 PATENTS ACT 1952-69 COMPLETE SPECIFICATION

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Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Publthed Priority This document Rntdf~ m I's I Il I 'ider viUig EL "inr on and is correct j p Related Art; Name of Applicant HOECHST AKTIENGESELLSCHAFT AddressofApplicant. 45 Bruningstrasse, D-6230 Frankfurt/Main 80, Federal Republic of Germany Actual inventor: Address for Service: ANDREAS LOTZ, GERHARD WOHNER, CHRISTIAN KLUC and ERICH

LUCK

EDWD. WATERS SONS, QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

Complete Specification for the Invention entitled: BACTERIA-LYSING ENZYME PRODUCT FORM STREPTOMYCETES, ITS PREPARATION AND ITS USE FOR PRESERVING CHEESE The following statement Is a full description of this Invontion, including the best method of performing it known to Us i, j- -1- I-c~ I~ I HOECHST AKTIENGESELLSCHAFT HOE 87/F 017 Dr.KH/mu Description: Bacteria-Lysing enzyme product from Streptomycetes, its preparation and its use for preserving cheese.

In French Patent 80 03,231, the use of Lysozyme-type products from animal and plant secretions for preventing late blowing in various cheese types is described. Late blowing manifests itself in the course of cheese ripening by metabolization of the lactic acid present with formation of gas, which leads to serious defects in texture.

SThe spores of the anaerobic microorganisms, which cause S 15 this late blowing, are lysed on germination by the said enzyme products.

The preparation of a bacteria-lysing enzyme product from Streptomycetes is known and has been described, for example, in *German .Offentegungs.sc.hr:iften 2,011.,935, .2,040,444, 2,146,597 and 3,440,735. However, this enzyme product obtained from Streptomycetes can not readily be used for preserving cheese.

Surprisingly, it has been found that the Streptomycetes enzyme product, when prepared, for example, according to German Offenlegungschrift 3,440,735, is associated with proteases and is difficult to separate from the Latter.

It has now been found that, by means of the bacterialysing, protease-free enzyme product from Streptomycetes, cheese products can be preserved more successfully than with corresponding products from animal and plant secretions. Not only is late blowing prevented more effectively, but it has also been found that the growth of vegetative Gram-negative organisms, for example E. coli, or even pathogenic bacteria, for example Salmonella, Shigella, Campylobacter or Listeria, is also inhibited by the enzyme product. This fact is of particular importance with regard to the preservation of cheeses produced from i r 2 raw milk. In the USA, some fatal poisonings have occurred as the result of the consumption of soft cheese which was contaminated with Listeria monocytogenes. Moreover, early blowing caused by E. coli can be suppressed by means of the enzyme product from Streptomycetes. In this way, cheese can thus be prepared at low initial contamination and its storability is therefore improved overall.

The invention thus relates to: 1. a bacteria-lysing enzyme product, obtainable by fermenting bacteria from the genus Streptomyces, which has a specific lytic activity of 20,000 to 60,000 units/mg of protein.

2. the process for preparing the bacteria-lysing enzyme product defined under 1, which comprises a) culturing a bacterium from the genus Streptomyces in a nutrient medium and b) isolating the said enzyme product, present in the supernatant'of." the :culture, by purification by means of ion exchanger chromatography, S 3. the use of a bacteria-lysing enzyme product, as defined under for preserving cheese products.

The invention is explained below in detail, especially in c i ts preferred embodiments. The invention is also defined in the patent claims.

In principle, any lysozyme-type product from Streptomycetes can be used according to the invention. In culture mddia of simple composition, a high yield of bacteria-lyzing enzyme product is achievable by means of Streptomycetes at a fermentation time of 1 to 7 days. Preferably, however, the bacteria-lysing enzyme product obtained according to German Offenlegungsschrift 3,440,735 from Streptomyces coelicolor DSM 3030 or its variants and mutants are used. Strzjtomc C.oal.o\or .'QS e o( cq ot X '-lq-Trsc.HI 77 or uqu 0, 3 An addition of sugar beet moLasses in a quantity of 5 to g, preferably 10 to 20 g, per Liter of culture medium has proved particularly advantageous for culturing Streptomyces coelicolor DSM 3030. A further increase in yield is obtained when 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 e preferred.

Since the bacteria-lysing enzyme product obtained from Streptomycetes is associated with proteases, it must first be f;red from the latter before use as a preservative in cheese. This separation can be carried out by ion exchanger chromatography, preferably on weakly acidic, macroporous cation exchangers such as, for example, crossl ink:ed .methac.ryl-atesi Ambe r-ite). The -desired enzyme product remains almost quantitatively bound to the exchanger rssin, whereas the major part of the proteases remains in the culture solution. The remaining residue of the proteases is eluted from the column by elution with a 0.05 to 0.5 molar salt solution, preferably NaCI solution.

The bacteria-lysing enzyme product is then desorbed from the column by means of a 0.5 to 2 molar salt solution, for example a solution of NaCL, Na acetate or Na lactate.

The product is desalinated and concentrated by subsequent membrane filtration, preferably an ultrafiltration, for example on cellulose acetate membranes having a permeability of 5,000 to 10,000 Dalton. The enzyme concentrate is stable on storage in this form and can thus already be used as a preservative. It can then still be dried, before it is used as a preservative in cheese. An enzyme product having a specific activity of about 20,000 to 60,000 units/mg of protein, preferably 30,000 to 40,000 units/mg of protein is obtained according to the invention.

r -4- The enzyme product according to the invention cin advantageously be added to the vat milk even before the addition of rennet. Although it is also possible to incorporate the enzyme into the finished cheese curd, uniform distribution in the curd is then more difficult.

The bacteria-lysing enzyme product can be used both as an aqueoius, concentrated solution and in various formulations.

I Thus, for example, the enzyme can be applied to a carrier, which has the advantage that, on coagulation of the milk, the enzyme remains almost quantitatively in the curd.

Coating of tne enzyme product also has the additional advantage that the enzyme is released slowly in the medium which is to be preserved, and that thus a longer-lasting activity is ensured. Milk proteins in a native state can be used as the carrier materials. The carrier and the enzyme are dissolved in water in a ratio of 20:1 to 200:1 and then sprayed together in commercially available spray driers. Aqueous solutions of native milk proteins such as, Ior..e l xamp-le,-c"ase.inates or'-hey proteins, or other usual foodstuff auxiliaries such as hydrocolloids, proteins, fats (mono-, di- and tri-glycerides), waxes and the like, can also be used for powder coating, for example by the known Wurster principle. The coating material and the enzyme are used in a ratio of 10:1 to 1:20. The coating process by the Wurster principle has been described in numerous patents, for example in US Patents 3,241,520, i 3,545,120, 3,698,133 or 3,950,891.

The dosage of the enzyme product can vary within wide Slimits. An excess is not harmful, but logically the dosage should be determined in accordance with the contamination of the milk. It is therefore advantageous first to establish the microbial contamination of the milk by methods known per se and then to add the appropriate quantity of the bacteria-lysing enzyme product, which is sufficient to prevent further propagation of harmful microorganisms. The concentrations can be chosen such that there are 1,000 to 15,000 units of enzyme product, preferably 1,000 to 10,000 units, especially 2,000 to 5,000 units in each 1 g of cheese product. After the addition and distribution of the bacteria-lysing enzyme product in the milk or even in the cheese curd, the furtoer procedure is as usuaL in cheese production. Starter cultures and ripening cultures, with which the milk is inoculated in the course of cheese production, are not damaged.

In principle, any types of cheese such as, for example, hard cheese, semi-hard cheese, semi-solid cheese, soft cheese and fresh cheese, can be treated with the bacterialysing enzyme product from Streptomycetes. Preferably, however, it is used in the production of hard cheese such as, for example, Emmental, Alpine cheese or Cheddar, Ssemi-hard cheese such as, for example, Edam, Gouda or Tilsit, and semi-solid cheese such as, for example, Wilstermarsch, butter cheese or Steinbuch.

Cheese products which contain the bacteria-lysing enzyme product from -Streptomyce-tes not"show-any-"spoilage phenomena after storage for 6 to 12 weeks, depending on the cheese type, even at higher temperatures up to 20 0

C.

The taste of the cheese produced in this way is not impaired. It should also be emphasized that these products can be prepared without the addition of potassium nitrate, or with only small quantities.

Bacteria-lysing enzyme products from Streptomycetes can, in principle, be produced at lower costs than the products mentioned which are obtained from animal or plant secre- Stions. Low-cost manufacture is a prerequisite for wide application of the enzyme in the cheese industry.

In contrast to the comparable known products, the Streptomycetes enzyme product is also active against Gram-negative spoilage-causing organisms in the milk. It is more effective against Gram-positive spore formers than the other products mentioned. For the same result, |i 1 6comparatively Less of the Streptomycetes enzyme product has to be used in cheese production than of the other I known lysozyme-type products.

In the examples which follow, the invention is explained in more detail. Percentage data are by weight, unless otherwise stated.

Example 1 Isolation and purification of the bacteria-Lysing enzyme product i After the fermentation according to Example 3 of German Offenlegungsschrift 3,440,735, a quantity of Na oxalate i. a 2+ Sequivalent to the Ca is added to the fermenter broth, in order to precipitate the calcium present in the nutrii ent medium. The Ca oxalate is then centrifuged off together with the fungus mycelium. The supernatant from i 20 tbthe culture:-risiadusted ,with:.ac eticaci.d to .pH i s300 ml of crosslinked methacrylate Amberlite I in the H form are equilibrated wit 0.05 moar Na ace- State buffer at pH 6.0. After the exchanger has been packed into a chromatography column, 10 Liters of culture supernatant are charged to the column at 2 to 3 BV/hour (BV bed voloume). The protease is removed by washing the exchanger u ith 3 to 5 BV of 0.05 molar Na acetate buffer (pH 6) and 5 to 10 BV of 0.15 mol of NaCI in a 0.05 molar Na acetate buffer (pH The bacteria-lysing enzyme product is then eluted with 3 to 4 BV of 1 mol NaCL in 0.05 molar Na acetate buffer (pH Before freeze-drying, the enzyme-containing solution is desalinated and concentrated by means of ultrafiltration (10,000 Dalton). The enzyme concentrate obtained shows no measurable protease activity and has a specific lytic activity of 40,000 units/mq of protein on Iverage.

Determination of the activity of bacteria-lysing enzyme 7 product: 0.2 ml of samples containing bacteria-lysing enzyme product are pipetted to 2.8 ml of a suspension of 0.2 mg of Micrococcus luteus ATCC 4698 (Boehringer Mannheim) per ml of 0.1 M sodium acetate buffer (pH and the decrease 4 in turbidity is determined at 25 0 C by measuring the extinctioin at 450 nm. 1 unit is defined as the decrease in extinction by 0.001 photometer scale units per minute.

1 Example 2 Use of the bacteria-lysing enzyme product as a preservac tive in cheese 100 g of starter (Streptococcus diacetylactis and Streptococcus cremoris, mesophilic mixed cultures from Wiesby, Nibull) and 50 ml of a 20% aqueous CaCI2 solution as well as s-carotene are added to 100 liters of vat nilk.

The mixture is brought to a temperature of 32 0

C.

g "of-the bacter-ia-lysiirg pr-oduct--according to Example 1 are dissolved in 2 liters of vat milk and added with stirring to the 100 liters of milk. After a further addition of 120 g of the abovementioned starter, the stirrer is switched off, in order to allow ripening for about 20 minutes. Ripening has finished when a pH value of 6.5 to 6.3 and a Soxlett-Henkel number of 7 to 8 have been reached.

ml of rennet are then added to the milk with stirring.

The coagulation time is 30 minutes. During this period, the stirrer is switched off.

The curd it then cut vertically and horizontally with a curd knife. In order to obtain a uniform curd size, the mixture is stirred again for about 30 minutes. After the whey (about 40 liters) has been skimmed off, the curd is washed for 30 minutes with 30 liters of warm water (6000.

After the washing whey has been separated off, the curd j Ii 'ii 8 is pressed under 3 bar for ai period of 3 to 4 hours and placed into a salt bath (20% NaCL) for 24 hours.

The subsequent ripening takes place for 6 weeks at 16 to 200C. Even with longer ripening of up to 12 weeks, no spoilage phenomena are to be observed.

Example 3 Fixing of the bacteria-lysing enzyme product on a carrier 1,000 g of a commercially available, non-denatured Na caseinate are dispersed in 49 liters of water. The pH value of this suspension is adjusted to pH 5.5 with lactic acid. 1,000 ml of a concentrate of the enzyme product prepared according to Example 1, having an activity of 1,000,000 units/mI, are then added. The solution is dried by means of a commercially available spray drier, which is fitted with an atomizer nozzle, at an air temperature of 80 0

C.

Example 4 1,000 g of an enzyme product (spray-dried) prepared according to Example 1 are introduced into a commercially available powder coater (fluidized-bed drier with a Wurster device and a bottom spray device). At an ambient air temperature of 20 to 30 C, the same quantity of a special fat of defined *eLting point 40 0 for example a cacao butter substitute, is sprayed on {rom the melt. The end product is still free-flowing.

Claims

2. A bacteria-lysing enzyme product as claimed in claim 1, which has a specific lytic activity of 30,000 to t 40,000 units/mg of protein.
3. A bacteria-lysing enzyme product as claimed in claim 1 or 2, obtainable by fermenting Streptomyces coelicolor DSM 3030 or its variants and mutants.
4. A process for preparing the bacteria-lysing enzyme product as claimed in claim 1 or 2, which comprises a) culturing a bacterium from the genus Streptomyces in a nutrient medium and b) isolating the said enzyme product, present in the supernatant of the culture, by purification by means of ion exchanger chromatography. c) Subsequently washing the column with 0.05 to molar salt solution until said enzyme product bound to the column is freed from protease. The process as claimed in claim 4, wherein Streptomyces coelicolor DSM 3030 is cultured.
6. The use of a bacteria-lysing enzyme product as claimed in one or more of claims 1 to 3 for preserving cheese products. L r- I ri r it i 2 rrdr -i;ii*ar" 9a
7. The use as claimed in claim 6, wnich comprises employing the enzyme product from Streptomyces coelicolor DSM 3030 or its variants and mutants.
8. The use as claimed in claim 6 or 7, wherein such a quantity of the enzyme product is added that there are 1,000 to 15,000 units thereof in lg of cheese product. I 3:31 BM KJS: SC
9. The use as claimed in one or more of claims 6 to 8, wherein the bacteri-kySing enzyme product is adsorbed on a carrier. The use as claimed in one or more of claims 6 to 8, wherein the bacteria-lysing enzyme product is encapsulated in a pulverulent form by means of an auxiliary suitable for foodstuffs.
11. The use as claimed in one or more of claims 6 to which comprises employing the enzyme product in hard cheese, semi-hard cheese and semi-soiid cheese. DATED this 21st day of January 198r. HOECHST AKTIENCESELLSCHAFT -oscIt Ag rass*sm*' EDWD, WATERS SONS PATENT ATTORNEYS QUEEN STREET MELBOURNE. VIC. 3Q000,