WO1995008275A1

WO1995008275A1 - Shelf stable product

Info

Publication number: WO1995008275A1
Application number: PCT/EP1994/002891
Authority: WO
Inventors: Johannes Petrus Paulus Maria Smelt
Original assignee: Unilever Plc; Unilever N.V.
Priority date: 1993-09-24
Filing date: 1994-08-30
Publication date: 1995-03-30
Also published as: CA2172244A1; JPH09502874A; HUT74962A; AU7614894A; ZA946804B; SK38296A3; EP0793424A1; CN1135165A; TW302272B; CZ86096A3; PL313616A1; HU9600718D0

Abstract

A process for producing a shelf stable product comprising (a) subjecting the product to a pressure of 10 to 1000 Mega-Pascal at a temperature of less than or equal to 60 °C for a period of 1 minute to 10 hours; and (b) inactivation of any remaining vegetative cells in the product; wherein a membrane destructive agent such as nisin is added to the product either prior to step (a) or step (b) or immediately after step (b).

Description

SHELF STABLE PRODUCT

Technical Field of the Invention

The invention relates to stabilisation of products which are vulnerable to spoilage or poisoning by microbial spores, especially ambient stable and extended chill shelf life products, in particular food products, and a process for the preparation thereof.

Background to the Invention

A problem with edible products is that often they are susceptible to spore outgrowth on storage and hence need to undergo a preservation system to extend their shelf life. Typical preservation systems are;

For ambient stable products

(i)Neutral pH plus sterilisation. Sterilisation typically involves a minimum heat treatment equivalent to 121°C for 3 minutes (F₀ = 3) ;

(ii)Low equilibrium pH (pH <_. 4.6) plus pasteurisation in order to prolong shelf-life and inactivate vegetative pathogens.

Both products produced using methods (i) and (ii) may have a high water activity (Aw of above 0.93) ; or

(iii) Low water activity (Aw) products (Aw <_. 0.93) produced by introducing a relatively high level of salt and/or sugar into the product followed by pasteurisation, in order to prolong shelf-life and inactivate vegetative pathogens. For extended chill shelf life products

It is generally accepted from a safety standpoint, neutral pH, high Aw products that are intended for extended shelf life at chill temperature require a process equivalent to 90°C for 10 minutes in order to inactivate non-proteolytic Clostridium botulinum. From a spoilage standpoint, in order to prevent the outgrowth of heat-resistant spoilage Bacilli, then a process equivalent to 95°C for 25 min is required.

All such preservation systems are often detrimental to product quality and thus for some time a novel preservation system has been sought which provides a shelf stable product with minimum loss of quality, for example product texture, colour and/or taste, which can be applied to edible products having a pH of more than 4.6 and a water activity of more than 0.93.

Surprisingly, it has been found that this can be achieved if the product is first subjected to high pressure conditions, a cell membrane destructive agent being present either before or after the high pressure treatment; then subjected to a treatment to inactivate any remaining vegetative cells which may be present due to germination of the spores and/or non-spore forming micro-organisms.

Disclosure of the Invention

Accordingly the present invention relates to a process for producing a shelf stable product comprising

(a) subjecting the product to a pressure of 10 to 1000 Mega-Pascal at a temperature of less than or equal to 60°C for a period of 1 minute to 10 hours; and

(b) inactivation of any remaining vegetative cells in the product ,-

wherein a membrane destructive agent is added to the product prior to step (a) or step (b) or immediately after step (b) .

By shelf-stable product is meant a product that has extended shelf-life either under ambient storage conditions (stable for > 3 months) or under chill conditions (stable for > 10 days) .

By vegetative cells is meant both germinated spores and non-spore forming micro-organisms.

Preferably the membrane destructive agent is added prior to step (a) :

In order to obtain the required shelf stable product it is necessary to either prevent any spores present in the product from germinating, or to encourage the spores to germinate and then kill the germinated spores.

Although the applicants do not wish to be bound by any theory, it is believed that the following occurs in the above steps. In step (a) the pressure conditions at moderate temperatures for 1 minute to 10 hours will allow the germination of the spores to occur. Preferably this is done in the presence of a membrane destructive agent. Surprisingly, it has been found that the combined application of the pressure conditions and the membrane destructive agent leads to a very high degree o spores to be germinated and a surprisingly low level of super-dormant spores. It is believed that ,this very effective germination and removal of super-dormant spores to a low level is unique for the combination of the pressure conditions and presence of membrane destructive agent and will not be found under other conditions, for example, the combined use of temperature and membrane destructive agent. Furthermore, the presence of the membrane destructive agent will effectively prevent the formation of active micro¬ organisms from the germinated spores.

In step (b) the vegetative cells are inactivated by, for example, heat treatment (conventional pasteurisation) , repetitive heat treatments, pressure treatment, repetitive pressure treatment, or by a combination thereof. This step will not only kill the non-spore forming micro-organisms, but will also kill the germinated spores. The net result is that spores are very effectively removed and no active micro-organisms are formed from the spores.

The conditions in step (a) are 10-1000 Mega-Pascal at a temperature of less than or equal to 60°C for a time between 1 minute to 10 hours.

Preferably the pressure in step (a) is from 50 to 400 Megapascal, more preferred 50 to 300 Megapascal, most preferred 50 to 250 Megapascal. Pressure may be generated by any method for high pressure generation, for example liquid pressure may be applied whereby the product is liquid in itself or is emersed in water after which the pressure of the water is raised. High pressure technology in general and its potential use is for example described by R.G. Earnshaw (Food Technology International Europe '92 pages 85-88) .

The temperature is preferably from 10 to 60°C, more preferred 20 to 60°C, most preferred 35 to 60°C? The time for step (a) is preferably from 10 minutes to 8 hours, more preferred 20 minutes to 5 hours, most preferred 20 minutes to 4 hours.

For the purpose of this specification the term cell membrane destructive agent has the meaning as known in the art: ie. any agent capable of affecting microbial membranes for example, lantibiotics, pseudorandom peptides, magainins, attacins, cecropins, defensin, eugenol, allecin, subtilin, Pep 5, epidermin, cinnamycin, Ro09-0918, duramycin and ancovenin. The amount of cell membrane destructive agent is preferably from 10 to 1000 ppm, more preferred 15 to 300 ppm, most preferred 25 to 150 ppm.

Especially preferred cell membrane destructive agents are so-called lantibiotics such as described in EP 427 912. Members of this group include nisin, and pediocin.

Within this class nisin is particularly preferred.

Another class of membrane destructive agents are pseudo¬ random peptides. For the purposes of the present specification the term 'pseudo-random' will be used to refer to both completely random peptides and to those in which no steps have been taken to inhibit or restrict peptide growth to single step elongation of the peptide chains so as to produce an ordered sequence of residues. These peptides, having far less structure than those found in nature, are known and have found applications as drug carriers or as reagents in immunological assay techniques.

Typically, the peptide comprises a co-polymer of at least one amino acid having an isoelectric point above 7 and at least one amino acid having a bulky functional group.

For the purposes of the present specification, bulky groups are those having a total of five or more carbons and heteroatoms. These include the aromatic groups derived from toluyl rings (as in phenylalanine and tyrosine) and indoles (as in tryptophan) , as well as sufficiently long side chains such as the guanidino group of arginine, and the amino group of lysine. Typically the amino acid having an isoelectric point above 7 is selected from the group comprising arginine, lysine, histidine and mixtures thereof.

Typically, the amino acid having an aromatic or other bulky functional group is selected from the group comprising arginine, tryptophan, tyrosine, phenylalanine and mixtures thereof.

It should be noted that arginine, and ornithine have a sufficiently high isoelectric point and sufficiently bulky functional group in the side chain that poly-arginine and poly-ornithine are effective peptides.

Particularly preferred peptides are those which comprise homopolymers of arginine and copolymers of lysine and phenylalanine, arginine and tryptophan and/or lysine and tryptophan. Mixed systems are also envisaged.

All of the above-mentioned amino acids are naturally occurring in the L chirality and consequently it is preferred that this form of the amino acid is used.

The molar ratio of amino acids of the two types is preferably in the range 10:1-1:10, more preferably 5:1-1:2 with an equal or predominant molar quantity of the basic amino acid being preferred.

Other amino acid residues can be present in the peptide, including non-standard amino acids such as ornithine, which is not an essential amino acid but does have an isoelectric point close to 10.

Accordingly it is preferred that the membrane destructive agent is selected from lantibiotics, pseudo-random synthetic peptides and mixtures thereof. Preferably the membrane destructive agent is nisin. The inactivation of the vegetative cells (step (b) ) can be carried out in any suitable way, for example by heat treatment to a temperature of 60 to 100°C for 1 to 100 minutes. Particularly preferred, however is that the inactivation is a high pressure sterilisation at a pressure of 300 - 1500 Megapascal, a temperature of 60°C or below and a time of 1 minute to 10 hours. If high pressure conditions are used in step (a) and in step (b) this will provide a surprisingly good product quality.

In an especially preferred embodiment of the invention the pressure during step (b) is at least 50 megapascal higher than the pressure during step (a) , more preferred more than 100 Megapascal higher e.g. 100 to 400 megapascal higher than the pressure in step (a) . Preferably the pressure in step (b) is between 350 and 500 Megapascal. The temperature during the high pressure inactivation in step

(b) is preferably from 5 to 50°C, more preferred 10 to 45°C, most preferred 15 to 40°C, for example ambient temperature. The time for high pressure inactivation in step (b) is preferably from 5 minutes to 8 hours, more preferred 10 minutes to 5 hours, most preferred 10 minutes to 4 hours.

Where appropriate step (a) and (b) may be repeated for even further reducing the number of spores in the products. For example the product may be subjected to 2 to 10 preservation cycles as described above. Preferably the number of cycles is from 2 to 4.

The preservation process of the invention may advantageously be applied to all products which tend to suffer from problems with spores. Examples of suitable products are food products, personal products and detergency products.

Advantageously the preservation process is applied to products, having a pH of more than 4.6, more preferred more than 4.6 and less than 10, most preferred more than 4.7 and less than 8. Also preferably the water-activity a_w of the food is more than 0.93, more preferred a_w is from 0.96 to 1.00, most preferred 0.97 to 1.00.

Since the process of the invention allows for the production of shelf stable products whereby only moderate temperatures are applied, the process of the invention is especially suitable for products to which a high temperature is detrimental to its quality e.g. products which are unstable or undergo undesired structural changes or form off-flavours when heated at temperatures normally used in preservation.

Examples of cosmetic products which may be subjected to the process of the invention are creams, lotions, tonics, toothpaste, lipstick, gels, shampoo and other hair products.

Examples of detergent products are liquid systems like liquid fabric washing detergents, household cleaners, abrasives, fabric conditioners and (semi-) solid detergents e.g. pastes and soap bars.

Preferably the process is used to provide shelf stable foodstuffs. Examples of suitable food products are spreads, in particular zero or extremely low fat spreads, dressings, dairy and non-dairy creams, toppings, processed cheese, pates, semi-hard cheese, sauces, sweet spreads, margarines, ice-cream, meat and fish products, bavarois, bakery and dough products, vegetables, fruit, soups, dairy products, beverages. In particular this process may be used to provide ambient stable sauces, soups and dressings.

Before, during or after steps (a) and (b) the edible products are preferably filled into a suitable package for further use. If the filling takes place before steps (a) and (b) it is not necessary to use aseptic filling since the package will be sterilised during steps (a) and (b) .

If, however the product is filled during or after steps (a) and (b) , then preferably aseptic filling conditions are applied.

The invention will now be illustrated by means of the following examples:

Example 1

Shows the effect of nisin added prior to pressure treatment on B.subtilis.

Preparation of B.Subtilis Spores

An overnight culture of Bacillus subtilis was made in BHI at 30°C. Appropriate aliquots were streaked on agar plates (plate count Agar (Difco) ) supplemented with 0.04 mg/L MgCl₂. The agar was incubated at 30°C for 3 to 5 days. Well sporulated cultures (>30% spores) were harvested by washing once in sterile distilled water.

Pressure Treatment

A spore concentration of from 10⁶ to 10⁷/ml in 5ml distilled water was used. The spores were subjected to a high pressure treatment of 200 mPa at 35°C for 180 min, followed by pasteurisation at 85°C for 5 min. A number or different concentrations (0-100 ppm) of nisin were added prior to high pressure treatment. After treatment the spores were disseminated into agar and incubated for 5 days at 30°C before counting. Results are expressed as a log reduction factor. Log Reduction Factor =

log (Starting Inoculum Colony forming units (cfu) finishing cfu in inoculum

Results were compared with the log reduction factor achieved when;

(a) no pressure treatment and no pasteurisation occurred

(b) no pressure treatment occurred ie. only pasteurisation

(c) pressure treatment only ie. no pasteurisation.

Results are shown in Table 1.

Comparative Example A

Shows the effect of lysozyme (not a membrane destructive agent) added prior to pressure treatment of B.subtilis.

Example 1 was repeated except a number of different concentrations (0-200 ppm) of lysozyme were added prior to high pressure treatment instead of nisin. Results given in Table 2 are expressed as a log reduction factor.

Elyample 7.

Shows the effect of nisin or pediocin addition at different stages in the process on C.Botulinum.

Preparation of C.botulinum spores

Clostridium botulinum type A strains ZK3, 62A, VII, type B strains 2345, bolus alba, 6 were used. A cocktail was made by mixing equal amounts of the spores together. Table 1

ppm nisin No Pressure Treatment Pressure Treatment in distilled water No Pasteurisation No Pasteurisation Pasteurisation Pasteurisation

0 0 0 2.18 2.66

10 0 0.07 2.33 3.88

10 35 0 0.05 2.37 5.48

100 0 0.12 2.43 5.48

15 Table 2

ppm of lysozyme No pressure treatment Pressure Treatment in distilled water and no pasteurisation

No pasteurisation Pasteurisation

20 0 0 2.1 2.3

10 2.5 2.9 50 2.8 3.3

IOΌ 2.7 3.2

200 2.6 3.2

25

Sporulation of proteolytic C.botulinum

Approximately 0.1ml of cooked meat stock (Difco) culture was inoculated into 10ml of liquid TPGS medium and incubated for 24 hour at 30°C.

TPGS medium

Tryptone (Difco) 5% bactopeptone (Difco) 0.5% glucose 0.2% soluble starch 0.2% cysteine 0.05% pH 6.8

sterilised 15 min at 120°C.

The entire culture was inoculated into 1000 ml TPGS. This

TPGS was then poured over the agar phase. The biphasic culture was incubated anaerobically for 5 to 7 days at 30°C.

Acrar Phase

Meat Extract (Liebig) 1.67% bactopeptone (Difco) 1% tryptone (Difco) 1% gelatin (Gelatine Delft) 1% agar 2%

sterilised 15 min at 120°C.

When sufficient spores were present (10-70% as observed by microscopic examination) spores were harvested by collecting the liquid phase.

The spore suspension was washed 3 times with distilled water by repeated centrifugation at about 5°C at 12000g for 10 min each time.

The spores were then treated in an ultrasonic bath after washing to ensure loose spores. The treated suspension was heated for 10 min at 80°C to ensure elimination of botulinu toxin and/or vegetative cells and enumerated.

Sporulation of non-proteolytic C.botulinum

Sporulation was carried out as described for proteolytic C.botulinum except that the treated suspension was heated for 30 min at 60°C to ensure elimination of botulinum toxin and/or vegetative cells before enumeration.

Enumeration of Spores

Dilutions were done in a peptone (0.1%) and physiological salt (0.85%) solution. Proteolytic spores were enumerated in pour plates containing TSA, cystein and egg yolk (0.05g/ml) . Incubation was at 30°C for 3 to 5 days. Non- proteolytic spores were enumerated in TPG and egg yolk containing pour plates.

Treatment

35 ppm nisin or 300 ppm Pediocin were added either prior to high pressure treatment (200 MPa at 45°C for 180 min) , prior to pasteurisation (protocol (i) - 85°C for 5 min,- protocol

(ii) - 95°C for 10 min) or after both pressure and pasteurisation treatments.

The C.botulinum was inoculated into CMM (cooked meat medium, Difco) to a final concentration of 10⁷/ml for the high pressure treatment and pasteurisation treatment . Recovery was carried out in Tryticase peptone glucose (TPG) (to enumerate C.botulinum) . The TPG agar plates were incubated anaerobically for 5 days at 30°C. Results, expressed as a log reduction factor are shown in Table 3.

Table 3

Treatment Nisin Pediocin

Membrane 4.5 4.4 destructive

P agent added

R prior to

0 pressure

T treatment 0 C Membrane 4.2 4.2 0 destructive L agent added prior to

(i) pasteurisation

Membrane 5.3 — destructive agent added after pasteurisation

Membrane 5.4 4.8

P destructive

R agent added

0 prior to

T pressure

0 treatment C 0 Membrane 5.2 4.5 L destructive agent added (ii) prior to pasteurisation

Membrane 5.8 -- destructive agent added after pasteurisation

Example 3

Shows the effect of nisin and high pressure on C.botulinum in cheese .

C.botulinum cocktail was prepared as detailed in Example 2.

Treatment

Spores of the cocktail of C.botulinum were mixed into a cheese having the following formulation;

Cheese formulation

% by weight

Skim milk powder 12.2

Milk protein concentrate 4.0 Butter 39.0

Salt 1.1

Na pyrophosphate 0.2

Na citrate dihydrate 0.4

Water 43.1

The final spore concentration was 10⁷/ml. Nisaplin was added to the cheese at a level of 0.05% by weight

(corresponds to 10 mg/kg pure nisin) .

The cheese was subjected to a number of different pressure treatments for 180 min as detailed in Table 4, followed by a pasteurisation treatment of 80°C for 10 minutes.

Results expressed as log reduction factor are given in Table 4. Table 4

Pressure Temperature Log Reduction Factor

0.IMPa 35°C 0.0

45°C 0.0

60MPa 35°C 0.4

45°C 1.0

200MPa 35°C 1.3

45°C 2.4

Example 4

Shows the effect of pressure and nisin on C.botulinum.

A C.botulinum cocktail was prepared as detailed in Example 2.

Treatment

Spores of the cocktail of C.botulinum were mixed into cooked meat medium (CMM, Difco) to a final concentration 10⁷/ml. The CMM contained 0, 10 or 35 ppm nisin. The 3 inoculated CMM varieties were subjected to different pressure treatments as detailed in Table 5. After pressure treatment the inoculated CMM varieties were subjected to one of the following pasteurisation protocols:

(np) no pasteurisation (i) 85°C for 5 min (ii) 95°C for 10 min

Recovery was done in TPG with the same level of nisin present as in the CMM. Results expressed as log Reduction factor are shown in Table 5.

Table 5

nisin no 0. IMPa, 90 min 200MPa, 90 min concentration treatment 50°C 50°C

(ppm) np (i) (ii) np (i) (ii)

0 0 0 0 0 0.7 1.1 2.7

10 10 0.5 1.1 0.8 1.3 1.7 2.6 3.3

35 1.0 0.8 0.3 1.4 1.7 2.3 3.5

15

20

25

Example 6

Shows effect of nisin on B.subtilis in 2 stages of pressure/post pasteurisation treatment.

B.subtilis spores were prepared as for Example 1.

A spore concentration of from 10⁶ to 10⁷/ml in 5ml distilled water was used.

Nisin at 35 ppm concentration was added prior to pressure treatment and/or pasteurisation treatment. The pressure treatment was 200MPa, at 35°C for 180 min. The pasteurisation treatment was 80°C for 5 min.

Results, expressed as a log reduction factor, are given in Table 7.

Table 7

nisin present nisin present log reduction pre-pressure post- factor treatment pasteurisation treatment

/ X 5.2

X / 5.3

/ / 5.3

Example 7

Shows a comparison of pasteurisation and pressure treatment to inactivate the vegetative cells.

B.subtilis spores were prepared as for Example 1.

A spore concentration of from 10⁶ to 10⁷/ml in 5 ml distilled water was used. The spores were subjected to a high pressure treatment of 60MPa at 35°C for 30 min followed by either

(i) Pasteurisation of 5 min at 85°C or

(ii) Pressure treatment at 400 MPa at 35°C for 10 min.

35 ppm nisin was added prior to high pressure treatment (step (a) ) . After treatment the spores were disseminated into agar and incubated for 5 days at 30°C before counting. Results are expressed as a log reduction factor and are shown in Table 8.

Table 8

Treatment log reduction factor

(i) High pressure plus >5.9 pasteurisation (ii) Repetitive High >5.9 Pressure

Example 8

Shows a comparison of pasteurisation and pressure treatment to inactivate the vegetative cells .

C.Botulinum cocktail was prepared as for Example 2.

Spores of the cocktail of C.botulinum were mixed into cooked meat medium (CMM, Difco) to a final concentration of 10⁷/ml. The CMM contained 35 ppm nisin. The spores were subjected to a high pressure treatment of 60MPa~at 35°C for 30 min followed by either

(i) Pasteurisation of 5 min at 85°C or

(ii) Pressure treatment at 400 MPa at 35°C for 10 min.

Recovery was done in TPG with 35 ppm nisin added. Results expressed as log reduction factor are shown in Table 9.

Table 9

Treatment log reduction factor

(i) High pressure plus 2.5 pasteurisation (ii) Repetitive high 2.4 pressure

Claims

1. A process for producing a shelf stable product comprising:

(b) inactivation of any remaining vegetative cells in the product,-

characterised in that a membrane destructive agent is added to the product either prior to step (a) or step (b) or immediately after step (b) .

2. A process according to claim 1 wherein the membrane destructive agent is added prior to step (a) .

3. A process according to claim 1 or 2 wherein the level of membrane destructive agent is from 10 to 1000 ppm.

4. A process according to any preceding claim wherein the level of membrane destructive agent is from 15 to 300 ppm.

5. A process according to any preceding claim wherein the level of membrane destructive agent is from 25 to 150 ppm.

6. A process according to any one of the preceding claims wherein the membrane destructive agent is selected from lantibiotics, pseudo-random synthetic peptides a^"hd mixtures thereof.

7. A process according to any preceding claim wherein the membrane destructive agent is nisin.

8. A process according to any preceding claim wherein in step (a) the pressure is from 50 to 400 Mega-pascal.

9. A process according to any preceding claim wherein in step (a) the temperature is from 20 to 60°C.

10. A process according to any preceding claim wherein in step (b) the vegetative cells are inactivated by heat treatment, repetitive heat treatment, pressure treatment, repetitive pressure treatment or a combination thereof.

11. A process according to any preceding claim wherein steps (a) and (b) are repeated from 2 to 10 times.

12. A process according to any preceding claim wherein the pH of the product is greater than 4.6.

13. A process according to any preceding claim wherein the water activity of the product is greater than 0.93.

14. A process according to any preceding claim wherein the product is selected from a food product, a personal product and a detergent product.

15. Shelf stable product obtainable by

(b) inactivating any remaining vegetative cells,*