WO1993018179A1

WO1993018179A1 - Bacteriocin from pediococcus cerevisiae

Info

Publication number: WO1993018179A1
Application number: PCT/EP1993/000514
Authority: WO
Inventors: Pieter Van Solingen; Jacobus Stark; Pieter Cornelis Langeveld
Original assignee: Gist-Brocades N.V.
Priority date: 1992-03-05
Filing date: 1993-03-05
Publication date: 1993-09-16

Abstract

The present invention discloses a bacteriocin obtainable from Pediococcus cerevisiae P18. Said bacteriocin is composed of proteinaceous material and has a broad range of activity against Listeria monocytogenes. The bacteriocin is inactive against the tested starter and surface culture strains. The invention further provides a method for obtaining the bacteriocin. Finally, the use of the bacteriocin is illustrated.

Description

BACTERIOCIN FROM PEDIOCOCCUS CEREVISIAE

Technical Field

The present invention is in the field of food and feed preservation. The present invention provides a microbial bacteriocin which is active against a broad range of Listeria without affecting a broad range of starter cultures and surface flora organisms.

Background of the invention

Food and feed as well as the food and feed industrial environment, is often contaminated by unwanted microorganisms. Widespread occurence of Salmonella and Campylobacter have been reported. The occurence^' of Listeria monocytogenes. which is often introduced along with raw materials, may be as high as that of the other bacteria, however the amount of people infected with this bacterium is much lower.- The seriousness of the problem is in the mortality rate of people infected with Listeria. A mortality rate of 30% after infection with Listeria monocytogenes has been reported. Several outbreaks of human listeriosis have been reported for example in Canada (Schlech W.F. et al. (1983) N. Engl. J. Med. 308 : 203-206), in the USA (Flemming D.W. et al. (1985) N. Engl. J.. Med. 212. : 404-407) and in Switzerland (Food Chem. News (1987) Dec. p. 7) . Listeria monocvtognes is now recognized as a major foodborne pathogen.

Listeria monocytogenes has the ability to withstand a large variety of environmental conditions associated with traditional methods of food preservation such as refrigeration temperatures (Wilkins, P.O., Bourgeois, R. , Murray, R.G.E. (1972) Can. J. Microbiol. 98.: 543-551), high salt concentrations (Seelinger, H.P.R., Jones, D. (1986) Genus Listeria, pp. 1235-1245, Bergey's Manual of Systematic Bacteriology, Vol. 2), and pH levels as low as 5.0 (Connor, D.E., Bracket, R.E. , Beuchat, L.R. (1986), Appl. Environ. Microbiol. .50.: 59-63) . Listeria monocytogenes has also been shown to survive under processing and storage conditions. Specifically in products such as hard salami (Johnson, J.L. , Doyle, M.P., Cassens, R.G., Schoeni, J.L. (1988), Appl. Environ. Microbiol. 54.: 497-501), cheeses (Ryser, E.T., Marth, E.H. (1987) , J. Food Prot. 50: 372-378) , and fermented milk (Schaack, M.M. , Marth, E.H. (1988), J. Food Prot. 51: 607-614) made from raw materials contaminated with the organism. These observations suggest that traditional, physical food and feed preservation processes generally do not prevent growth of Listeria monocytogenes. Effective measures need to be taken to minimize the contamination of food and feed products.

Chemical preservation poses its own problems. The concentration of sodium benzoate (El-Shenawy, M.A. , Marth, E.H. (1988) , J. Food Prot. 51: 525-530) , sorbic acid (El- Shenawy, M.A. , Marth, E.H. (1988), J. Food Prot. 51.: 842- 847) _r and nitrite (Junttilla, J. , Him, J. , Hill, P. , Nur i, E. (1989) , J. Food Prot. 52.: 158-161) permitted in foodstuff is too low to prevent growth of Listeria monocytogenes.

Other anti-Listeria agents have been found in nature. Lactic acid bacteria are able to inhibit other microorganisms by producing organic acids, peroxide or more specifically inhibitory substances such as- antibiotics or bacteriocins. From the standpoint of dairy products and their manufacture antibiosis is beneficial in some instances such as inhibition of pathogenic or spoilage bacteria, but detrimental in others such as inhibition of lactic starter cultures and other microorganisms, that contribute to ripening processes and organoleptic properties in general.

Bacteriocins are inhibitory- molecules produced by a wide range of bacteria. Examples of well known bacteriocins are Nisin from Lactobacillus lactis (Hurst, A. (1983) In A.L. Branen and P.M. Davidson (eds) Antimicrobials in Foods, Marcel Dekker Inc., New York) and Pediocin A from Pediococcus pentosaceus (Daeschel, M.A. , Klaenhammer, T.R. (1985), Appl. Environ. Microbiol. 5_0: 1538-1541). Both are broad spectrum bacteriocins and it is therefore not surprising that these bacteriocins were also active against some Listeria monocytogenes strains.

EP 326062 describes a method for inhibiting Listeria using a bacteriocin. The bacteriocin is isolated from a Pediococcus acidilactici. EP 453719 discloses the amino acid sequence of this bacteriocin.

Pediococci have an important role in several meat and vegetable fermentations. They are mainly used as starter cultures. An advantage of their use is that they restrict the growth of (selected) other bacteria by producing specific bacteriocins.

Bacteriocins all have a specific spectrum of bacteria against which they are active. Generally it is preferable to use bacteriocins which work against all unwanted bacteria without affecting the starter cultures or the desired surface flora bacteria. Such a preferred bacteriocin has not been reported to date.

Summary of the invention

The present invention provides a bacteriocin obtainable from Pediococcus cerevisiae P18. This bacteriocin is active against a wide range of tested Listeria strains, specifically against, Listeria innocua. Listeria ivanovii. Listeria welshimeri and Listeria monocytogenes strains.

It is another aspect of the invention that said bacteriocin does not affect the tested starter cultures and surface flora cultures.

In another aspect the invention discloses a method for using the bacteriocin in' the preservation of food, feed, cosmetics or pharmaceutical preparations. Specifically, soft en semi-hard cheeses, vegetables and processed meats are well preserved.

The present invention further provides a method for obtaining the said bacteriocin.

Description of the figures

Figure 1 shows the effect of bacteriocin P18 on Listeria monocytogenes in cheese expressed as the logarithm of colony forming units per g. Figure 2 shows the effect of bacteriocin P18 on Listeria monocytogenes in pate as the logarithm of colony forming units per g. Figure 3 shows the effect of bacteriocin P18 on Listeria monocytogenes in milk as the logarithm of colony forming units per g.

Detailed description of the invention

The present invention describes a novel bacteriocin obtainable from Pediococcus cerevisiae P18. The bacteriocin can be obtained by growing Pediococcus cerevisiae P18 in MRS- broth. After a suitable time the cellular material is removed i.e. by centrifugation. The supernatant contains the bacteriocin, which can be further purified using known techniques such as ammonium-sulphate precipitation, gel filtration and HPLC. The bacteriocin of the present invention was further purified by filter-sterilisation and concentration. The filtrate or supernatant was concentrated between 8 and 15 fold. When these concentrated solutions were used in an agar diffusion test (as described below) and no inhibition zone could be detected it was assumed that the bacteriocin was not active against the plated indicator strain. Minimal inhibitory concentrations (MIC) were also determined using serial dilutions of the bacteriocin against both Listeria and starter/surface cultures in icrotiter plates.

Further characterisation of the active component of the supernatant of the Pediococcus culture showed that the bacteriocin is a protein. After treatment with pronase no residual activity of the bacteriocin could be detected. Other proteases tested include papain, proteinase K, α-chymotrypsin which were all found to destroy the activity of the bacteriocin. Trypsin, lysozym, α-amylase and β-amylase were found to have no effect on the bacteriocin activity.

The present bacteriocin was also found to be highly thermostable. After heating a sample of the supernatant of a Pedicoccus culture at 100°C for 3 min 100% of the activity was recovered, after 20 min. at 120°C 25% of the initial activity was recovered.

The novel bacteriocin described here, was called Bacteriocin P18 and it is highly specific against Listeria strains, especially Listeria monocytogenes. Bacteriocin P18 inhibits growth of Listeria monocytogenes without interfering with the growth and activity of other bacteria, beneficial to processes of manufacture and ripening of fermented food products, such as Lactobacilli. Streptococci and Brevibacteriu linens under the given conditions.

The present invention discloses a bacteriocin characterised in that it is active against a broad range of Listeria strains. We use the term broad since the bacteriocin turned out to be active against all the tested Listeria monocytogenes strains and against Listeria innocua. Listeria ivanovii and Listeria welshimeri. Specifically, it was shown that the bacteriocin of this invention is active against the following Listeria strains which have all been deposited at the Centraal Bureau voor de Schimmelcultures in Baarn, the Netherlands on March 2, 1992, under the given deposition numbers and against the type strain DSM 20600; Listeria monocytogenes LMD 92.18 to LMD 92.29. Wherein LMD 20 and 23 have later been characterized as Listeria innocua. The following is a further characterisation of the Listeria monocytogenes strains.

Strain Source Characteristics

#1 Listeria monocytogenes LMD 92.22 cheese serovar 6a #2 Listeria innocua LMD 92.20 cheese serovar 6b #3 Listeria innocua LMD 92.23 cheese serovar 4b #4 Listeria monocytogenes LMD 92.21 cheese serovar l-2a #5 Listeria monocytogenes DSM 20600 rabbit serovar 1-2a #6 Listeria monocytogenes LMD 92.18 cow brain #7 Listeria monocytogenes LMD 92.19 calf faeces #8 Listeria monocytogenes LMD 92.24 patient serovar l-2a #9 Listeria monocytogenes LMD 92.25 patient serovar 1-2c #10 Listeria monocytogenes LMD 92.26 patient serovar 4b #11 Listeria monocytogenes LMD 92.27 patient serovar 6a #12 Listeria monocytogenes LMD 92.28 patient serovar l-2b #13 Listeria monocytogenes LMD 92.29 patient serovar 4a #14 Listeria innocua DSM 20649 #15 Listeria ivanovii DSM 20750 #16 Listeria welshimeri DSM 20650

The present bacteriocin is further characterized in that it does not show any acitivity against a series of starter and surface cultures used in cheese making or meat preparation. Specifically, the following starter cultures have been tested Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris,

Lactococcus lactis subsp. lactis biovar. diacetylactis, Leuconostoc mesenteroides subsp. cremoris,

Geotrichum candidu , Penicillium candidum, Saccharomvces cerevisiae. Saccharomvces unisporum and Streptococcus thermophilUs A Visbyvac^R (Labor Wiesby, Niebϋll, Germany)•. As an example of a surface culture Brevibacterium linens (Kernhem cheese) was used. Also, against this strain no activity was detected.

The present bacteriocin can be used in a wide range of applications. Specifically, in food and feed preservation. Food most often associated with contamination of Listeria monocytogenes are milk based cheeses, raw milk, ice-cream and cottage cheeses and vegetables like lettuce. Specific cheeses are soft, and semi-hard cheeses particularly. Brie, Camembert, Roquefort, Munster, Kernhem, Vacherin Mont d^,Or and Danish blue.

Processed meat particularly pate is also associated with Listeria infection. Food or feed treated with machinery and which is not heat-treated thereafter is particularly prone to infection. Meats like beef, pork, poultry and fish are also easily contaminated. The protein of the present invention can advantageously be used to prevent this contamination. This is illustrated by examples concerning Brie and other cheeses (like Camembert) , pate and milk.

Apart from food and feed products medicinal, veterinary products and pharmaceutical preparations can easily be contamined. Finally, cosmetics can be contaminated also in these cases the present bacteriocin can advantageously be used.

The present invention discloses a method for preventing growth of Listeria monocytogenes on food, feed, cosmetics or pharmaceutical preparations which comprises: adding an effective amount of a bacteriocin into or upon a food, wherein the bacteriocin is obtainable from Pediococcus cerevisiae P18. The method can also be applied to the other products mentioned above and which are susceptible to contamination with Listeria strains such as Listeria monocytogenes. L. innocua. L. ivanovii and L. welshimeri. Furthermore, the invention provides food, feed, cosmetics and pharmaceutical preparations wich have been treated with bacteriocin.

Example 1

Isolation of bacteriocin from Pediococcus

Strain

Pediococcus cerevisiae P18 was deposited at Centraal Bureau voor de Schimmelcultures in Baarn, the Netherlands on February 11, 1992, under number CBS 125.92.

Bacteriocin production

Pediococcus cerevisiae P18 was grown in 100 ml shake flasks containing 20 ml of MRS-broth (Oxoid, Basingstoke, England) for 20 hours at 30°C and 250 rpm in a Shaking Incubator (New Brunswick Scientific, Edison, NJ, USA) . The culture was centrifuged for 5 minutes at 1800 g at room temperature. The supernatant was filter-sterilized using a 0.2 μm filter (Minisart type 165.34, Sartorius, Goettingen, Germany). The culture broth was concentrated using the Amicon Centricon™ microconcentrator system type Centricon-3, following the instructions of the manufacturer (Grace Co., Danvers, MA, USA) .

Bacteriocin assay Bacteriocin activity was determined by two different agar diffusion methods. a) . Bacteriocin solutions were filter-sterilized and 15 μl aliquots were pipetted in wells (4 mm across) punched out of the agar layer. The plates were incubated for 24 hours at 30^βC. The lowest concentration of bacteriocin, that gives detectable inhibition using Listeria monocytogenes LMD 92.27 as indicator strain was defined as 1 arbitrary unit per ml (AU/ml) . (Used in Example 2) . b) . Bacteriocin producing cells were spotted on the surface of Brain Heart Infusion Agar (BHI) (Difco laboratories,

Detroit, Mi, USA) containing 10⁶ cells per ml of an indicator bacterium. (Used in Example 3) . Nature of the anti-Listeria activity

The anti-Listeria activity in the culture broth of Pediococcus cerevisiae P18 -can be inactivated by proteolytic enzymes. After incubation with 1 mg/ml Pronase (Boehringer, Mannheim, Germany) for 1 hour at 37^βC no activity could be detected. Therefore the anti-Listeria activity is of proteinaceus nature and will be referred to as Bacteriocin P18.

Other proteases tested include papain, proteinase K, α- chymotrypsin which were all found to destroy the activity of the bacteriocin. Trypsin, lysozym, α-amylase and β-amylase were found to have no effect on the bacteriocin activity.

Bacteriocin concentrates were heated for a certain time, cooled and the residual anti-Listeria activity was measured in the agar diffusion test. Results indicated in Table I show the high stability of this bacteriocin.

Table 1

nd = not determined Example 2

Activity of Bacteriocin P18 against various Listeria monocytogenes strains

Thirteen (13) Listeria monocytogenes strains were tested as indicator organisms in the agar diffusion test. Aliquots of 15 μl filter-sterilized culture broth of Pediococcus cerevisiae PI8 (32 AU/ml) were pipetted in the wells. The results are shown in Table 2.

diffusion plates containing various bacterial strains as starter culture or surface culture in cheese making. A list of the organisms is given in Table 3. Inhibition zones around single colonies were measured. Table 3

Starter cultures

Probat M4 Visbyvac^R (Labor Wiesby, Niebύll, Germany) containing the following strains:

Lactococcus lactis subsp. lactis Lactococcus lactis subsp. cremoris

Lactococcus lactis subsp. lactis biovar. diacetylactis Leuconostoc mesenteroides subsp. cremoris

Streptococcus thermophilus A Visbyvac^R (Labor Wiesby, Niebύll, Germany)

Surface culture Brevibacterium linens (Kernhem cheese)

No detectable inhibition zone was found in the agar diffusion test using the strains mentioned in Table 3 as indicator strains. Under the same conditions, a clear inhibition zone of 4 mm was found around a Pediococcus cerevisiae P18 colony using Listeria monocytogenes LMD 92.27 as an indicator strain.

Example 4 Activity of bacteriocin P18 against various Listeria strains and against strains used in cheese making. using a microtiterplate assay

The strains mentioned in Table 5 and 6 were grown in

500 ml shakeflasks containing 100 ml of a medium

(composition, see Table 4) for 24 hours at 30"C and 250 rpm in a shaking incubator (New Brunswick Scientific, Edison, NJ,

USA) .

A serial dilution in the CM medium of the bateriocin P18 was made (256 to 0.00025 AU/ml) and brought in the wells of the microtiterplates. Then the strains were inoculated in a concentration of 10³ cells/ml. The microtiterplates were incubated during 17 hours at 30"C. The results are summarised in Table 5 and 6.

Table 4 CM medium

The components were diluted in 1000 ml deminiralisated water.

Table 5

Table 6

From Table 5 it can be seen that the different Listeria strains are inhibited using less than 1 AU/ml and often much less of bacteriocin. Table 6 shows that for different starter cultures more than 256 AU/ml of bacteriocin are required.

Example 5

Activity of Bacteriocin P18 against Listeria monocytogenes on soft cheese

Fresh Brie cheese, directly from the brine bath, was cut in pieces of 6 by 6 cm. The surface of the cheese was inoculated with a cocktail of 5 Listeria monocytogenes strains (Table 2, strain #1 to #5) at 10² cfu/cm². After 30 minutes at room temperature 400 μl of various dilutions of Pediococcus cerevisiae P18 culture broth was spread over the surface. The blocks of cheese were placed in a plastic box at a relative humidity of 95% and incubated for 8 days at 14"C. To determine the number of viable Listeria monocytogenes cells the samples were divided in 2 parts. To each part 2 volumes (v/w) 0.85% NaCl solution were added. The samples were homogenized for 2 minutes in a Stomacher Homogenizer (Steward and Co. Ltd, London, England) . Serial dilutions of the suspensions were plated on PALCAM-Listeria-Selective Medium (Merck, Darmstadt, Germany) . The plates were incubated overnight at 30°C. The results are shown in Table 7.

Table 7

Concentration Bacteriocin P18 Number of viable (AU/ml) Listeria monocytogenes

0 1. 85 x 10^s

1 1. 25 x 10⁵

3 1. 65 X 10⁴

10 5. 65 X 10³

30 1. 40 x 10³

100 6. 50 X 10²

Example 6

Activity of bacteriocin P18 against Listeria monocytogenes in a camembert cheese model

The cheese model was executed in the following way. 250 ml of pasteurised milk was brought into a beaker of 400 ml and incubated in a waterbath with a temperature of 35^βC. After one hour the following components were added:

- 750 Ul 3.3% CaC12 ' 2 H-0

- 2.5 ml starter culture (culture of Lactococcus lactis subsp. lactis #3, subsp. cremoris #5 and subsp. diacetylactis #10 in skim-milk)

- Penicillium candidum #14, 10⁵ spores/ml milk

- Listeria monocytogenes cocktail, 100 cfu's/ml, consisting of strain #1, #2, #3, #4 and #5 (Table 5)

- 50 units P18 bacteriocin/ml milk.

After three hours the milk was curdled, by adding 375 ul Maxiren^® to the milk. After one hour the curdled milk was cut with a cheese knife. A half hour later the whey was removed and the curdled milk was put into a cheese-press. The next day the cheese was salted with 1% sodium-chloride and placed into a container with a moisture content of 95%. The incubation temperature was 14^βC. After zero, two, five, eight, twelve and fourteen days the number of cfu's of Listeria monocytogenes was determined by plate-counting on PLACAM-Listeria-Selective Medium. The results are shown in Figure 1.

Example 7

Distribution of bacteriocin P18 in the curdled milk and the whey

Using the test described in Example 5, the activity of the bacteriocin was determined in milk, curdled milk and in whey by the Bacteriocin assay a) .

The results are shown in Table 8. It can be seen that the bacteriocin mainly can be found in the curdled milk, which is where its activity is wanted.

Table 8

Example 8

Activity of Bacteriocin P18 against Listeria monocytogenes in pate

Pate was divided in 12 pieces of 5 gram each. The surface of the pate was inoculated with a cocktail of 5 Listeria monocytogenes strains (Table 2, strain #1 to #5) at 180 cfu/gram. After an incubation period of 2 hours at 14°C and 2 hours at 5°C the bacteriocin P18 (50 AU/gram) was spread over the surface of 6 of the pieces. The other 6 pieces were used as a control. The pieces were incubated at 5°C. After 0, 1, 2, 3, 7 and 10 days respectively the number of cfu of Listeria monocytogenes was determined by using the following method: a complete piece of pate was homogenized in saline solution as described in Example 4. Serial dilutions of the suspensions were plated on PALCAM-Listeria-Selective Medium (Merck, Darmstadt, Germany) . The plates were incubated overnight at 30*C. The results are shown in Figure 2.

Example 9

Activity of Bacteriocin P18 against Listeria monocytogenes in milk

A milk sample was inoculated with Listeria monocytogenes LMD 92.27 (10² cells/ml). After incubation for half an hour at 14"C the sample was divided in two portions of 60 ml each. To one of the portions Bacteriocin P18 was added to a concentration of 4.5 AU/ml. The other portion was used as a control. The incubation temperature was 14"C. After 0, 16, 24 and 42 hours respectively the number of cfu of Listeria monocytogenes was determined by plate-counting on PALCAM-Listeria-Selective Medium. The results are shown in Figure 3.

Claims

1. A microbial bacteriocin characterised in that it is active against at least three of the following Listeria monocytogenes strains LMD 92.18 to LMD 92.29 Listeria innocua LMD 20 and LMD23 and Listeria monocytogenes DSM 20600.

2. A bacteriocin according to claim 1 further characterised in that it is not active against starter cultures or surface flora cultures.

3. A bacteriocin according to claim 1 or 2 characterized in that it is active against, Listeria monocytogenes LMD 92.18 to LMD 92.29 and Listeria monocytogenes DSM 20600 and against Listeria innocua. Listeria ivanovii and Listeria welshimeri.

4. A bacteriocin characterised in that it is not active against Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris,

Lactococcus lactis subsp. lactis biovar. diacetylactis, Leuconostoc mesenteroides subsp. cremoris.

Streptococcus thermophilus A Visbyvac^R (Labor Wiesby, Niebύll, Germany) and Brevibacterium linens (Kernhem cheese)

5. A bacteriocin obtainable from Pediococcus cerevisiae P18 deposited under number CBS 125.92.

6. A method for preventing growth of Listeria strains on food, feed, cosmetics and pharmaceutical preparations which comprises: adding an effective amount of a bacteriocin to food, feed, cosmetics or pharmaceutical preparations wherein the bacteriocin is obtainable from Pediococcus cerevisiae P18.

7. A method according to claim 6 characterised in that the food is selected from the group comprising; soft, and semi-hard cheeses particularly. Brie, Camembert, Roquefort, Munster, Kernhem, Vacherin Mont d'Or and Danish blue or processed meat particularly pate.

8. Food, feed, cosmetics or pharmaceutical preparations treated with a bacteriocin according to claims 1-5.

9. A process for obtaining a bacteriocin comprising,

- growing of Pediococcus cerevisiae P18 under conditions wherein the bacteriocin is produced;

- separating the cells from the supernatant; - and optionally further purifying the bacteriocin.