Reduction of the concentration of gram-negative bacteria in a fermented food product by the combination of red wine extract and a culture comprising at least one bacteriocin-producing lactic acid bacterial strain

Abstract

The present invention relates to a method of reducing the concentration of Gram-negative bacteria in a food product, the method comprising adding a red wine extract produced from red wine by dealcoholization, concentration and drying, and a culture comprising at least one class IIa bacteriocin-producing lactic acid bacterial strain to a food product, ripening the food product, and storing the food product at a temperature of at the most 15 C. until a concentration less than 1 E-1 of Gram-negative bacteria. In a preferred embodiment of the invention, Gram-negative bacteria already present are eliminated. In one embodiment of the invention, the Gram-negative bacteria are Salmonella ssp. The bacteriocin-producing culture may comprise at least one of Lactobacillus curvatus (DSM 18775) and Pediococcus acidilactici (DSM 28307). The invention further relates to a kit for reducing the concentration of Gram-negative bacteria in a food product comprising a red wine extract produced from red wine by dealcoholization, concentration and drying, and a culture comprising at least one class IIa bacteriocin-producing lactic acid bacterial strain.

Claims

1. A method of reducing the concentration of Gram-negative bacteria in a food product, comprising: adding to a food product a red wine extract produced from red wine by dealcoholization, concentration and drying, and a culture comprising at least one class IIa bacteriocin-producing lactic acid bacterial strain, ripening the food product, and storing the food product at a temperature of at most 15 C. until a concentration of Gram-negative bacteria of less than 1 CFU per 10 g food product is reached, wherein the Gram-negative bacteria are selected from Aeromonas caviae; Aeromonas hydrophila; Aeromonas sobria; Campylobacter jejuni; Citrobacter ssp.; Enterobacter ssp.; Escherichia coli enteroinvasive strains; Escherichia coli enteropathogenic strains; Escherichia coli enterotoxigenic strains; Escherichia coli O157:H7; Klebsiella ssp.; Plesiomonas shigelloides; Salmonella ssp.; Shigella ssp.; Vibrio cholerae; and Yersinia enterocolitica, wherein the method reduces the concentration of the Gram-negative bacteria in the food product to a greater extent than adding the class IIa bacteriocin-producing lactic acid bacterial culture and not the red wine extract.

2. A method according to claim 1, wherein the method reduces the presence of Gram-negative bacteria present in the food product at the start of the method.

3. A method according to claim 1, wherein the Gram-negative bacteria are selected from Escherichia coli enteroinvasive strains; Escherichia coli enteropathogenic strains; Escherichia coli enterotoxigenic strains; and Escherichia coli O157:H7.

4. A method according to claim 1, wherein the Gram-negative bacteria are Salmonella ssp.

5. A method according to claim 1, wherein the culture comprises at least one class IIa bacteriocin-producing strain selected from Carnobacterium maltaromaticum, Carnobacterium pisicola, Carnobacterium divergens, Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus sakei, Lactococcus lactis, Leuconostoc carnosum, Leuconostoc gelidium, Pediococcus acidilactici, Pediococcus pentosaceus.

6. A method according to claim 1, wherein the culture comprises at least one of Lactobacillus curvatus (DSM 18775) and Pediococcus acidilactici (DSM 28307).

7. A method according to claim 1, wherein the fermented food product is a dairy product, a meat product, a vegetable product, a fruit or a grain.

8. A method according to claim 7, wherein the fermented food product is a spreadable sausage.

9. A method according to claim 7, wherein the fermented food product is a sliceable sausage.

10. A fermented food product obtained by a method of claim 1.

11. A fermented food product obtained by a method of claim 2.

12. The fermented food product of claim 11, wherein the product is a dairy product, a meat product, a vegetable product, a fruit or a grain.

13. The fermented food product of claim 11, wherein the product is a spreadable sausage.

14. The fermented food product of claim 11, wherein the product is a sliceable sausage.

15. A method according to claim 2, wherein the Gram-negative bacteria are selected from Escherichia coli enteroinvasive strains; Escherichia coli enteropathogenic strains; Escherichia coli enterotoxigenic strains; and Escherichia coli O157:H7.

16. A method according to claim 2, wherein the Gram-negative bacteria are Salmonella ssp.

17. A method according to claim 2, wherein the culture comprises at least one class IIa bacteriocin-producing strain selected from Carnobacterium maltaromaticum, Carnobacterium pisicola, Carnobacterium divergens, Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus sakei, Lactococcus lactis, Leuconostoc carnosum, Leuconostoc gelidium, Pediococcus acidilactici, Pediococcus pentosaceus.

18. A method according to claim 2, wherein the culture comprises at least one of Lactobacillus curvatus (DSM 18775) and Pediococcus acidilactici (DSM 28307).

19. A method according to claim 2, wherein the fermented food product is a dairy product, a meat product, a vegetable product, a fruit or a grain.

20. A kit for reducing the concentration of Gram-negative bacteria in a food product, comprising: a red wine extract produced from red wine by dealcoholization, concentration and drying, and a culture comprising at least one class IIa bacteriocin-producing lactic acid bacterial strain, wherein the combination of the red wine extract and the culture inhibits growth of Gram-negative bacteria selected from Aeromonas caviae; Aeromonas hydrophila; Aeromonas sobria; Campylobacter jejuni; Citrobacter ssp.; Enterobacter ssp.; Escherichia coli enteroinvasive strains; Escherichia coli enteropathogenic strains; Escherichia coli enterotoxigenic strains; Escherichia coli O157:H7; Klebsiella ssp.; Plesiomonas shigelloides; Salmonella ssp.; Shigella ssp.; Vibrio cholerae; and Yersinia enterocolitica to a greater extent than the culture alone.

Description

LEGENDS TO FIGURES

(1) FIG. 1: Development of pH during ripening (3 days) and storage under vacuum at 15 C. (35 days) until the end of shelf life (38 days). Grey line is grobe Teewurst prepared by applying Staphylococcus xylosus (DSM 28308), Lactobacillus curvatus (DSM 18775) and Pediococcus acidilactici (DSM 28307) and black line is grobe Teewurst prepared by the same culture to which 0.5% red wine extract has been added.

(2) FIG. 2: Development of Salmonella cell count during ripening (3 days) and storage under vacuum at 15 C. (35 days) until the end of shelf life (38 days). Grey line is grobe Teewurst prepared by applying Staphylococcus xylosus (DSM 28308), Lactobacillus curvatus (DSM 18775) and Pediococcus acidilactici (DSM 28307) and black line is grobe Teewurst prepared by the same culture to which 0.5% red wine extract has been added.

(3) FIG. 3: Development of pH in the batches prepared as described in Example 3.

(4) FIG. 4: Development of Salmonella spp. cell count for the batches prepared as described in Example 3.

DEPOSITED STRAINS

(5) The Lactobacillus curvatus strain CHCC9720 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under the accession number DSM 18775 with a deposit date of Nov. 9, 2006 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

(6) The Pediococcus acidilactici CHCC4073 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under the accession number DSM 28307 with a deposit date of Jan. 30, 2014 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

(7) The Staphylococcus xylosus strain CHCC5680 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under the accession number DSM 28308 with a deposit date of Jan. 30, 2014 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

(8) For the above-identified deposited microorganisms, the following additional indications apply:

(9) As regards the respective Patent Offices of the respective designated states, the applicants request that a sample of the deposited microorganisms stated above only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn.

(10) Embodiments of the present invention are described below, by way of non-limiting examples.

EXAMPLES

Example 1: Salmonella Challenge Test

(11) The effect of a combination of a bacteriocin-producing culture and red wine extract towards Salmonella was shown in a challenge test which was performed according to the following description at appropriate facilities.

(12) Materials and Methods:

(13) A standard meat mince for grobe Teewurst (see exemplarily shown standard recipe) was supplemented with Staphylococcus xylosus (DSM 28308), Lactobacillus curvatus (DSM 18775) and Pediococcus acidilactici (DSM 28307) with a cell count concentration in a range of 1.0E+6 cfu/g to 1.0E+7 cfu/g of meat for each strain.

(14) TABLE-US-00001 TABLE 1 Ingredient Addition [%] Total [kg] Gammon piece or silverside of pork 50 12,500 Neck fat 50 12,500 Meat base 100 25,000 Dextrose 0.35 0.088 Pepper white 0.20 0.050 Paprika 0.10 0.025 Sodium ascorbate 0.04 0.010 Mace 0.04 0.010 Cardamom 0.02 0.005 Paprika extract 0.01 0.003 Nitrite curing salt (0.5%) 2.40 0.600

(15) The meat mince was subsequently inoculated by 5.0E+01 cfu/g of three different S. enterica subsp. typhimurium strains (DSM554), a wild isolate from raw fermented sausage and a wild isolate from fresh pork meat (strains TZL-038 and TZL-039 from the strain collection of TZL-MiTec GmbH) (Technologietransfer Zentrum Lippe). In addition to that the meat mince was supplemented with 24 g/kg of nitrite curing salt (NPS).

(16) The prepared meat mince was divided into 2 batches. Into one of the batches 5 g/kg of red wine extract (697808 Red Wine Extract powder) commercially available from Chr. Hansen A/S was added. To the control batch no red wine extract was added. Both batches of meat mince were filled into standard casings with a diameter of 50 mm and a length of 230 mm with a target filling weight of approx. 285 g.

(17) For ripening the prepared spreadable sausages were transported to the laboratory of meat technology of the Hochschule Ostwestfalen-Lippe. The target weight for end of ripening was 250 g. The ripening was performed according to table 2.

(18) TABLE-US-00002 TABLE 2 Ripening program Duration Temperature Humidity Aeration Weight loss 24 h 25 C. 80% Medium 4-5% 12 h 22 C. 80% Medium 14 h Smoke 75% Medium 9-10% 12 h 18 C. 75% High Target: 12%

(19) In the following the sausages were analyzed in regard to the cell count of Salmonella (day 0=raw material, day 3=end of ripening, day 10, day 17, day 24=mid of shelf life, day 31, day 38=end of shelf life) by the procedure described in BVL L 00.00-20 Salmonellen (Bundesamt fr Verbraucherschutz and LebensmittelsicherheitUntersuchung von LebensmittelnHorizontales Verfahren zum Nachweis von Salmonella spp. in Lebensmitteln (bernahme der gleichnamigen Norm DIN EN ISO 6579, Ausgabe Oktober 2007)).

(20) Results:

(21) pH:

(22) The pH of the different batches was determined during the whole process at different time points (day 0, 3, 5, 10, 17, 24, 31 and 38). FIG. 1 shows the course of pH of both batches, the control batch where only the bacteriocin-producing culture was added and the sample batch where the red wine extract was added additionally.

(23) Cell Count Salmonella:

(24) FIG. 2 shows the development of the Salmonella cell count for both the control batch where only the bacteriocin-producing culture was added and the sample batch where the red wine extract was added additionally. As evident from the figure there is a clear reduction of the cell count of Salmonella when adding red wine extract in addition to the bacteriocin-producing culture compared to the control batch without the addition of red wine extract. From day 24 all samples (25 g) were negative for Salmonella when using the combination of bacteriocin-producing culture and red wine extract.

(25) Conclusion:

(26) FIG. 2 illustrates that from day 24 until the end of shelf life S. typhimurium could not be detected anymore in sausages produced by the combination of red wine extract with the culture comprising Staphylococcus xylosus (DSM 28308), Lactobacillus curvatus (DSM 18775) and Pediococcus acidilactici (DSM 28307) whereas Salmonella was still present in sausages produced with the culture only.

Example 2: Bacteriocin Activity Test

(27) The aim of this test is to quantitatively determine the activity of group IIa bacteriocins produced by a culture comprising at least one bacteriocin-producing lactic acid bacterial strain by using a rapid microtiter assay which is a modification and combination of the assays described by Budde et al., 2003.

(28) It is generally recognized that group IIa bacteriocins inhibit Lactobacillus sakei NCFB 2714, hence this strain is often used as indicator organism in assays for detection of class IIa bacteriocins. The present microtiter assay gives a quantitative measure of the bacteriocin activity of class IIa bacteriocins expressed as Arbitrary Units (AU/ml) based on the number of two-fold dilutions of a culture supernatant causing 50% growth inhibition of the indicator organism Lactobacillus sakei NCFB 2714.

(29) The culture fermentate of a culture comprising at least one lactic acid bacterial strain putatively producing a bacteriocin is prepared by inoculating MRS broth with the bacteriocin-producing strain or culture to be tested to reach an OD.sub.600 of 0.01-0.02 (OD.sub.600 after subtraction of the blank value i.e. non inoculated MRS) which corresponds to a cell concentration of about 1E+07 cfu/ml. The culture is inoculated overnight (about 18 h) at 30 C. The cell suspension is centrifuged at 4500 g for 15 min at 5 C. The supernatant is removed and adjusted to pH 6.00.1 by using NaOH or 0.2N HCL followed by filter sterilization (0.2 m).

(30) The microtiter assay is prepared by first preparing the indicator solution strain by diluting 0.2 mL of the Lactobacillus sakei NCFB 2714 strain in 18 mL MRS broth. In each well 50 l MRS-broth is added except for the first well of each row and the wells from the rows G and H and 50 l of supernatant is added to the first and the second wells of the two first rows. From the well of the second row, 50 l is transferred to the next well and this step is repeated. The content of each well has to be thoroughly mixed with the pipette before transferring liquid to the next well. Then 150 l of the indicator strain is added to each well except row H (the blank). 10 l of proteinase K is added in each well of row G. The microtiterplate is read in the microreader by measuring the OD.sub.600 every 30 min during 20 hours at 30 C.

(31) Calculation of Bacteriocin Activity

(32) The OD.sub.600 (0 h) is subtracted from The OD.sub.600 (20 h). The activity of class IIa bacteriocins is reported in Arbitrary Units (AU/ml) defined as the reciprocal of the highest two-fold dilution showing 50% growth inhibition, calculated as 50% of the turbidity obtained for the indicator organism in the absence of bacteriocin solution.

Example 3: Salmonella Challenge Test in a Teewurst Meat Model System

(33) The effect of a combination of two class IIa bacteriocin-producing cultures B-LC-20 (Pediococcus acidilactici) and F-LC (Staphylococcus xylosus, Pediococcus acidilactici, and Lactobacillus curvatus) and a red wine extract (RWE) towards Salmonella spp. in particular towards S. enterica serovar Typhimurium DSM11320 was tested in a challenge test performed in a meat model system based on a traditional Teewurst formulation. DSM11320 is a non-pathogenic S. enterica strain which is classified as risk class I according to TRBA 466 (Technische Regeln fr biologische ArbeitsstoffeEinstufung von Prokaryonten (Bacteria and Archaea) in Risikogruppen). The challenge test was performed according to the following description at appropriate facilities.

(34) Materials and Methods:

(35) A standard meat mince for feine Teewurst (see table 3) was produced according to the production process described (see table 4) and supplemented with dextrose (C*PharmDex 02010, Cargill Deutschland GmbH, Cerestarstrasse 2, D-47809 Krefeld) and RWE (697808 Red Wine Extract powder) and the cultures T-D-66 (Lactobacillus plantarum, and Staphylococcus carnosus) or B-LC-20 (Pediococcus acidilactici) and F-LC (Staphylococcus xylosus, Pediococcus acidilactici, and Lactobacillus curvatus) according to the batch description (see table 5). The applied cultures are commercially available from Chr. Hansen A/S and were dosed according to the standard recommendation of Chr. Hansen. The batches 1-6 were subsequently inoculated with S. enterica serovar Typhimurium DSM11320 with a target cell count of 1.0E+5 cfu/g of meat.

(36) TABLE-US-00003 TABLE 3 Standard recipe for Teewurst Ingredient Pork silverside (0 C.) 6.0 kg 60% Pork back fat (0 C.) 4.0 kg 40% Casings Nitrite Salt (0.5%) 26 g/kg 2.6% Dextrose According to batch description White pepper 2 g/kg 0.2% Paprika powder sweet 2 g/kg 0.2% Sodium ascorbate 0.5 g/kg 0.05% Mace 0.5 g/kg 0.05% Cardamom 0.2 g/kg 0.02% Total 10 kg 100%

(37) TABLE-US-00004 TABLE 4 Meat preparation and production process Raw material PI and PVII mincing to 3 mm hole plate preparation Store in chiller (0 C.) till production Chopping and filling Addition of meat and fat process Addition of spices, starter culture- and protective culture at 1000 U/min After 10 rounds addition of nitrite salt Chopping at 4000 U/min till 9 C. Stuffing in 200 g beaker

(38) TABLE-US-00005 TABLE 5 Batch description Batch Dextrose Dextrose number Description RWE (5 g/kg) (4 g/kg) (1 g/kg) 1 T-D-66 + DSM11320 X 2 T-D-66 + DSM11320 X X 3 T-D-66 + DSM11320 X X 4 B-LC-20 + F-LC + X DSM11320 5 B-LC-20 + F-LC + X X DSM11320 6 B-LC-20 + F-LC + X X DSM11320

(39) The prepared beakers were fermented and ripened according to the temperature profile shown in FIG. 3 mimicking a standard process for the production of Teewurst. The meat model system was not subjected to a drying procedure.

(40) Results:

(41) pH:

(42) The development of pH is shown in FIG. 3. The pH of the different batches was determined during the first 100 hours of the process by continuous measurement. In the following the pH was only measured after 124 h, 268 h, 436 h and 916 h in 3 beakers per batch by 3 individual measurements for each.

(43) Cell Count of Salmonella spp.:

(44) FIG. 4 shows the development of the Salmonella spp. cell count for the relevant batches. As evident from the figure there is a clear reduction of the cell count of Salmonella spp. when adding red wine extract (RWE) in addition to the class IIa bacteriocin-producing cultures B-LC-20 (Pediococcus acidilactici) and F-LC (Staphylococcus xylosus, Pediococcus acidilactici, and Lactobacillus curvatus) (batch 5 and 6) compared to the control batch 4 where only the class IIa bacteriocin-producing cultures and no RWE was added. The most significant reduction of Salmonella spp. can be seen for batch 5. It is also evident from FIG. 4 that the combination of a standard non-bacteriocin producing culture (T-D-66) with the RWE is not leading to a significant reduction in cell count of Salmonella spp. and that no difference can be observed in comparison to control batch 1 were no RWE was added but only the non-bacteriocin producing culture.

(45) Conclusion:

(46) The results of above described experiment confirm previous findings that the combination of a class IIa bacteriocin-producing culture and a RWE is able to reduce Salmonella spp. significantly. As evident from FIG. 4 the application of any single component of the concept neither the RWE alone respectively in combination with a standard non-bacteriocin producing culture (batch 2 and 3) nor the class IIa bacteriocin-producing cultures without the addition of the RWE (batch 4) leads to a similar reduction of DSM11320. It is also evident that the addition of RWE to a standard non-bacteriocin producing culture (batch 2 and 3) is not having any added value in regard to the reduction of Salmonella compared to control batch 1 where only the non-bacteriocin producing culture is added.

(47) However, it is also evident that the more significant pH drop by the addition of RWE is having an influence on the reduction of Salmonella spp. but it can be clearly seen by comparing batch 6 and batch 2/3 which show a similar final pH, that the pH drop alone cannot explain the more pronounced reduction of Salmonella in batch 6. From this comparison it is clearly evident that only the combination of a class IIa bacteriocin producing culture with the RWE is leading to a significant reduction of Salmonella spp. in the Teewurst model system.

REFERENCES

(48) Budde et al., Leuconostoc carnosum 4010 has the potential for use as a protective culture for vacuum-packed meats: culture isolation, bacteriocin identification, and meat application experiments, International Journal of Food Microbiology, Vol. 83, Issue 2, 15 Jun. 2003, 171-184 BVL L 00.00-20 Salmonellen (Bundesamt fr Verbraucherschutz and LebensmittelsicherheitUntersuchung von LebensmittelnHorizontales Verfahren zum Nachweis von Salmonella spp. in Lebensmitteln (bernahme der gleichnamigen Norm DIN EN ISO 6579, Ausgabe Oktober 2007)) Eijsink et al., Comparative studies of class IIa bacteriocins of lactic acid bacteria, Applied and Environmental Microbiology, 1998, 3275-3281 Ennahar et al., Class IIa bacteriocins: Biosynthesis, structure and activity, FEMS Microbiology Reviews, 24, 2000, 85-106 Papagianni et al., Determination of bacteriocin activity with bioassays carried out on solid and liquid substrates: assessing the factor indicator microorganism, Microbial Cell Factories, 2006, 5:30 Vermeiren et al., Evaluation of meat born lactic acid bacteria as protective cultures for the biopreservation of cooked meat products, International Journal of Food Microbiology, 96, 2004, 149-164