Method for reducing the content of pathogenic organisms present in food materials

Abstract

The present invention relates to a method for reducing the concentration of pathogenic organisms such as Listeria spp. in fermented food products. The method comprises the steps of: (i) providing a food material, (ii) mixing said food material with a starter culture, (iii) mixing the food material with at least one adjunct culture in form of a bacteriocin-producing Pediococcus species, (iv) subjecting the mixture provided in step (iii) to a fermentation process, said fermentation process being con-ducted at conditions that are sub-optimal for growth of the bacteriocin-producing Pediococcus species in order to provide a limited acidification and allow for a high production of bacteriocin, and obtain a fermented food product. Furthermore, the invention relates to the use of bacteriocin-producing Pediococcus species as an adjunct culture for securing microbial safety of fermented food products.

Claims

1. A fermented food product produced by a method comprising: (i) mixing a food material with (a) a starter culture and (b) at least one adjunct culture comprising Pediococcus acidilactici that produces a bacteriocin; (ii) fermenting the food material at a sub-optimal temperature for growth of the Pediococcus acidilactici of 30 C. or below; and (iii) ripening the food material; wherein during fermentation the acidification caused by the Pediococcus acidilactici is about 0.5 pH unit or less, and wherein growth of Listeria monocytogenes during fermentation and/or ripening is suppressed, and wherein the pH of the fermented food product is at most 0.5 pH units lower than that of a fermented food product produced by the same method except without the Pediococcus acidilactici adjunct culture.

2. The fermented food product of claim 1, wherein the bacteriocin is selected from the group consisting of pediocin, bavaracin, sakacin, curvacin, leucosin, and plantaracin.

3. The fermented food product of claim 1, wherein at least 75% of the L. monocytogenes are killed, inactivated, or inhibited by the end of ripening.

4. The fermented food product of claim 1, wherein the method further comprises a drying process performed under sub-optimal conditions for growth of the P. acidilactici.

5. The fermented food product of claim 1, wherein the fermenting is performed under further sub-optimal growth conditions for the P. acidilactici provided by at least one parameter selected from water activity, relative humidity, atmospheric composition, curing salts, added nutrients, and other additives.

6. The fermented food product of claim 1, wherein the method reduces counts of Listeria monocytogenes by at least 2 log cfu/g of fermented food by the end of ripening.

7. The fermented food product of claim 1, wherein the fermented food product is selected from the group consisting of cheese, sausage, ham, fish, and vegetables.

8. The fermented food product of claim 1, wherein the fermented food product is sausage.

9. A fermented food product produced by a method comprising: (i) mixing a food material with (a) a starter culture and (b) at least one adjunct culture comprising Pediococcus acidilactici that produces a bacteriocin and (ii) fermenting the food material at a sub-optimal temperature for growth of the Pediococcus acidilactici of 30 C. or below, wherein during fermentation the acidification caused by the Pediococcus acidilactici is about 0.5 pH unit or less, and wherein growth of Listeria monocytogenes during fermentation is suppressed, and wherein the pH of the fermented food product is at most 0.5 pH units lower than that of a fermented food product produced by the same method except without the Pediococcus acidilactici adjunct culture.

10. The fermented food product of claim 9, wherein the bacteriocin is selected from the group consisting of pediocin, bavaracin, sakacin, curvacin, leucosin, and plantaracin.

11. The fermented food product of claim 9, wherein at least 75% of the L. monocytogenes are killed, inactivated, or inhibited by the end of ripening.

12. The fermented food product of claim 9, wherein the method further comprises a drying process performed under sub-optimal conditions for growth of the P. acidilactici.

13. The fermented food product of claim 9, wherein the fermenting is performed under further sub-optimal growth conditions for the P. acidilactici provided by at least one parameter selected from water activity, relative humidity, atmospheric composition, curing salts, added nutrients, and other additives.

14. The fermented food product of claim 9, wherein the method reduces counts of Listeria monocytogenes by at least 2 log cfu/g of fermented food by the end of ripening.

15. The fermented food product of claim 9, wherein the fermented food product is selected from the group consisting of cheese, sausage, ham, fish, and vegetables.

16. The fermented food product of claim 9, wherein the fermented food product is sausage.

17. A method for producing a fermented food product, comprising: fermenting a food material with at least one adjunct culture comprising Pediococcus acidilactici that produces bacteriocin, wherein during fermentation the acidification caused by the Pediococcus acidilactici is about 0.5 pH units or less.

Description

(1) The invention is further illustrated in the following non-limiting examples and in the drawings, where

(2) FIG. 1A illustrates the pH-development during ripening of sausages applied with or without B-LC-20 together with a Chr. Hansen BACTOFERM fast fermenting starter culture F1. Sausages were fermented at 24-20 C. for 3 days, followed by ripening at 18 to 16 C. for 11 days.

(3) FIG. 1B illustrates the pH-development during ripening of sausages applied with or without B-LC-20 together with a Chr. Hansen BACTOFERM fast fermenting starter culture F-SC-111. Sausages were fermented at 24-20 C. for 3 days, followed by ripening at 18 to 16 C. for 11 days.

(4) FIG. 2A illustrates pH-development during ripening of sausages applied with or without B-LC-20 together with a Chr. Hansen BACTOFERM traditional fermenting starter culture T-SPX. Sausages were fermented at 24-20 C. for 4 days, followed by ripening at 18 to 14 C. for 17 days.

(5) FIG. 2B illustrates pH-development during ripening of sausages applied with or without B-LC-20 together with a Chr. Hansen BACTOFERM traditional fermenting starter culture T-SC-150. Sausages were fermented at 24-20 C. for 4 days, followed by ripening at 18 to 14 C. for 17 days.

EXAMPLES

Example 1

(6) Influence of B-LC-20 on pH Development in Sausage Mince.

(7) The influence of the adjunct culture on acidification is demonstrated and a summary of the pH/time profiles encountered when applying B-LC-20 to sausage mince. Table 1 shows the pH development during the fermentation period as determined every second day and table 2 shows the pH development as determined continuously from 0 to 68 hours.

(8) TABLE-US-00001 TABLE 1 pH development in sausage mince determined each second day Code Day 0 Day 2 Day 4 Day 6 Control (starter 5.79 0.04 4.81 0.03 4.80 0.04 4.79 0.01 culture) Control + 5.79 0.01 4.77 0.01 4.77 0.01 4.80 0.04 B-LC-20

(9) TABLE-US-00002 TABLE 2 pH development in sausage mince determined by continuous measurement Code/Hours 0 10 20 30 40 50 60 68 Control (starter 5.62 5.71 5.58 5.16 4.97 4.86 4.79 4.77 culture) Control + 5.62 5.70 5.55 5.12 4.91 4.80 4.76 4.76 B-LC-20

(10) The results show that there is no significant influence on the final pH of adding the adjunct culture in form of B-LC-20 together with the control culture as compared to adding the control culture alone. B-LC-20 was added in a concentration of 1.110.sup.7 CFU/g mince. Control culture was added in a total lactic acid bacteria concentration of 3.310.sup.6 CFU/g.

(11) It is shown that there is a slight influence of B-LC-20 during the first 20-60 hours, but the maximum difference between the two curves is 0.06 pH-unit which is well within the normal batch to batch variation encountered in real sausage productions.

(12) The control starter culture consists of a blend of lactobacilli, pediococci, micrococci and staphylococci.

Example 2

(13) Influence of B-CL-20 on Listeria Reduction

(14) Summary of Trials

(15) Two independent trials to assess the behavior of Listeria in sausages along the ripening process were performed. Three batches were manufactured in each trial. One with the control starter culture non active against Listeria and two batches with the control starter culture together with an antilisteria starter culture added at two different concentrations (B-LC-20, Chr. Hansen A/S). Each batch was inoculated with a cocktail of five Listeria monocytogenes strains (approx. 10.sup.3 CFU/g) at the time of manufacturing.

(16) Ripening was done in an adapted versatile environment test chamber Sanyo Model MLR-350H, with a fermentation period for 72 h at 14 C. with 80% RH.

(17) At the selected times three sausages were sampled from each batch for determining Listeria and Lactic acid Bacteria counts, pH and weight loss.

(18) The behavior of Listeria was similar in both trials, showing important differences between the control batch (A) and the batch added the antilisteria containing cultures (B and C) where Listeria diminished 2 log cfu/g (trial 1) and 3 log cfu/g in trial 2. No significant differences in the Listeria counts between batches B and C were observed.

(19) In conclusion the above result points out that the bacterial culture B-LC-20 proved to be a suitable culture for sausages manufactured according to the present formulation, showing additional Listeria reduction after the fermentation period and till the end of the ripening as compared to the control starter culture alone.

(20) Methodology

(21) A Listeria challenge test in dry sausages along the ripening process was designed according to the following protocol. Two independent trials (trial 1 and trial 2) were performed at Institut de Recerca I Tecnologia Agroalimentries (IRTA), Monells, Spain. The anti-Listeria activity of the Lactic acid Bacteria culture B-LC-20 at two different concentrations were tested.

(22) Bacterial Cultures

(23) Chr. Hansen anti.listeria culture B-LC-20 and the control starter culture was stored frozen (20 C.) until use.

(24) Listeria monocytogenes: Two strains were from the strain collection of IRTA, i.e. strain CTC1011 and CTC1034; and three strains were supplied by Chr. Hansen (P01, P05, P15). Each strain was separately grown in the IRTA standard medium TSBYE and stored frozen (20 C.). Viable counts were determined before each trial in order to calculate the appropriate dilution to reach the expected inoculation in the meat mixture (approximately 10.sup.3 CFU/g).

(25) Manufacturing

(26) Three batches (12 Kg each) were manufactured.

(27) Batch A Control (starter culture)+Listeria monocytogenes cocktail

(28) Batch B Control (starter culture)+B-LC-20 low concentration

(29) Batch C Control (starter culture)+B-LC-20 high concentration

(30) The bacterial cultures were added separately at the time of mixing. First was added L. monocytogenes cocktail (in 20 ml saline solution), followed by the starter culture and the adjunct culture.

(31) Ripening Conditions

(32) Fermentation and drying of sausages were done in an adapted versatile environmental test chamber Sanyo Model MLR-350H.

(33) Fermentation was carried out for 72 h at 24 C. with RH>90%.

(34) After the fermentation period the conditions were adjusted for drying until day 29 at 14 C. with 80% RH.

(35) Analyses

(36) During fermentation and drying, pH and weight loss were measured in 3 marked sausages per batch, daily during the first week and with an interval of 3-4 days during the last 3 weeks.

(37) Microbial Analyses

(38) Three different sausages from each batch (A, B, C) were nalaysed at each sampling time (days: zero (after 4 hours), 2, 7, 14 and 29). Each sample consisted of 25 grams of a previous homogenized sausage.

(39) The determination of Listeria was done at each sampling time, except at day 0, by Most Probably Number technique (MPN) in Fraser Broth Base (Oxoid)+Half Fraser selective supplement (Oxoid) (48 hours, 37 C.) followed by confirmation of the positive tubes in Palcam Listeria Selective Agar Base (Merck)+Selective Supplement att. Van Netten et al. (Merck) (48 hours, 37 C.).

(40) Listeria counts at time zero were done by spreading the appropriate dilutions in Palcam supplemented agar plates and incubating at 37 C. for 72 hours. Lactic acid bacteria counts were performed at each sampling time in MRS agar (72 hours at 30 C. under anaerobic conditions).

(41) TABLE-US-00003 TABLE 1 Listeria spp. And Lactic Acid Bacteria counts along the ripening of dry sausages in trial 1. Batch Time (days) Listeria spp. Lactic Acid Bacteria A 0 3.31 0.02 6.47 0.11 A 2 2.69 0.33 8.85 0.05 A 7 3.15 0.19 8.93 0.07 A 14 2.45 0.18 8.73 0.06 A 29 2.34 0.35 8.60 0.01 B 0 3.25 0.04 7.50 0.06 B 2 2.24 0.12 8.73 0.08 B 7 1.77 0.35 8.78 0.17 B 14 1.18 0.39 8.61 0.03 B 29 0.89 0.69 8.51 0.02 C 0 3.22 0.10 7.77 0.05 C 2 2.05 0.41 8.68 0.07 C 7 1.48 0.26 8.75 0.20 C 14 1.43 0.51 8.52 0.04 C 29 0.54 0.16 8.41 0.09 Values are the average of triplicate samples expressed as log cfu/g standard deviation.

(42) Batches were inoculated as follows:

(43) Batch A (control starter culture, 3.010.sup.6 CFU/g), Batch B (control starter culture+B-LC-20 low concentration (2.910.sup.7 CFU/g mince)), Batch C (control starter culture+B-LC-20 high concentration (5.610.sup.7 CFU/g mince)).

(44) All the batches were inoculated with a cocktail of 5 different Listeria monocytogenes strains (CTC1011, CTC1034, P01, P05, P15).

(45) TABLE-US-00004 TABLE 2 pH and weight loss along the ripening of dry sausages in trial 1. Batch Time (days) pH Weight loss % A 0 5.82 0.01 NA A 1 5.57 0.04 1.54 0.73 A 2 4.90 0.09 2.96 1.49 A 3 4.67 0.04 5.79 1.13 A 4 4.60 0.01 10.36 0.79 A 7 4.69 0.01 17.04 0.60 A 11 4.75 0.01 21.66 0.54 A 14 4.71 0.01 24.31 0.34 A 18 4.86 0.02 27.04 0.38 A 24 4.75 0.01 30.23 0.33 A 29 4.99 0.13 31.89 0.36 B 0 5.79 0.03 NA B 1 5.50 0.05 1.22 0.64 B 2 4.92 0.01 2.46 0.76 B 3 4.61 0.03 4.24 0.74 B 4 4.49 0.01 8.71 1.15 B 7 4.55 0.02 15.92 0.71 B 11 4.58 0.01 21.71 0.33 B 14 4.49 0.17 25.02 0.65 B 18 4.69 0.01 28.36 0.39 B 24 4.63 0.01 31.27 0.58 B 29 4.75 0.01 32.89 0.61 C 0 5.83 0.01 NA C 1 5.37 0.01 1.32 0.49 C 2 4.89 0.03 2.67 0.53 C 3 4.59 0.02 5.104.89 0.03 C 4 4.49 0.01 9.66. 0.33 C 7 4.55 0.01 16.69. 0.26 C 11 4.61 0.01 22.57 0.30 C 14 4.57 0.02 25.57 0.28 C 18 4.69 0.01 28.60 0.07 C 24 4.62 0.01 31.56 0.25 C 29 4.74 0.03 33.15 0.26 NA = non applicable Values are the average of triplicate samples standard deviation.

(46) TABLE-US-00005 TABLE 3 Listeria spp. and Lactic Acid Bacteria counts along the ripening of dry sausages in trial 2. Batch Time (days) Listeria spp. Lactic Acid Bacteria A 0 3.40 0.01 6.41 0.15 A 2 2.56 0.54 8.83 0.04 A 7 2.68 0.36 8.75 0.04 A 14 1.86 0.19 8.76 0.03 A 29 1.56 0.35 8.39 0.14 B 0 3.39 0.03 7.25 0.07 B 2 2.04 0.12 8.56 0.10 B 7 1.13 0.26 8.58 0.11 B 14 1.16 0.20 8.35 0.08 B 29 0.33 0.17 8.33 0.08 C 0 3.67 0.05 7.79 0.02 C 2 1.87 0.19 8.50 0.08 C 7 1.56 0.35 8.47 0.07 C 14 0.84 0.20 8.52 0.12 C 29 0.23 0.23 8.34 0.04 Values are the average of triplicate samples as log cfu/g standard deviation.

(47) Batch A (control starter culture, 2.610.sup.6 CFU/g), Batch B (control starter culture+B-LC-20 low concentration (1.510.sup.7 CFU/g), Batch C (control starter culture+B-LC-20 high concentration (5.910.sup.7 CFU/g).

(48) All the batches were inoculated with a cocktail of 5 different Listeria monocytogenes strains (CTC1011, CTC1034, P01, P05, P15).

(49) TABLE-US-00006 TABLE 4 pH and weight loss along the ripening of dry sausages in trial 2. Batch Time (days) PH Weight loss % A 0 6.10 0.07 NA A 1 5.83 0.02 3.09 1.27 A 2 5.04 0.03 4.73 1.13 A 3 4.86 0.03 7.30 1.39 A 4 4.90 0.01 10.03 0.89 A 7 4.91 0.01 14.81 0.76 A 11 4.95 0.01 19.19 0.53 A 14 4.94 0.01 21.14 0.48 A 18 4.98 0.01 24.08 0.66 A 24 5.04 0.01 27.41 0.44 A 29 5.04 0.01 29.92 0.43 B 0 5.97 0.02 NA B 1 5.52 0.07 3.79 1.48 B 2 4.98 0.02 5.17 1.03 B 3 4.72 0.03 7.90 1.70 B 4 4.72 0.01 11.31 1.06 B 7 4.71 0.03 16.53 0.95 B 11 4.74 0.01 21.84 0.38 B 14 4.75 0.01 23.96 0.39 B 18 4.80 0.02 27.24 0.40 B 24 4.84 0.02 30.90 0.20 B 29 4.89 0.02 33.26 0.13 C 0 5.99 0.01 NA C 1 5.49 0.04 2.77 0.59 C 2 4.93 0.01 4.16 1.16 C 3 4.68 0.01 6.33 0.75 C 4 4.70 0.01 9.985 0.48 C 7 4.68 0.02 15.84 0.60 C 11 4.70 0.01 21.29 0.61 C 14 4.74 0.01 23.52 0.51 C 18 4.79 0.02 26.65 0.62 C 24 4.84 0.02 30.37 0.58 C 29 4.83 0.02 32.69 0.67 NA = non applicable Values are the average of triplicate samples standard deviation.
Discussion

(50) Two independent trials to assess the behavior of Listeria in sausages along the ripening process were performed. Three batches were manufactured in each trial. One control with starter culture non active against listeria and two batches with the same starter culture plus an antilisteria adjunct culture added at two different concentrations (B-LC-20) in each batch. Each batch was inoculated with a cocktail of five Listeria monocytogenes strains (approx. 103 cfu/g) at the time of manufacturing.

(51) In both trials and after 2 days of fermentation Listeria diminished. The counts in batch B and C were lower than in batch A. These differences between the control batch A and the batches inoculated with the antilisterial cultures increased until the end of ripening. In trial 1, by the end of the ripening, Listeria diminished 1 log cfu/g in batch A while in batch B and C Listeria decreased more than 2 logs cfu/g. In trial 2 by the end of ripening Listeria diminished 1.8 (log cfu/g) in batch A, and more than 3 logs in batch B and C.

(52) Lactic acid bacteria counts reached the maximum after 2 days of fermentation with similar values at the end of the process in each batch (around 108 cfu/g) in both trials. The curve of pH was similar for the different batches in both trials. The minimum pH was recorded after 4 days in trial 1 and after 3 days in trial 2 despite the pH at time zero was higher in trial 2. The pH-drop in the control batches were in both trials similar to the pH-drop in the batch with added adjunct culture. pH was between 1.22-1.24 in batch A after 3-4 days of fermentation, and between 1.26-1.34 in batches B and C. The small differences were probably caused by the extra glucose added with the adjunct culture pouch. Weight loss showed similar profile in both trials with no differences between the lots.

(53) Conclusion

(54) The culture B-LC-20 proved to be a suitable protective adjunct culture for fermented sausages manufactured according to the present formulation, showing additional Listeria reduction after the fermentation period and till the end of ripening, compared to a control starter culture alone.

(55) In general, addition of extra inoculum of lactic acid bacteria reduces the time to on-set of fermentation (the lag phase) and thereby speed up the overall acidification rate. When increasing the inoculum by 10 times (from 5.Math.10.sup.5 to 5.Math.10.sup.6 CFU/g) acidification lag phase for a typical North European type fermented sausage was halfed and the time to reach pH of 5.3 and 4.9 reduced by 25 and 30%, respectively.

(56) Addition of B-LC-20 to the sausage recipe in Example 2, trial 1 resulted in increased inoculation level of total lactic acid bacteria of approx. 15 times, from 3.Math.10.sup.6 to between 3.Math.10.sup.7-6.Math.10.sup.7. It was expected that the lag phase would have been reduced considerably and the time to reach pH 4.9 reduced by at least 30%. This expected reduction did not take place, pH reached 4.9 after 2 days for all three batches, i.e. addition of an adjunct culture such as B-LC-20 to the existing recipe did not speed up acidification time as expected. In example 1, table 1 and 2 acidification speed did not increase significantly, either.

(57) Therefore, the inventors of the present invention surprisingly found that the use of an adjunct culture such as B-LC-20 provides a unique anti-listerial reduction for fermented sausages since it was found that Pediococcus acidilactici is a strong producer of pediocin (which destroys Listeria monocytogenes) at European fermentation temperatures (<26 C.) while not being a strong acidifier at this temperature.

(58) The reduction of Listeria is primarily caused by pediocin produced by an adjunct culture such as B-LC-20 in the food material during the fermentation and drying process. The effect of pediocin is a well known phenomenon in the literature.

(59) However, the uniqueness of the adjunct culture (B-LC-20) and the method disclosed herein is that the food manufacturer can use the adjunct culture together with the normal acidification culture since it does not alter the overall acidification profile and the quality of the product significantly. As mentioned above, the acidification profile is of utmost importance for the sensory quality. Thus, the manufacturer does not need to change his present recipe or processing conditions, but will get the advantage of reduction in Listeria numbers.

Example 3

(60) Influence of the Adjunct Culture on the Acidification Profile of Different Fermented Sausages

(61) The influence of the adjunct culture B-LC-20 on the acidification profile of four types of sausages fermented with different starter cultures are demonstrated in FIGS. 1 and 2. In all cases, the addition of the adjunct culture in the sausage mince together with the starter culture did not influence the acidification profile of the sausages significantly. In addition, internal sensory evaluations showed that the sensory quality of the sausages was unchanged by addition of B-LC-20.

(62) TABLE-US-00007 Organisms Inoculation, total LAB F-1 Pediococcus pentosaceus 5 10.sup.6 CFU/g mince Staphylococcus xylosus F-SC-111 Lactobacillus sakei 1 10.sup.7 CFU/g mince Staphylococcus carnosus T-SPX Pediococcus pentosaceus 5.5 10.sup.6 CFU/g mince Staphylococcus xylosus T-SC-150 Lactobacillus sakei 1 10.sup.7 CFU/g mince Staphylococcus carnosus

LITERATURE

(63) Foegeding, P. M.; Thomas, A. B.; Pilkington, D. H.; Klaenhammer, T. R. 1992. Enhanced control of Listeria monocytogenes by in situ-produced pediocin during dry fermented sausage production, Applied and Environmental Microbiology, Vol. 58(3), page 884-890. Tjener, K., Stahnke, L. H., Andersen, L., Martinussen, J. 2003. A fermented meat model system for studies of microbial aroma formation. Meat Science, 66(1), 211-218. Utility model BA 1994 00266