# Patent

## Title
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 conducted 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.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a Continuation Application of U.S. Ser. No. 11/578,331 filed Aug. 6, 2007 which is the U.S. National Phase of PCT/DK2005/000259, filed Apr. 15, 2005, which claims priority from Danish Patent Application No. PA 2004 00596 filed Apr. 15, 2004. The entire subject matter of each of these applications is incorporated by reference.


FIELD OF INVENTION
The present invention relates to the field of improving microbial safety in the production of food products. In particular the present invention relates to microbial strains useful for reducing the amount of pathogenic organisms e.g., Listeria when added to a fermented food products, such as a fermented meat product
PRIOR ART
During the production of fermented food products, such as e.g. sausage products, a starter culture is most often applied in order to control the fermentation process instead of relying on the natural developing flora. Commonly, the starter culture comprises a combination of one or more lactic acid bacteria (LAB) and one or more species from the Micrococcaceae and Staphylococcaceae families. During the fermentation process the lactic acid bacteria primarily produce lactic acid whereby pH drops to the desired pH-value depending on the culture and the processing conditions (temperature, sugar type/content etc.) and the food product produced.
Whereas the lactic acid bacteria are mainly responsible for the acid formation, the Micrococcaceae spp. and Staphylococcaceae spp. are responsible for enhancing the flavour formation by producing non-volatile and volatile compounds through various biochemical reaction steps. Additionally, the Micrococcaceae spp. and Streptococcoceae spp. are responsible for the speed and intensity of colour formation in particular in fermented sausage types.
Micrococcaceae spp. and Streptococcoceae spp. are very sensitive to low pH as their growth is drastically slowed down when pH is reduced to a pH below 5.0. In e.g. the manufacturing of dried sausages it is essential for the flavour and colour formation of the meat products that the acidification profile is well-controlled and is not altered from batch to batch. In particular, a fast pH-lowering may impair the quality and result in a less mature and less complex flavour profile that will force the food product manufacturer to ripen the food product for a longer period of time to reach the same flavour intensity (Tjener et al., 2003).
In manufacturing of fermented food products presence of pathogenic organisms like Listeria monocytogenes may be a problem if the raw materials are contaminated. During the production of e.g. fermented sausages Listeria monocytogenes will normally decrease in numbers during the fermentation and ripening period, primarily, due to the formation of lactic acid, the resulting drop in pH and due to the reduction in water activity caused by the subsequent drying process. However, quite often, a considerable number of Listeria monocytogenes survives. This may cause a serious safety problem as consumption of infected food may give rise to lethal listerial infections (listeriosis).
In order to reduce the presence of pathogenic micro-organisms in the food product certain bacteriocin producing lactic acid bacteria including Pediococcus strains and certain Lactobacillus strains have been added to the starter culture to produce bacteriocins some of which kill and/or inactivate the pathogenic organisms and accordingly reduce their concentration in the product.
Foegeding et al. (1992) disclose the effectiveness of pediocin produced in situ by Pediococcus acidilactici as an antilisterial component. However, the fermentation of sausages was conducted at 38° C. which caused an extensive acid production and thus a very fast drop in pH. As mentioned above a rapid pH-lowering impairs the general quality of the product and results in a less mature and less complex flavoured profile. Thus, the adverse influence of Pediococcus acidilactici, on the general quality of the resulting product renders the method unsuitable for food fermentations where the above-mentioned conditions and features apply.
Utility model BA 1994 00266 discloses a lactic acid bacterial starter culture comprising a selected bacteriocin-producing Pediococcus spp. and a selected bavaricin-producing Lactococcus useful for inhibiting pathogenic organisms e.g. Listeria in meat products including fermented meat products.
Evidently, Pediococcus spp. are often not well qualified as starter cultures in the manufacturing of food products although the species are known for their potential for reducing the amount of pathogenic organisms e.g. Listeria. 
Thus, there is a persisting need in the industry to be able to reduce the amount of pathogenic organisms and at the same time obtain optimal characteristics of the fermented food product e.g. acidification profile, flavour and colour development.
SUMMARY OF THE INVENTION
Accordingly, in interesting aspects, methods for A) manufacturing of a fermented food product and B) for reducing the concentration of Listeria spp (in particular Listeria monocytogenes in a fermented food product are provided. Said methods comprise the steps of:

    
    
        (i) providing a food material,
        (ii) mixing the 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 obtained in step (iii) to a fermentation process, said fermentation process being conducted 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 the fermented food product.
    
    


In another aspect of the present invention a fermented food product obtainable by a method of the present invention is provided.
In a further aspect, the present invention provides, bacteriocin-producing Pediococcus species for use as an adjunct culture for securing microbial safety of a fermented food product, wherein said culture, when added to a food fermentation process, is being subjected to conditions that are sub-optimal for growth of said species, thereby producing bacteriocin without significantly affecting the acidification profile of the fermentation.
DETAILED DISCLOSURE
Prior to a discussion of the detailed aspects and embodiments of the invention a definition of specific terms used herein is provided.
As used herein, the term “fermentation” or “food fermentation” refers to the process of biochemical changes e.g. an acidification in animal and/or plant material (i.e. a food matrix), involving activity of live microbial cells under aerobic and/or anaerobic conditions to obtain a food product of desired quality.
The term “adjunct culture” is to be understood as a microbial culture that can be added to a food matrix and produce a bacteriostatic and/or bacteriocritic product (e.g. bacteriocins and antibiotics) without adversely affecting the desired fermentation profile of said food matrix. Preferably, the adjunct culture does not adversely affect the acidification profile during manufacturing of the food product.
It follows that “conditions sub-optimal for growth” are to be understood as growth conditions allowing the adjunct culture, when added to the food matrix, to act as described above.
The term “starter culture” refers to a preparation containing microbial cells that is intended for inoculating a food matrix to be subjected to fermentation. The starter culture is intended for providing the desired change in the characteristics of the food matrix during fermentation (e.g. a desired acidification). Typically, a starter culture will proliferate during the fermentation process.
A “bioprotective agent” is to be understood as a live organism that exerts its bioprotective effect when added to a food matrix without adversely affecting the food matrix.
The bioprotective effect is defined as an effect accomplished by the production of a bacteriostatic and/or bacteriocritic product whereby the presence and/or activity of undesired organisms e.g. Listeria monocytogenes is inhibited and/or diminished.
In the present context, the term “microorganism” is used in its normal meaning. Thus, in its broadest meaning the term “microorganism” is intended to cover algae, protozoa, viruses, bacteria and fungi. Preferred microorganisms are bacteria and fungi, in particular bacteria, such as lactic acid bacteria.
The expression “lactic acid bacteria (LAB)” designates a group of Gram positive, catalase negative, non-motile, microaerophilic or anaerobic bacteria which ferment sugar with the production of acids including lactic acid as the predominantly produced acid, acetic acid, formic acid and propionic acid. The industrially most useful lactic acid bacteria are found among Lactococcus species, Streptococcus species, Enterococcus species, Lactobacillus species, Leuconostoc species, Pediococcus species and Bifidobacterium species.
Commonly used starter culture strains of lactic acid bacteria are generally divided into mesophilic organisms having optimum growth temperatures at about 30° C. and thermophilic organisms having optimum growth temperatures in the range of about 40 to about 45° C. Typical organisms belonging to the mesophilic group include Lactococcus lactis, Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactobacillus casei subsp. casei and Lactobacillus paracasei subsp. paracasei. Thermophilic lactic acid bacterial species include as examples Streptococcus thermophilus, Pediococcus acidilactici, Enterococcus faecium, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus. 
Also the strict anaerobic bacteria belonging to the genus Bifidobacterium including Bifidobacterium bifidum and Bifidobacterium longum are commonly used as dairy starter cultures and are generally included in the group of lactic acid bacteria. Additionally, species of Propionibacterium are used as dairy starter cultures, in particular in the manufacture of cheese. Additionally, organisms belonging to the Brevibacterium genus are commonly used as food starter cultures.
Another group of microbial starter cultures is fungal cultures, including yeast cultures and cultures of filamentous fungi, which are particularly used in the manufacture of certain types of cheese and beverage. Examples of currently used cultures of fungi include Penicillium roqueforti, Penicillium candidum, Geotrichum candidum, Torula kefir, Saccharomyces kefir and Saccharomyces cerevisiae. 
In the production and storage of fermented food products a persisting problem is the potential contamination of the food material by pathogenic organisms such as Listeria spp. In order to overcome this problem the inventors of the present invention surprisingly found that by applying at least one adjunct culture in form of a bacteriocin-producing Pediococcus species to the food material it is possible to reduce the amount of pathogenic organisms without influencing the fermentation profile and hence the desired sensorial quality of the food product. This effect is obtained by subjecting the bacteriocin-producing Pediococcus species to fermentation conditions that are sub-optimal for growth of the Pediococcus species. Typically, such conditions will be optimal for growth of the starter culture and most surprisingly it was found that such conditions allow for a high production of bacteriocin by the Pediococcus species. Accordingly, the present invention allows the food manufacturer to select and use the recipes and processing conditions securing optimal development e.g. acidification of the starter culture and at the same time secure optimal food safety by adding the adjunct culture of the present invention at any suitable point in time during the fermentation process.
As mentioned above, the starter culture and the adjunct culture may be added to the food material in any order. The time lapsed between the addition of the first of the starter culture and the adjunct culture to the addition of the second is 0 seconds, e.g. at the most 10 seconds, such as at the most 30 seconds, e.g. at the most 1 minute, such as at the most 5 minutes, e.g. at the most 10 minute, such as at the most 60 minutes, e.g. at the most 300 minute, such as at the most 600 minutes, e.g. at the most 1 day, such as at the most 2 days, e.g. at the most 3 days, such as at the most 4 days, e.g. at the most 6 days.
In a preferred embodiment of the present invention the starter culture is added at the same time or before the adjunct culture is added to the food material.
The invention is successful in reducing and/or inhibiting the amount and/or activity of any pathogenic organism sensitive to bacteriocins produced by Pediococcus spp., such as pediocin, in particular Listeria spp., such as Listeria monocytogenes. 
It has been shown that the reduction in pathogenic organisms may be provided by mixing the food material containing the pathogenic organisms with a bacteriocin-producing Pediococcus species. Suitable Pediococcus species include Pediococcus pentosaceus and Pediococcus acidilactici, In particular, the Pediococcus acidilactici strain B-LC-20 (DSM 10313) marketed by Chr. Hansen A/S under the trademark SafeProTM is preferred. It is however contemplated that other bacteriocin-producing Pediococcus species may provide the same advantageous characteristics and effects as those illustrated herein.
The effect of the bacteriocin-producing Pediococcus species is most probably due to the tendency of such Pediococcus species to produce bacteriocins capable of killing, inactivating and/or inhibiting pathogenic organisms. Until now it has not been realized that the ability of the Pediococcus species to inhibit pathogenic organisms e.g. Listeria not necessarily needs to cause a general increase in acidification when added as an adjunct culture. The optimal growth temperature for Pediococcus species such as Pediococcus acidilactici is about 40° C. or even higher. However, most starter cultures used for food fermentations develop optimally, i.e. result in the desired sensorial quality of the product, at temperatures below 30° C. Thus, previously it was not always suitable to include Pediococcus species in order to control e.g. Listeria as a reasonable compromise between optimal conditions for the starter vs. optimal conditions for the Pediococcus strain could not be established. The present invention will allow the food manufacturer to freely select desired starter culture organisms and perform food fermentation at conditions that are optimal for the desired development of the food product. At the same time the food safety can be secured by adding the adjunct culture of the present invention without adversely influencing the fermentation profile.
Obviously, when it is realized by the skilled person that production of bacteriocin by Pediococcus species is not linked to an increase in acidification activity it will be possible to test conditions that favor the bacteriocin production without any significant acid production. Such conditions, with an impact on growth of microbial organisms, are well known by the person skilled in the art. They include but are not limited to water activity, atmospheric conditions, Relative Humidity (RH), nutrients such as carbon source, nitrogen source etc. and other additives such as minerals, vitamins etc. Thus, it is within the scope of the present invention that conditions other than temperature, which is used in the examples provided herein, can be used to obtain a sub-optimal growth of the bacteriocin-producing Pediococcus species and thus, obtain the effect of the present invention.
In a preferred embodiment of the present invention the bacteriocin produced by the Pediococcus species is selected from the group consisting of Class II bacteriocins, including bacteriocins such as pediocin, bavaracin, sakacin, curvacin, leucosin and plantaricin.
In one aspect, the present invention provides a method (A) for the manufacturing of a fermented food product.
In another aspect, the present invention provides a method (B) for reducing the concentration of Listeria spp. in a fermented food product
In the present context the term “reducing the concentration” relates to a reduction in the amount of a pathogenic organism. A reduction may be provided by killing, inactivating or inhibiting the activity of the pathogenic organism. In an embodiment of the present invention 100% of the pathogenic organism are killed, inactivated or inhibited, such as at least 90%, e.g. at least 75%, such as at least 50%, e.g. at least 40%, such as at least 30%, e.g. at least 25%, such as at least 20%, e.g. at least 10%, such as at least 5%, e.g. at least 1%.
In certain applications, a “stabilization” of the pathogenic organisms that may be present in the food matrix will be sufficient to render the food safe. Thus, the adjunct culture secures that the pathogenic organisms that are present in the food matrix do not increase in numbers.
Said methods (A and B) comprise the steps of:

    
    
        (i) providing a food material,
        (ii) mixing the 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 conducted at conditions that are sub-optimal growth of the bacteriocin-producing Pediococcus species in order to provide a limited acidification effect abut allow for a high production of bacteriocin, and obtain the fermented food product.
    
    


The fermented food product may be subjected to a drying process simultaneously with the fermentation process in step (iv) and/or subsequent to the fermentation process in step (iv) to obtain a dry fermented food product.
Several food products may be produced by the method according to the present invention provided that the food material is fermented. Examples of fermented food products include, but are not limited to dairy products such as various cheese products, fermented meat product, such as sausages e.g. spreadable and dried sausages and ham, fermented fish and fermented vegetables.
The fermented food product is manufactured by providing a food material which is subjected to a fermentation process and optionally the fermented food product is subjected to a drying process in order to provide a dry fermented food product.
In order to reduce the concentration of the pathogenic organisms, it is desired that this reduction can be provided without significantly altering the quality of the final food product, i.e. the food producer may apply the culture to his present or preferred recipe without otherwise changing the recipe or processing conditions. To obtain the desired effect a culture of bacteriocin-producing Pediococcus species is applied to a food material as an adjunct culture, which is separated from the starter culture. In the present context “adjunct culture” is a culture that is added to the food material or joined with the starter culture, but which does not form part of the starter culture, i.e. the adjunct culture is an additional culture not attempted to “produce” the fermented food product, but to supply an extra technological advantage; in this case a killing, inactivating or inhibiting effect towards pathogenic organisms. In the present context “adjunct culture” and “bacteriocin-producing Pediococcus species” are used interchangeably and adjunct culture is used to further illustrate the specific characteristics of the bacteriocin-producing Pediococcus species.
The manufacturing of the fermented food product is being controlled and performed by the starter culture alone. The starter cultures is responsible for the development of a non-limiting group of quality parameters such as acidification, reduction in water binding and water activity, general appearance, colour, texture, odour, aroma, taste, flavour and other sensorial and technological parameters. Thus, minimal, or preferably no, influence on the quality parameters from the adjunct culture is provided.
In order to limit or eliminate the influence of the bacteriocin-producing Pediococcus species on the quality parameters the fermentation process is conducted at conditions sub-optimal for growth of the bacteriocin-producing Pediococcus species as described hereinbefore.
In a specific embodiment of the present invention the optional drying process is conducted at conditions that are sub-optimal for growth of the bacteriocin-producing Pediococcus species in order to provide a limited acidification effect and allow for a high production of bacteriocin.
In the present context the term “limited acidification” relates to the influence of at least one adjunct culture on acidification. In a preferred embodiment of the present invention the limited acidification provides a difference in pH-value caused by the adjunct culture of 0.5 pH-unit or less, such as 0.25 pH-unit or less, e.g. 0.1 pH-unit or less, such as 0.25 pH-unit or less, e.g. 0.075 pH-unit or less, such as 0.06 pH-unit or less, e.g. 0.05 pH-unit or less, such as 0.04 pH-unit or less, e.g. 0.03 pH-unit or less, such as 0.02 pH-unit or less, e.g. 0.01 pH-unit or less.
In a preferred embodiment of the present invention the sub-optimal growth conditions are provided by changing at least one of the parameters selected from the group consisting of temperature, water activity, RH, atmospheric composition, curing salts, added nutritions, such as the carbon source, additives, such as the chemical acidulent glucono-delta-lactone or various water binding additives and the amounts of bacteria.
In order to provide sub-optimal growth conditions in respect of the adjunct culture during the fermentation process the temperature in the fermentation process is equal to or below 30° C., such as equal to or below 28° C., e.g. equal to or below 26° C., such as equal to or below 24° C.
In order to provide sub-optimal growth conditions during the drying process in respect of the adjunct culture the temperature in the drying process is equal to or below 30° C., such as equal to or below 25° C., e.g. equal to or below 20° C., such as equal to or below 15° C., such as equal to or below 10° C., e.g. equal to or below 5° C.
When mixing the adjunct culture with the food material which either comprises the starter culture or which is subsequently mixed with a starter culture, the concentration of the at least one adjunct culture increases the inoculation level of total lactic acid bacteria at most 1000 times, e.g. at most 500 times, such as at most 100 times, e.g. at most 50 times, such as at most 10 times, e.g. at most 8 times, such as at most 5 times, e.g. at most 4 times, such as at most 3 times, e.g. at most 2 times.
In a preferred embodiment of the present invention the at least one adjunct culture is added in a concentration in the range of 102-1010 CFU/g product, e.g. in the range of 102-109 CFU/g product, such as in the range of 103-109 CFU/g product, e.g. in the range of 104-109 CFU/g product, such as in the range of 102-108 CFU/g product, e.g. in the range of 102-107 CFU/g product, such as in the range of 103-107 CFU/g product, e.g. in the range of 104-107 CFU/g product, such as in the range of 106-107 CFU/g product, e.g. in the range of 106-107 CFU/g product such as in the range of 106 CFU/g product, e.g. in the range of 103-106 CFU/g product, such as in the range of 102-104 CFU/g product.
In presently preferred embodiment of the invention the adjunct culture is added in a range of 5×106-9×107 CFU/g product.
In a preferred embodiment of the present invention the food material and/or the dry fermented food product is analysed for the content of pathogenic organisms. If the content of the organisms exceeds a predetermined acceptable level the adjunct culture may be added to the food material in order to kill, inactivate or inhibit the pathogenic organisms.
In the case the food material is analysed for the content of pathogenic organisms and that it is established that the content exceeds a predetermined acceptable level the adjunct culture may be added directly to the food material to kill, inactivate or inhibit the pathogenic organisms.
In the case the dry fermented food product is analysed for the content of pathogenic organisms and that it is established that the content exceeds a predetermined acceptable level, the batches of food materials subsequently produced are mixed with the adjunct culture to kill, inactivate or inhibit the pathogenic organisms.
Under conditions of the method of the invention the bacteriocin-producing Pediococcus species can be added to a food fermentation without adversely affecting the fermentation profile. Thus, the species can be used as adjunct cultures for securing microbial safety of fermented food product.
Naturally, the characteristics used to describe the method of the invention will also apply when the strain is used as an adjunct culture as described above.
In specific embodiments, the adjunct culture is provided in a suitable package. Such packages may be e.g. a pouch, a tetra-pak, a can and any other suitable means described in the art for containing microbial species.
Preferably, the package or corresponding marketing material is provided with instructions indicating the fermentation conditions that are sub-optimal for growth of the bacteriocin-producing Pediococcus species.
Further, the adjunct culture may be provided in any suitable form e.g. in a frozen or freeze dried form.
In a preferred embodiment the adjunct culture is a freeze-dried preparation of B-LC-20 (DSM 10313) provided by Chr. Hansen A/S under the trademark SafeProTM.
The isolated strain is useful for the purposes of the invention described herein. Additionally, it is contemplated that the bacteriocin-producing Pediococcus species may be used as a bioprotective agent for improving safety of all food products.
Although, the invention focuses on the application of the adjunct culture during a food fermentation it is within the scope of the present invention that the adjunct culture can be added to a food matrix that are not subjected to a fermentation process.
A sample of the strain has been deposited according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The deposit was made on 24 Oct. 1995 under the accession number DSM 10313.



The invention is further illustrated in the following non-limiting examples and in the drawings, where
FIG. 1, A and B illustrate the pH-development during ripening of sausages applied with or without B-LC-20 together with a Chr. Hansen BactofermTM fast fermenting starter culture. Sausages were fermented at 24-20° C. for 3 days, followed by ripening at 18 to 16° C. for 11 days, and
FIG. 2. A and B illustrate pH-development during ripening of sausages applied with or without B-LC-20 together with a Chr. Hansen BactofermTM traditional fermenting starter culture. Sausages were fermented at 24-20° C. for 4 days, followed by ripening at 18 to 14° C. for 17 days.



EXAMPLES
Example 1
Influence of B-LC-20 on pH Development in Sausage Mince
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.







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
















TABLE 2







pH development in sausage mince determined


by continuous measurement









Hours















Code
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









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 1.1×107 CFU/g mince. Control culture was added in a total lactic acid bacteria concentration of 3.3×106 CFU/g.
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.
The control starter culture consists of a blend of lactobacilli, pediococci, micrococci and staphylococci. 
Example 2
Influence of B-CL-20 on Listeria Reduction
Summary of Trials
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. 103 CFU/g) at the time of manufacturing.
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.
At the selected times three sausages were sampled from each batch for determining Listeria and Lactic acid Bacteria counts, pH and weight loss.
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.
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.
Methodology
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 Agroalimentáries (IRTA), Monells, Spain. The anti-Listeria activity of the Lactic acid Bacteria culture B-LC-20 at two different concentrations were tested.
Bacterial Cultures
Chr. Hansen anti.listeria culture B-LC-20 and the control starter culture was stored frozen (−20° C.) until use.
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 103 CFU/g).
Manufacturing
Three batches (12 Kg each) were manufactured.
Batch A Control (starter culture)+Listeria monocytogenes cocktail
Batch B Control (starter culture)+B-LC-20 low concentration
Batch C Control (starter culture)+B-LC-20 high concentration
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.
Ripening Conditions
Fermentation and drying of sausages were done in an adapted versatile environmental test chamber Sanyo Model MLR-350H.
Fermentation was carried out for 72 h at 24° C. with RH>90%.
After the fermentation period the conditions were adjusted for drying until day 29 at 14° C. with 80% RH.
Analyses
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.
Microbial Analyses
Three different sausages from each batch (A, B, C) were analysed 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.
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.).
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).







TABLE 1







Listeria spp. And Lactic Acid Bacteria counts


along the ripening of dry sausages in trial 1.















Lactic Acid



Batch
Time (days)
Listeria spp.
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.






Batches were inoculated as follows:
Batch A (control starter culture, 3.0×106 CFU/g), Batch B (control starter culture+B-LC-20 low concentration (2.9×107 CFU/g mince)), Batch C (control starter culture+B-LC-20 high concentration (5.6×107 CFU/g mince)).
All the batches were inoculated with a cocktail of 5 different Listeria monocytogenes strains (CTC1011, CTC1034, P01, P05, P15).







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.













TABLE 3







Listeria spp. and Lactic Acid Bacteria counts


along the ripening of dry sausages in trial 2.















Lactic Acid



Batch
Time (days)
Listeria spp.
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.






Batches were inoculated as follows:
Batch A (control starter culture, 2.6×106 CFU/g), Batch B (control starter culture+B-LC-20 low concentration (1.5×107 CFU/g), Batch C (control starter culture+B-LC-20 high concentration (5.9×107 CFU/g).
All the batches were inoculated with a cocktail of 5 different Listeria monocytogenes strains (CTC1011, CTC1034, P01, P05, P15).







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

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.
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.
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. ΔpH 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.
Conclusion
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.
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 5·105 to 5·106 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.
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 3·106 to between 3·107-6·107. 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.
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.
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. 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
Influence of the Adjunct Culture on the Acidification Profile of Different Fermented Sausages
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.














Organisms
Inoculation, total LAB


















F-1
Pediococcus pentosaceus
5 × 106 CFU/g mince



Staphylococcus xylosus


F-SC-111
Lactobacillus sakei
1 × 107 CFU/g mince



Staphylococcus carnosus


T-SPX
Pediococcus pentosaceus
5.5 × 106 CFU/g mince  



Staphylococcus xylosus


T-SC-150
Lactobacillus sakei
1 × 107 CFU/g mince



Staphylococcus carnosus

## Claims

1. A method for suppressing the growth of Listeria monocytogenes in a fermented food product, comprising:
(i) providing a food material;
(ii) mixing the food material with a starter culture that (A) comprises one or more lactic acid bacteria and (B) provides a desired change in at least one characteristic of the food material during fermentation, wherein the characteristic comprises, acidification profile, reduction in water binding, reduction in water activity, general appearance, color, or flavor;
(iii) mixing the food material with at least one adjunct culture comprising a Pediococcus acidilactici that produces a Class II bacteriocin;
(iv) subjecting the food material containing the starter culture and adjunct culture to a fermentation process at a temperature of not more than 30° C., wherein the fermentation process
(A) results in acidification of the food material, of which the acidification caused by the adjunct culture is 0.5 pH-unit or less; and
(B) allows for a production of the Class II bacteriocin and reduces counts of Listeria monocytogenes by at least 2 log cfu/g of fermented food product by the end of ripening.

2. The method of claim 1, wherein the fermentation process is conducted at a temperature of not more than 25° C.

3. The method of claim 1, further comprising a drying process simultaneously with the fermentation process in step (iv) and/or subsequent to the fermentation process in step (iv) to obtain a dry fermented food product.

4. The method according to claim 1, wherein the acidification caused by the adjunct culture is 0.25 pH-unit or less.

5. The method of claim 4, wherein the fermentation process is conducted at a temperature of not more than 28° C.

6. The method of claim 1, wherein the addition of the adjunct culture increases the number of total lactic acid bacteria at most 1000 times compared to a comparable starter culture without the adjunct culture.

7. The method of claim 1, wherein the adjunct culture is added in a concentration in the range of 102-1010 CFU/g food material.

8. The method of claim 1, wherein the adjunct culture is added in a concentration of 2×107 CFU/g food material.

9. The method of claim 1, wherein the fermented food product is a fermented dairy product.

10. The method of claim 1, wherein the bacteriocin-producing Pediococcus acidilactici strain is Pediococcus acidilactici strain DSM 10313.

11. The method of claim 1, wherein the starter culture further comprises at least one bacteria selected from the genera Micrococcus and Staphylococcus.

12. The method of claim 1, wherein the starter culture comprises at least one organism selected from the group consisting of Lactococcus Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paracasei, Bifidobacterium bifidum, Bifidobacterium longum, Propionibacterium spp. Brevibacterium spp. Penicillium roqueforti, Penicillium candidum, Geotrichum candidum, Torula kefir, Saccharomyces kefir, and Saccharomyces cerevisiae.

13. The method of claim 1, wherein the flavor comprises odor, aroma, or taste.

14. The method of claim 1, wherein the fermentation process reduces counts of Listeria monocytogenes by at least 1.75 log cfu/g of fermented food product after 14 days of ripening.