WO2007003917A1

WO2007003917A1 - Probiotic lactobacillus plantarum or pentosus starter strains

Info

Publication number: WO2007003917A1
Application number: PCT/GB2006/002440
Authority: WO
Inventors: Trine D. Klingberg; Brigitte B. Budde; Mogens Jacobsen; Lars Axelsson; Kristine Naterstad
Original assignee: Matforsk As; Gilde Norge Ba; The University Of Copenhagen; Scan Foods Ab; Rutherford, Jodie
Priority date: 2005-07-01
Filing date: 2006-06-30
Publication date: 2007-01-11
Also published as: GB0513556D0

Abstract

The present invention relates to an isolated Lactobacillus plantarum or pentosus strain which has the ability to act as a starter culture, has protective activity and which is a probiotic. Cultures of the strain and food products inoculated with the strain are encompassed by the invention. The invention further relates to the use of the strains as a starter/probiotic/protective culture, methods for producing a fermented food product, compositions and kits comprising the strains and the strains for use in therapy.

Description

PROBIOTIC LACTOBACILLUS PLANTARXM OR PENTOSUS STARTER STRAINS

The present invention is concerned with new Lactobacillus plantarum or pentosus strains which have probiotic, starter and protective activities and cultures and food products comprising such strains. Uses of the strains in the fermentation of meat, dairy, cereal and vegetables are further covered by the present invention, as are uses as a probiotic, protective or a starter culture, and in medical treatment.

Lactic acid bacteria (LAB) have several properties which are important for the food industry. For example, some lactic acid bacteria can firstly act as probiotic cultures, which are live microbial food ingredients which have beneficial effects on the host (Fuller, 1989). Beneficial effects which have been reported include the ability to prevent intestinal infections by competitive exclusion (Forrester et al., 2001; Lee et al., 2003), stimulate the immune system to decrease allergy in susceptible individuals (Kalliomaki et al., 2001 ; Pochard et al., 2002), suppress intestinal inflammatory diseases (Rembacken et al., 1999; Gronchetti et al., 2000, 2003) and shorten the period of diarrhoea initiated by rotavirus (Majamaa et al., 1995; Guandalini et al., 2000; Rosenfeldt et al., 2002). Further possible beneficial effects of probiotic cultures include the reduction of serum cholesterol and suppression of cancer by blocking or removing carcinogens.

It is presumed necessary for probiotic cultures to be alive to exert a positive effect on the health and well being of the host and hence the presence of an adequate number of live cells is important for a probiotic culture. Of further importance is the ability of a probiotic culture to survive through the upper part of the intestinal tract and hence high tolerance to bile and low pH is necessary. The probiotic culture should also be able to adhere to the intestinal epithelial cells thereby excluding pathogenic bacteria and reaching the underlying lymphoid system to mediate beneficial immune effects. Dairy products, for example fermented milk and yoghurt, are often used as food carriers for probiotic cultures. Some lactic acid bacteria may also act as protective cultures, which kill or inhibit the growth of food deteriorating or pathogenic bacteria. Lactobacillus curvatus and Lactobacillus sake have for example been shown to inhibit many types of food poisoning bacteria and Lactobacillus sake has further been shown to reduce the numbers of Staphylococcus aureus in sausages (Hammes et al., 1990).

Although lactic acid bacteria are Generally Recognised As Safe (GRAS), the specification of origin, non-pathogenicity and antibiotic resistance characteristics of lactic acid bacteria to be used in the food industry should also be assessed (Salminen et al., 1998; Mattila Sandholm et al., 1999; Saarela et al., 2000).

Finally, some lactic acid bacteria can act as starter cultures for fermented foods such as fermented sausages, yoghurt or sour dough bread. Such cultures are required to have several further properties, including desirable technological, sensory and safety properties. For example, starter cultures for producing fermented sausages or other fermented meat products should be well adapted to the conditions of the fermented meat to become dominant in the final product since fermented meat products contain a natural high background microbiota. The culture should also be able to produce sufficient lactic acid to ensure the safety, texture and flavours of fermented meat products which they are producing.

The main fermented meat products consumed in the Western world are fermented sausages which have their origin in Mediterranean countries. Such fermented sausages are cured meat products which are shelf stable (without cooling) and are often consumed without the application of a heating process. The sausages are produced by a ripening process which involves fermentation and the reduction of the water content by drying. In traditional fermentation, bacteria, yeasts and fungi naturally present contribute to the ripening process. In the last decades, lactic acid bacteria have been used as starter cultures for the fermentation.

Lactobacillus curvatus and Lactobacillus sake, have been regularly used as starter cultures for fermenting meat products and are sold as commercial starter culture preparations. These cultures have been shown to result in high sensorial quality. Other bacterial species such as L. plantarum, L. brevis, L. alimentarius, L. casei, L. farciminis and L. viridescens have also been used as starter cultures (Hamnes et al., 1990). Known starter cultures for meat fermentation have however not been shown to possess both of the further activities of being protective and probiotic. In particular, starter cultures used for meat fermentation have previously not had probiotic properties, which are known to be beneficial. Hence, fermented meat products currently are unexploited as regards their potential to provide probiotic cultures when consumed.

Strains of Lactobacillus plantarum or Lactobacillus pentosus have now been identified which advantageously possess all three properties of being a starter culture, protective and probiotic. Hence, probiotic properties can now be given to fermented meat products. This is particularly advantageous due to the beneficial effects of probiotic cultures on health and hence the present invention now allows fermented meat products to be used to pass on such health benefits.

Hence, the present invention provides a purified or isolated Lactobacillus plantarum or Lactobacillus pentosus strain wherein said strain has the ability to act as a starter culture, has protective activity and is probiotic.

The phrase "the ability to act as a starter culture" as used herein is defined as the ability of a strain to acidify or decrease the pH of a sample, e.g. a particular food product, to an appropriate extent. For example, the starter culture can reduce the pH to an extent to trigger fermentation. Preferably, the strain decreases the pH by producing sufficient quantities of lactic acid. More preferably the pH is reduced to or below 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5 or 3.0. Most preferably the pH is reduced to or below 5.1 or 5.2, for example to between 4.5 and 5.2, preferably between 4.8 and 5.1 or 4.8 and 5.0, or 4.8 and 4.9. Appropriate tests to assess the decrease in pH can readily be determined by a person skilled in the art.

A suitable test might thus involve adding the strain to a food product in an appropriate concentration and monitoring the pH of the food product for an appropriate amount of time, for example until the pH has stabilised at a final level. For example, for meat products, the strains can be added at an appropriate concentration, for example of the order of 10⁶ to 10⁷ CFU/g and the mixture incubated at an appropriate temperature while monitoring the pH at appropriate intervals, e.g. every hour, until no further changes take place. The pH can be monitored using pH electrodes (for example from Xerolyt, Mettler-Toledo, Process, Analytical Inc.) which can be inserted into the sample whose pH is to be measured. pH can be recorded using an automatic logging system (Intab AA C-2, Interface- Teknit AB, Sweden) which can be controlled by a software package (e.g. Easy View 2-12, Interface-Teknik AB, Sweden).

Preferably, said acidification (reduction in pH) to the appropriate level takes place in less than 65 hours, for example less than 60, 55, 50 or 45 hours. More preferably said acidification takes place within 40 hours of starting the incubation (adding the starter culture), for example within 35 or 30 hours. Most preferably said acidification takes place within 25 hours of starting the incubation (adding the starter culture). The reduction in pH preferably starts to occur only a few hours after the incubation of the starter culture. Hence, the reduction in pH may commence 1, 2, 3, 4, 5, 6, 7 or 8 hours after incubation.

Thus, in a preferred embodiment of the invention the strains of the invention decrease the pH of a food product, for example a fermented meat sausage, to a level below 5.1, for example between 4.8 and 4.9, within 25 hours of starting the incubation. In a further preferred embodiment, the phrase "the ability to act as a starter culture" as used herein also includes the ability of a strain to dominate in the fermented product and/or the ability to give desirable sensory features (or not to produce any undesired sensory features) in a fermented product.

Hence, the strain preferably is able to become dominant in the final product, e.g. the fermented product. A dominant strain is one which is present or has the ability to become present in greater numbers or at a greater proportion than other microflora or microorganisms in the product and/or at one or more stages in the fermentation process. Particularly, a dominant strain should be competitive with or should be able to suppress other microflora which may be present either intentionally, endogenously or fortuitously in the product and preferably a dominant strain should be present in numbers at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% greater than other microorganisms or microflora which are present.

A dominant strain and therefore the ability of a strain to dominate can readily be identified or assessed by methods known in the art. For example, a sample of the fermented product or food product, preferably the final food product, containing a particular strain, can be homogenised and plated out in serial dilutions on to agar plates. Individual colonies can then be analysed using methods such as Randomly Amplified Polymorphic DNA (RAPD) and fragment profiles investigated in order to identify clones and assess the dominance.

The ability of a strain to give desirable sensory features (or to not produce any undesired sensory features) involves the strain having the ability to sufficiently decrease the pH of a product, e.g. of a meat product e.g. of a fermented sausage.

The reduction of the pH is also crucial for the safety and texture of the end products. Preferably, the strain decreases the pH by producing sufficient quantities of lactic acid. More preferably the pH is reduced to or below 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5 or 3.0. Most preferably the pH is reduced to or below 5.1 , for example to between 4.5 and 5.2, 4.8 and 5.1 or 4.8 and 5.0, or 4.8 and 4.9.

Sensory descriptive profiles (flavour profiles) of final products can also be assessed by trained assessors (for example as described in Hagen et al, 2000, Meat Science 55:161-168), where one or more attributes such as odour intensity, acidic odour, colour tone of fat, whiteness, overall colour tone, colour intensity, overall flavour intensity, fat flavour, maturity flavour, acidic taste (fruity acid), spicy flavour, garlic flavour, smoke flavour, metallic flavour, sweet taste, sour taste, (acetic acid), salt taste, bitter taste, rancid flavour, hardness, tenderness, juiciness, saltiness and graininess are evaluated. The flavour profile can be determined using an unstructured line with end points (1-9) where 1 denotes low intensity and 9 high intensity for the characteristics described above. Differences in sensory score can be evaluated using Tukey's test for pairwise comparison of means using Minitab for windows release 12.1 (Minitab Inc., PA, USA).

Preferably, strains of the present invention which have the ability to give desirable sensory features, are able to produce flavour profiles in final products which are not significantly different from or are almost identical or identical to (e.g. as assessed by the above described Tukey's test) those produced by commercial starter cultures which are currently used in the production of a particular fermented product, e.g. a fermented meat product. Suitable commercial starter cultures for comparison would be known and readily available to a person skilled in the art, depending on the product concerned. Preferably, when the product is a fermented meat product, the flavour profiles produced are almost identical or identical to those obtained when commercial starter cultures such as Lactobacillus curyatus HJ5 and Staphylococcus carnosus Mill are used.

Alternatively viewed, the strains of the present invention preferably have the ability to give improved sensory features to final products compared with commercial starter cultures. Hence, particularly preferred is that when the product is a meat product, the flavour profile is improved to that obtained with a commercial starter culture.

Preferably, strains of the invention are also able to give the products a desired texture. When the product is a meat product, the desired texture is hard, springy, cohesive or resilient or a mixture of these depending on the particular product. The texture of sausages can be evaluated using Texture Profile Analysis (TPA) using a Texture Analyser TA - XT2 (Stable Micro Systems). A bite size sample can be compressed to 50% of its original height, twice, in a reciprocating motion in order to imitate jaw action. Parameters such as hardness, springiness, cohesiveness, resilience, gumminess and chewiness can be measured.

Preferably said ability to act as a starter culture refers to this ability to act as a starter culture in meat products and the ability to stimulate or induce fermentation in meat products. Thus, a preferred embodiment of the invention provides strains of bacteria as defined herein for use in fermentation and in particular for use in fermentation of meat products, although the present invention further provides strains of bacteria for use in fermentation of dairy, cereal and vegetable products. The term "probiotic" as used herein refers to a strain which has a positive effect on the health and well being of a host when ingested by said host, for example by improving intestinal microbial balance in a host. Preferably, a probiotic strain should possess one or more of the following abilities; the ability to inhabit or survive the gastrointestinal (GI) tract in vivo, the ability to suppress or inhibit growth of microorganisms unwanted in the GI tract, the ability to adhere to the GI tract (GIT) and the ability to strengthen the resistance of the epithelial cell layer to pathogenic microorganisms . Appropriate tests to determine the presence or absence of these abilities of a strain could be readily carried out by a person skilled in the art. For example, the ability of a strain to inhabit or survive the GI tract in vivo can readily be investigated in vitro by assessing the acid resistance of the strain and/or the bile tolerance of the strain. Preferably therefore, a probiotic culture should be able to survive through the upper part of the GIT and hence should have a high tolerance to low pH. More preferably, the strains should be able to survive or be resistant to pHs of at or less than 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0 or 1.5. Most preferably the strains should be able to survive pH conditions of at or around 2.5. The time period for survival is preferably 0.5, 1, 2, 3, 4, 5 or 6 hour, preferably more than 0.5, 1, 2, 3, 4, 5 or 6 hours. An appropriate test for strains of the present invention is thus to assess their ability to survive at a pH of 2.5 for a period of 1 hour, more especially at least 1 hour. Strains which can survive these conditions are believed to be able to survive through the upper part of the GI tract and are preferred. Methods of testing for such survival could readily be determined by a person skilled in the art and any appropriate method might be used. However, for example, the selected strains might be inoculated into growth medium adjusted with hydrochloric acid to obtain a final pH of 2.5 at a concentration of 10⁶ CFU/ml and following exposure for 1, 2, 3 and 4 hours the survival rates could be determined as the number of viable cells on agar after anaerobic incubation for 48 hours at 37⁰C. Hence, by survival or resistance is meant that some viable cells of the strains are present after exposure to low pH as defined above for the time periods described above. Preferably the log unit reduction is less than 4 after one hour exposure to low pH as defined above. For example, where log 7.0 cfu/ml cells are exposed, cell numbers can be reduced by 3.6 log unit after 1 hour. Survival of the cells can be assessed by any method known in the art, including plating a sample incubation on MRS agar and determining the number of colonies present (and hence viable cells) after 24 hours of incubation.

Preferably, the strains should be bile tolerant. The bile tolerance of strains can readily be measured in vitro by assessing the survival of strains in the presence of oxgall (bile from cow). Hence, preferably strains can survive for 0.5, 1, 2, 3, 4, 5 or 6 hours in vitro in the presence of bile or oxgall, where survival is defined as described above in relation to pH survival. More preferably the strains of the invention can grow in the bile condition, and hence the number of viable cells after exposure is greater than the number before exposure. The amount of bile or oxgall to be used in such tests can readily be determined by a person skilled in the art. However, an appropriate concentration to be used might be 0.1, 0.2, 0.3, 0.4 or 0.5% w/v and an appropriate survival time might be 1 hour. Thus, strains which can survive in 0.3% w/v oxgall for 1 hour are preferred.

Methods of testing for such survival could readily be determined by a person skilled in the art and any appropriate method might be used. However, for example, the selected strains might be inoculated into growth medium containing 0.3% w/v oxgall at a concentration of 10⁶ CFU/ml and following exposure for 1, 2, 3 and 4 hours the survival rates could be determined as the number of viable cells on agar after anaerobic incubation for 48 hours at 37⁰C. Particularly, strains may be inoculated onto MRS agar and following 24 hours incubation, viable cell numbers may be determined as colony forming units.

In vivo tests are also available to assess the ability of a strain to inhabit or survive the GIT. An appropriate test might for example involve human intervention studies, where a freeze dried strain can be consumed and fecal samples collected before and periodically after consumption can be plated out onto MRS agar and viable colonies counted. Such tests are described in more detail in Examples 11 and 12 of the present application. A probiotic culture can also have the ability to inhibit or suppress the growth of microorganisms unwanted in the GI tract (GIT), i.e. have an antimicrobial effect. Hence, the strains of the invention preferably have the ability to suppress the growth of microorganisms unwanted in the GIT, such as enteric pathogens or bacteria associated with food poisoning. Particularly, the strains may suppress or have an antimicrobial effect against one or more of Bacillus cereus, Shigella flexneri, Yersinia enterocolitica, Salmonella typhimurium, Listeria monocytogenes or Escherichia coli. More preferably the strains can suppress the growth of Listeria monocytogenes and/or Escherichia coli. Especially preferably the strains can suppress the growth of each of Bacillus cereus, Shigella flexneri, Yersinia enterocolitica, Salmonella typhimurium, Listeria monocytogenes and Escherichia coli. Suppression or inhibition of growth can be regarded as the ability to reduce the growth of pathogenic microorganisms below that which would occur in the absence of the strains. Preferably the growth of the pathogenic bacteria is reduced by 10, 20, 30, 40, 50, 60, 70, 80 or 90% or is reduced by an amount which is statistically significant. More preferably the strains of the invention can totally inhibit the growth of pathogenic microorganisms such as those discussed above or can reduce and preferably significantly reduce the numbers of pathogenic microorganisms. Ability to suppress or inhibit growth of pathogenic microorganisms can be measured or tested by agar spot tests as described below (e.g. using an agar diffusion model system as described in Example 7), or appropriate in vivo tests can be carried out. Alternatively viewed therefore, the reduction of growth of pathogenic bacteria can be measured using the zone of inhibition which occurs around a colony. Preferably the zone of inhibition is at least 0.5, 1, 1.5, 2.0, 2.5, 3 or 3.5 mm. More preferably the zone of inhibition is at least 1-2 mm.

A probiotic culture can also have the ability to adhere to the GIT tract, i.e. can adhere to epithelial cells and preferably intestinal epithelial cells in order to stimulate the underlying lymphoid system to mediate beneficial immune effects as well as exclude pathogenic bacteria. Adhesion capacity can readily be measured in vitro for example by incubating strains with an appropriate epithelial or epithelial-like cell line (for example a human colon cacinoma cell line such as Caco-2 cells T84 cells or HT29 cells) for a period of time, washing cells to remove non-adherent bacteria and determining the number of viable adherent bacteria by plating onto agar. The adhesion capacity can then be described as the percentage of bacteria adhered to the epithelial cells in relation to the total number of bacteria added. Adhesion capacity can also be measured using in vivo tests which are described in detail in Examples 11 and 12 of the present application. Such tests involve the consumption of the strains of interest and the collection of fecal samples before and after consumption and during a "wash out" period. The samples are plated onto agar and the number of colonies of the strain which are reisolated during the "wash out" period indicate colonization of the GIT. Thus, preferably the strains of the present invention can adhere to epithelial cells or epithelial-like cells, for example, Caco-2 cells, in vitro.

Preferably, the adhesion capacity of cells is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25%. More preferably the adhesion capacity is greater than 7%, 10% or 18%. Cells with such levels of adhesion capacity are considered as highly adhesive compared with very low adhesive cells e.g. E. coli B44 which have adhesive capacities of less than 5%. Generally, cells with an adhesion capacity of at least 5% are regarded as having the ability to adhere to intestinal epithelial cells and are preferred. As a comparison, cells with an adhesion capacity similar to or preferably greater than the adhesion capacity of E. coli B44 are regarded as having the ability to adhere to intestinal epithelial cells and are preferred.

A probiotic culture can further have the ability to strengthen the resistance of the epithelial cell layer, preferably the intestinal epithelial cell layer, to pathogenic microorganisms such as those described above, preferably Listeria monocytogenes. Preferably, the probiotic culture can increase the transepithelial electrical resistance (TER) of the epithelial cells probably as a result of an increase of the tight junctions. Most preferably, the TER is increased as a result of an increase of the expression of proteins which are associated with the tight junctions, such as ZO-I TER can be increased by over 10, 20, 30, 40, 50, 60, 70, 80 or 90% over periods of incubation with the strain of for example 6, 12, 18, 24, 30, 36, 48 or 54 hours. Preferably TER is increased by over 60% after 24 hours of incubation. Levels of TER can be measured in vitro using methods well known in the art such as Millicell - ERS Electrical Resistance System (Millipore, Bedford, MA). Such methods might conveniently involve measuring the effect on TER of incubating polarised mono-layers of an appropriate epithelial or epithelial-like cell line (for example a human colon cacinoma cell line such as Caco-2 cells T84 cells or HT29 cells) or the non-intestinal tumongenic porcine junal epithelial cell line IPEC- J2) with a strain which is to be tested. Appropriate in vivo tests are also known in the art to measure epithelial permeability, for example measuring the amount of carbohydrate (sucrose) in the urine of patients (Gotteland et al, 2001, Alimentary Pharmacological Therapy, 15: 11 -17). Thus, preferably the strains of the present invention can enhance or increase TER in vitro or in vivo. Further, in a most preferred embodiment, the probiotic culture can prevent or attenuate or delay a decrease in TER induced by a pathogenic organism, particularly Listeria monocytogenes. The probiotic activities described above generally refer to the ability of a strain to have a probiotic effect in mammals, for example in pigs, poultry, dogs and other domestic animals, although preferably in humans.

The term "protective activity" as used herein refers to the antimicrobial activity of the strains, where the strains of the invention have the ability to suppress or inhibit the growth or activity of one or more potentially food deteriorating or pathogenic microorganisms present in a food product. Hence, the strains of the invention preferably have the ability to suppress or inhibit the growth or activity of one or more food borne pathogens which are usually associated with food poisoning. Particularly, the strains may suppress the growth or have an antimicrobial effect against one or more of Bacillus cereus, Shigella flexneri, Yersinia enterocolitica, Salmonella typhimurium, Listeria monocytogenes, Escherichia coli or Campylobacter. More preferably the strains can suppress the growth of Listeria monocytogenes and/or Escherichia coli. Most preferably the strains can suppress the growth of each of Bacillus cereus, Shigella flexneri, Yersinia enterocolitica, Salmonella typhimurium, Listeria monocytogenes and Escherichia coli.

By suppressing or inhibiting the growth, the strains of the invention can reduce the growth of one or more of the pathogenic microorganisms below levels which would occur in the absence of the strains. Preferably, the growth of the pathogenic bacteria is reduced by 10, 20, 30, 40, 50, 60, 70, 80 or 90%, or is reduced by an amount which is statistically significant. Alternatively viewed, suppression of growth can be measured using the inhibition zone which occurs around spotted cultures, as described further below. More preferably, the strains of the present invention can totally inhibit the growth of pathogenic microorganisms or can reduce and preferably significantly reduce the numbers of the pathogenic microorganisms in comparison to samples which have not been exposed to the strains of the invention. Hence, the presence of the strains of the invention may result in the death of the pathogenic microorganisms. Any reduction or inhibition as described herein includes any measurable reduction or inhibition when the parameter in question in the presence of a strain is compared with the equivalent parameter in the absence of a strain. Preferably the reduction or inhibition will be a statistically significant one. Methods of determining the statistical significance of differences in parameters are well known and documented in the art. For example herein a parameter is generally regarded as significant if a statistical comparison using an appropriate statistical test such as a Student t-test shows a probability value of <0.05. The antimicrobial activity of the strains of the invention can conveniently be measured or tested using an agar spot test or an agar diffusion model system (Schillinger and Lϋcke, 1989 - see also Example 7), wherein pathogenic bacteria are overlayed onto agar plates onto which have been spotted cultures of the strains of the invention. The antimicrobial activity can be assessed by the measurement of the inhibition zone around the spotted cultures. Preferably, strains of the invention will have an inhibition zone of at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5mm or more. More preferably the zone of inhibition is at least 1-2 mm.

In a preferred embodiment of the invention, a strain comprises all of the above described probiotic activities, together with the ability to act as a starter culture and protective ability.

In a further preferred embodiment, the strains are sensitive to the majority of antibiotics. Sensitivity to antibiotics can be measured using methods which are well known and standard in the art, for example using an E-test (VIVA, Diagnostika GmbH, Kδln, Germany) according to the instructions of the manufacturer, where minimal inhibitory concentration values higher than breakpoints proposed by the Scientific Committee for Animal Nutrition or by Danielsen and Wind (2003) are considered to be resistant against the antibiotics. In such an E-test, strains to be tested are placed onto agar, contacted with the appropriate antibiotic and inhibition zones monitored after incubation for an appropriate amount of time, e.g. after 24 hours. The minimal inhibitory concentration is defined as the lowest concentration of the antibiotics which inhibit growth of the strains. Strains are preferably sensitive to ampillicin, quinupristin/dalfopristin, tetracyclin, gentamycin, erythromycin, trimethoprism, linezolid, rifampicin, chloramphenicol and clindamycin. Strains may however be resistant to vancomycin, kanamycin, ciprofloxacin and streptomycin under the condition used. In preferred embodiments any antibiotic resistance displayed by the strains of the invention is intrinsic (naturally occurring resistance) rather than an acquired resistance mediated by for example plasmids or transposons, as said intrinsic resistance is considered to represent a minimal risk for spread to other organisms, for example food borne pathogens or enteric pathogens.

In a particularly preferred embodiment the Lactobacillus plantarum strain of the invention is MF1298 (deposited on 22 December 2004 in DSMZ (Deutsche Sammtung von Mikroorganismen und Zellkulturen GmbH) under the Budapest treaty, deposit number - DSM 16997), or MF 1291 (deposited on 17 May 2005 in DSMZ under the Budapest treaty, deposit number DSM 17320), or has the identifying characteristics of one of these strains, or is a strain derived from one of these strains or the Lactobacillus pentosus strain of the invention is MFl 300 (deposited on 17 May 2005 in DSMZ under the Budapest treaty, deposit number

DSM 17321), or has the identifying characteristics of this strain or is a strain derived from this strain.

By "identifying characteristics" is meant, for example, the properties and characteristics discussed herein for the strains of the invention, including in the experimental examples.

Therefore, preferably a strain of the invention (1) has the ability to act as a starter culture and can acidify or decrease pH in a food product by an appropriate amount, can preferably give desirable sensory features (or does not give undesirable sensory features) and preferably can dominate in a fermented product; (2) is protective and can suppress growth or activity of one or more unwanted microorganisms in food; and (3) is probiotic and can preferably inhabit or survive in the GIT, can preferably suppress or inhibit growth of unwanted microorganisms in the GIT, can adhere to the GIT and can preferably strengthen resistance of the epithelial cell layer to pathogenic microorganisms. Particularly preferred is a strain which can acidify or decrease pH in a food product to at or below 6.5 (more preferably at or below 5.1) within 25 hours, can dominate in the fermented product by being present in greater numbers than other microflora or microorganisms, does not give undesirable sensory properties to a food product, can suppress growth or activity of one or more potentially food deteriorating or pathogenic microorganisms (for example as discussed elsewhere herein), can survive in the GIT in vivo and is resistant to pH of less than 5.0 (and preferably a pH of 2.5 or less) and to bile of 0.3% w/v, can inhibit the growth of microorganisms unwanted in the GIT (for example one or more of Bacillus cereus, Shigella βexneri, Yersinia enterocolitica, Salmonella typhimurium, Listeria monocytogenes and Escherichia coli), can adhere to epithelial-like cells in vitro (preferably with an adhesion capacity of at least 5%) or survive through the human GIT. Preferably the strain can strengthen the resistance of the epithelial cell layer and increase TER (preferably by over 10%) or increase expression of ZO-I proteins which are associated with the tight junctions.

More preferably, a strain of the invention has the above described characteristics and is further able to act as a starter culture in meat products and/or induce fermentation in meat products. More preferably a strain of the invention is sensitive to most antibiotics, most preferably to ampicilin, quinupristin/dalfopristin, tetracyclin, gentamycin, erythromycin, trimethoprism, linezolid, rifampicin, chloramphenicol and clindamycin. (Further details and methods of testing for the above described properties are described elsewhere herein.) Thus, the present invention also provides the Lactobacillus plantarum strain

MFl 298 or MF 1291, or a mutant or variant thereof having the functional characteristics or identifying characteristics as defined above. The present invention further provides the Lactobacillus pentosus strain MF 1300 or a mutant or variant thereof having the functional characteristics or identifying characteristics as defined above. Bacterial species belonging to Lactobacillus plantarum or Lactobacillus pentosus can be identified according to their 16S rDNA sequence and/or by a multiplex PCR method e.g. according to Torriani et al, 2001, Appl. Environ. Microbiol., 67, 3450-3454.

The preferred strains MF1298, MF1291 or MF1300 and mutants or variants thereof can be recognised by having a closely related, essentially corresponding, or identical genetic fingerprint. Preferably, the term genetic fingerprint refers to a banding pattern produced when the DNA of a strain is digested with one or more restriction enzymes and then separated and visualised for example by electrophoresis. Preferably pulse field gel electrophoresis is used and preferably separation is carried out in agarose gels (for example 1% agarose gels). More preferably the genetic fingerprints of the strains MF1291, MF1298 and MF1300 are as shown in Figure 10 and Figure 11. The genetic fingerprints of the strains shown in Figure 10 and Figure 11 refers to the banding pattern formed by pulse field gel electrophoresis of DNA obtained from the strains and digested with Ascl enzyme. Hence strains possessing the genetic fingerprints of MF1298, MF1291 and/or MF 1300 in Figure 10 or Figure 11 are also covered by the present invention. Other strains, of L. plantarum or L. pentosus (e.g. the other strains as shown in Figure 10 or Figure 11) or other strains of closely related Lactobacilli which do not possess the required functional activities of the strains of the invention may show differences in the banding pattern of the genetic fingerprint and therefore do not have a closely related or an essentially corresponding banding pattern to the strains of the invention. Preferably, a closely related, essentially corresponding genetic fingerprint is one where the banding pattern is more than 90% similar or identical in for example a PFGE cluster analysis. More preferably the banding pattern is 95, 96, 97, 98 or 99% similar or identical.

Methods for producing and assessing a genetic fingerprint are well known in the art and one particular method is exemplified in the present application (Example 16). Hence, DNA from bacteria can be digested with Ascl enzyme and then submitted to pulse field gel electrophoresis using an agarose gel (e.g. a 1% agarose gel), which can then be stained and the banding pattern observed under UV light. Alternatively, the invention provides mutants or variants having an essentially corresponding or closely related REA (Restriction Enzyme Analysis) pattern, which refers to a banding pattern formed by electrophoresis on agar gel when chromosomal DNA has been cleaved with EcoRI or other restriction enzyme. Preferably the mutants or variants have an REA pattern which is 90, 95, 96, 97, 98 or 99% similar or identical to MF1298, MF1291 and MF1300. Again, closely related strains of Lactobacillus with differences in the REA pattern may not have the functional characteristics required for the invention as previously defined above. Strains possessing the identical REA patterns as MF1298, MF1291 and MF1300 are also covered. Methods for carrying out REA are well-known in the art, for example in Stahl et al (Int. J. Syst. Bacteriol., 40, 189-193, 1990).

Most preferably, the Lactobacillus plantarum strain is MF 1298, or a mutant or variant thereof. A further embodiment of the invention includes cultures of the strains of the invention. Such cultures can be maintained on solid or liquid media or can be frozen or freeze dried:

General growth conditions of the strains includes anaerobic growth in MRS broth (Oxoid) at temperatures of between 20-40°C, preferably at 37°C. Cultures of the strains can be frozen or freeze dried using techniques known in the art.

Preferably cultures which are freeze dried retain their probiotic, protective and starter culture activities as defined above and also remain viable. Frozen or freeze- dried strains of the invention form a yet further aspect.

The present invention further provides food products, preferably probiotic food products, comprising one or more Lactobacillus strains of the invention, wherein the isolated strains have been added to the food product. Hence, alternatively viewed, food products which have been inoculated with one or more of the strains described above are encompassed. Alternatively viewed, food products and preferably meat products comprising more than 10⁵, 10⁶ or 10⁷ CFU/g of one or more of the strains of the invention are also encompassed. Particularly preferred are sausages comprising more than 10⁵, 10⁶ or 10⁷ CFU/g of one or more of the strains of the invention.

Further, food products and preferably meat products comprising one or more Lactobacillus strains of the invention are encompassed where a commercial starter strain is not present in the product. Commercial starter strains include strains which are commercially available at the date of filing this application, and some are described elsewhere herein.

Preferably, the food product comprising the one or more strains is a meat, dairy, cereal or vegetable product. More preferably, the food product is a meat product, most preferably selected from sausage and ham.

The food product comprising the one or more strains of the invention can be a fermented food product and particularly preferred is a fermented meat product, most preferred, a fermented sausage. Particularly preferred is a Scandinavian type fermented sausage. Other food products include cultured milk products, pickled vegetables and fermented cereals.

The present invention further covers the above described Lactobacillus plantarum oτpentosus strains for use in food products for human or animal consumption, e.g. for use as a starter culture for the fermentation of food products for human or animal consumption, and/or for use as a probiotic culture and/or for use as a protective culture. More preferably, the strains are for use as a starter culture for the fermentation of meat and most preferably for the fermentation of sausage.

Alternatively, the strains are provided for use as a starter culture for the fermentation of other food products, such as dairy, cereal or vegetable products and/or for use as a probiotic culture, and/or for use as a protective culture in such products. Thus, the strains of the invention for use as a starter culture and/or a probiotic culture and/or as a protective culture are also encompassed by the present invention.

Thus, the present invention further provides the use of the above described Lactobacillus plantarum oxpentosus strains in food products for human or animal consumption, e.g. the use of said strains as a starter culture for the fermentation of food products for human or animal consumption, and/or for use as a probiotic culture and/or for use as a protective culture. More preferably, the strains of the invention are used as a starter culture for the fermentation of meat and most preferably for the fermentation of sausage. Alternatively, the strains of the invention can be used as a starter culture for the fermentation of other food products, such as dairy, cereal or vegetable products and/or for use as a probiotic culture, and/or for use as a protective culture in such products.

Thus, the use of the strains of the invention as a starter culture and/or a probiotic culture and/or a protective culture are also encompassed by the present invention. Said uses are generally carried out in vitro, i.e. outside the human or animal body, for example in the food product per se.

Thus, the present invention further provides methods for fermenting food products comprising the addition of one or more of the strains of the invention to the food product in appropriate concentrations and under appropriate conditions to induce fermentation. Similar methods can also be used to confer probiotic and/or protective activities to a food product.

The invention further provides a method for producing a food product comprising the steps of inoculating, mixing or combining the food product with one or more strains of the invention.

The invention further provides a method for producing a fermented food product comprising the steps of inoculating, mixing or combining the food product with one or more strains of the invention and incubating the food product for a time and under conditions necessary for fermentation to occur. Preferably the method is for producing a meat product and in particular a fermented meat product, although the methods may also be used to produce other types of food products such as dairy, vegetable or cereal products, e.g. cultured milk products, pickled vegetables and fermented cereals.

Preferably the meat or other food product is inoculated with at least 10 cfu/g, e.g. 10⁶ to 10⁷ cfu/g of the strain. Alternatively, the meat or other food product is inoculated with an amount of bacterial cells such that the cells multiply and persist in concentrations exceeding 10 to 10 viable cells/g product, preferably throughout the entire shelf life of the food product, i.e. at the time of consumption. The inoculated meat product can be fermented for 1, 2, 3, 4 or 5 days (preferably 3 days), preferably at 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28°C, most preferably at 24⁰C

The method of producing a fermented food or meat product can also comprise the additional steps of drying the product after fermentation.

Drying preferably is carried out for 5, 10, 15, 20, 25, 30 or 35 days. The temperature at which the product is dried is preferably, 10, 12, 14, 16, 18, 20, 22, 24 or 26⁰C. Methods for producing food products and in particular fermented food products using strains of bacteria and in particular Lactobacillus strains of bacteria, are well known and standard in the art and vary depending on the type of product which is being produced. Thus, the conventional steps of making such products, e.g. the selection of a single meat or different meats, as appropriate, chopping to the appropriate size and mixing the meat in the desired proportions with the appropriate additional ingredients such as sugar, spices, seasoning, etc., are carried out according to any of these conventional methods, except that one or more strains of the invention are added to the food products in addition to or instead of the previously used bacterial strains. Thus, one or more strains of the invention can be used as the only bacteria added to the food product, or in combination with different strains of bacteria which are normally used in producing the product in question. If combinations of strains of the invention are used then generally the strains are used in equal amounts (50% of each). However other proportions e.g. 10%/90%, 20%/80%, etc. can be used.

Food products obtained or obtainable by such methods form a yet further embodiment of the invention.

The present invention also encompasses a process for isolating the strains of the invention described previously wherein the strains are isolated from a food product and/or the human GIT and are cultured using a suitable nutrient medium and are selected for their ability to act as a starter culture, to be protective and probiotic.

Preferably, the strains are isolated from a meat product, more preferably from a fermented sausage. Suitable tests which can be employed to select suitable strains have been previously described and several are further described in the Examples.

As described previously, the strains of the invention are probiotic and protective and preferably have the ability to strengthen the resistance of the epithelial cell layer to pathogenic microorganisms. The invention therefore also provides a composition, preferably a pharmaceutical composition, comprising one or more Lactobacillus plantarum or pentosus strains, cultures or food products of the invention, optionally together with at least one pharmaceutically acceptable carrier, diluent or excipient Preferably said compositions comprise freeze dried strains of the invention, optionally together with an appropriate diluent such as water, A further preferred composition comprises a food product fermented with the freeze dried strains of the invention.

The invention also covers a strain of the invention or a culture, composition or food product comprising said strain for use in therapy.

The amount of strain of the invention present in such compositions can be readily determined depending on the nature of the disease or condition to be treated. For example, preferred doses would involve the administration of greater than 10 bacteria (cfu) per day and the amount present in the compositions can be determined accordingly. For a probiotic effect, the preferred dose of bacteria is greater than 10 cells (cfu)/day, more preferably greater than 10⁹ or 5 x 10⁹ cfu/day and most preferably greater than 10¹⁰.

By "pharmaceutically acceptable" is meant that the ingredients must be compatible with other ingredients of the composition as well as physiologically acceptable to the recipient.

The pharmaceutical compositions may be formulated according to any of the conventional methods known in the art and widely described in the literature. Thus, the strains may be incorporated, optionally together with other active substances, with one or more conventional carriers, diluents and/or excipients, to produce conventional preparations which are suitable or can be made suitable for oral, subcutaneous, intramuscular or intravenous administration such as powders, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, ointments, sterile injectable solutions, sterile packaged powders, and the like. Preferably the pharmaceutical composition is prepared in a form appropriate for oral administration to a patient.

Examples of suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, maltose, glucose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, aglinates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, water, water/ethanol, water/glycol, water/polyethylene, glycol, propylene glycol, methyl cellulose, methylhydroxybenzoates, propyl hydroxybenzoates, tal, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable mixtures thereof. The compositions may additionally include lubricating agents, wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.

The improvements seen in patients treated in accordance with the present invention may be immediate (e.g. after a few days), or may be seen after a few weeks or a few months depending on the individual patient. Once the initial improvement is seen, continued improvement over the subsequent weeks and months may also occur. Treatment can be continued for as long as is desired or is necessary.

Alternatively viewed, the invention covers a method of treating a disease or condition in a mammal which method comprises administering to said mammal an effective amount of a strain of the invention, or a culture, composition or food product comprising said strain.

Further, also provided is the use of a Lactobacillus strain of the present invention or a culture, composition or food product comprising said strain in the manufacture of a composition or medicament for use in therapy. Possible diseases or conditions (or the symptoms thereof) which may be treated in accordance with the present invention include any disease or condition which is suitable for treatment with probiotic bacteria, e.g. the present invention can be used to prevent or treat intestinal infections (thought to work by competitive excursion), to reduce serum cholesterol, to suppress or prevent cancer (for example by blocking or removing carcinogens), to stimulate the immune system (which can be used for example to decrease allergic responses in susceptible individuals or to suppress intestinal inflammatory diseases or unregulated inflammation in the GIT, for example during active periods of inflammatory bowel diseases) to reduce the risk of sepsis with bacterial infections such as complications following abdominal surgery, or to treat any disease associated with increased permeability of the intestinal epithelium or reduced epithelial barrier function, for example diseases associated with altered light function structure or function, e.g. associated with reduced expression or function of light function proteins such as ZO-I . Examples of pathogens which can cause such effects and can hence be treated are Salmonella dublin, Salmonella enterica, E, coli, Yersinia pseudotuberculosis, Vibrio cholerae and Listeria monocylogenes. Thus, the strains of the invention can be used to treat gastrointestinal disorders and intestinal infections.

Other diseases or conditions include the prevention or treatment of any disease involving the presence of pathogenic bacteria in the GIT, for example food poisoning, diarrhoea (and in particular diarrhoea initiated by rotaurus). Preferred diseases to be treated are those caused by microorganisms such as Bacillus cereus, Shigella flexneri, Yersinia enterocolitica, Salmonella typhinium, Listeria monocytogenes, and Escherischia coli. Other conditions which can be treated or prevented include atopic eczema or allergies. Due to the fact that the strains, food products, etc., of the invention are probiotic, the therapies as described herein include the treatment of a mammalian subject in order to induce a beneficial effect on the health of said mammalian subject. Thus, "treatment" in accordance with the present invention includes the improvement in health or well being of the patient, hi addition, said "treatment" can be carried out on a healthy individual in order to produce beneficial effects on health. Thus, the terms "therapy" and "treatment" as used herein include prophylactic treatment of the diseases and conditions described herein.

A further embodiment of the invention is a kit comprising one or more strains of Lactobacillus plantarum oτpentosus of the present invention, wherein preferably said kits are for use as a starter culture for fermentation, and/or as a probiotic culture and/or as a protective culture. Preferably said kits are for use in the methods and uses of the invention as described elsewhere herein, for example for use in generating food products with probiotic and/or protective properties.

The non-limiting examples which follow serve to illustrate the invention in more detail and may be read with reference to the Figures.

Figures Figure 1

Adhesion capacity to Caco-2 cells for selected dominant NSLAB and strains from a culture collection. The selected dominant NSLAB included the homofermentative strains that lowered pH below 5.1 in a meat model and were able to grow at 37⁰C following freeze-drying. Strains used as controls include

Lactobacillus rhamnosus GG and Escherichia coli B44, previously found to show high and low adhesion to Caco-2 cells, respectively. The adhesion capacity is expressed as the percentage of adhered bacteria (cfu/ml) in relation to the added bacteria (approx. 10⁶ cfu/ml). Measurements were conducted three times (three different passages) with duplicate determinations (n=6). The error bars indicate the standard deviations.

Figure 2

Acid production in the meat model by Lactobacillus plantarum MF 1291 ( ), MFl 298 (D), MFl 300 (Δ) or the commercial starter culture Lactobacillus curvatus HJ5 (♦) and with no Lactobacillus added (■). All the meat batters were added Staphylococcus carnosus Mill (10 cfu/g).

Figure 3 Viable cell count (CFU/g) of three of the selected strains (MF1291, MF1298 and MF 1300) strains during fermentation (three days at 24^°C), drying (25 days at 16^°C) and storage (28-50 days at 5 C) of a sausage fermented either by MFl 291, MF1298, MF1300 or the commercial starter culture Lactobacillus curvatus HJ5 in combination with Staphylococcus carnosus (Mill). Four replicates were performed, except for HJ5, MF 1291 and MF 1300 with only a single determination at day 0. The error bars indicate standard deviations.

Figure 4

Flavour profile of sausage fermented either by MF1291 (D), MF1298 ( ), MFl 300 (Δ) or by the commercial starter culture Lactobacillus curvatus HJ5 (■) in combination with Staphylococcus carnosus (Mill) at day 28 corresponding to the ready-to-sell date. Figure 5

Re-isolation of culture MFl 298 from faeces of 17 test-persons after being consumed as a freeze-dried culture. The culture was consumed from day 1-18 followed by a wash out period from day 19-30.

Figure 6

Re-isolation of culture MF 1298 from faeces of 17 test-persons after being consumed as sausage fermented with MFl 298. The culture was consumed from day 1-18 followed by a wash out period from day 19-30.

Figure 7

Caco-2 cells exposed to culture MF1298 (Δ) or alone (0).

Figure 8

Caco-2 cells exposed to Listeria monocytogenes alone (Δ) or MF 1298 1 hour prior to Listeria monocytogenes (A). Caco-2 cells alone (■) represent the control.

Figure 9 An immunoblot of the expression of ZO-I protein associated with tight junctions of polarized Caco-2 cells following 12 hours of exposure to MFl 298 or L. monocytogenes.

Figure 10 Pulsed Field Gel Electrophoresis of 7 Lactobacillus plantarum and one Lb. pentosus strains (Lane 1 = molecular weight marker (Lambda concatemer), 2 = Lb. plantarum 1, 3 = Lb. plantarum 2, 4 = Lb. plantarum ATCC8041, 5 = Lb. plantarum 3, 6 = Lb. plantarum NC8, 7 = Lb. pentosus MF1300, 8 = Lb. plantarum MF1291, 9 = Lb. plantarum MFl 298, 10 = molecular weight marker (yeast chromosome)).

Figure 11 PFGE (Pulsed Field Gel Electrophoresis) of 7 Lactobacillus plantarum strains and one Lb. pentosus strain (Lane 1 = molecular weight marker (yeast chromosome), 2 = Lb. plantarum ATCC8041, 3 = Lb. pentosus MF1300, 4 = Lb. plantarum 1, 5 = Lb. plantarum 2, 6 = Lb. plantarum NC8, 7 = Lb. pentosus MF1291, 8 = Lb. plantarum MF1298, 9 = Lb. plantarum ATCC8014, 10 = molecular weight marker (lambda concatemer)).

EXAMPLES

Example 1 - Meat products

The dominant non-starter lactic acid bacteria (NSLAB) of (i) 11 different Norwegian fermented sausages (ii) three different Swedish fermented sausages and (iii) one Norwegian cured ham were isolated. Generally, the Norwegian types of fermented sausages are drier than the Swedish types, which have a shorter maturation period. The Norwegian types differ from each other mainly due to their different ingredients (e.g. mutton meat or blood) and spices. Commercial starter cultures were applied for the manufacture of these products except for the cured ham. The processing times including fermentation and drying were 20-40 days for the Norwegian fermented sausages and 5-6 days for the Swedish fermented sausages. At the end of processing the sausages were vacuum-packed and stored at 8°C. NSLAB were isolated from the Norwegian fermented sausages and cured ham three weeks following the end of processing. NSLAB from the Swedish fermented sausages were isolated immediately, one and two weeks following the end of processing.

Example 2 - Isolation and identification of meat isolates

In order to identify the dominant NSLAB of the meat products, 1O g of the meat product was homogenized in 90 ml peptone water (0.9% (w/v) NaCl, 0.1% (w/v) peptone) for 1 min using a stomacher (Stomacher, Lab-Blender 400, Seward Medical UAC House, London, UK). Appropriate serial dilutions were plated onto MRS agar (Oxoid, Basingstoke, England) and incubated anaerobically (Anaerogen, Oxoid) for 48 h at 25⁰C. Approximately, 6-9 colonies from the highest sample dilution were selected and grown in MRS-broth (Oxoid). The isolates from each sample were further characterised by Randomly Amplified Polymorphic DNA (RAPD) using a single HEX-labelled 9-bp primer: 5^Λ-ACGCGCCCT-3^Λ as described by Johansson et al. (1995). The PCR fragment profiles were separated by capillary gel electrophoresis on the ABI PRISM 3100 Genetic Analyzer using the Genescan-500-ROX (Applied Biosystems, Foster City, CA, USA) as internal lane standard. Using the same primer two PCR profiles were made from two DNA preparations originating from a single colony. Fragment profiles were analysed using Gel Compare II (Applied Maths, Gent,

Belgium). For the analysis, fragment sizes of 50-500 bp were applied. The band search was performed using a minimum profiling of 3% and a minimum area of 0.3%. A position tolerance of 1% was used for band matching. The Pearson correlation was used for the distance calculations. The cluster analyses (user manual, GelCompare II, Applied Maths) was performed by the provided unweighted pair group method with arithmetic averages (UPGMA) and WARD algorithms. Strains were considered identical when a similarity above 90% was obtained.

Dominant NSLAB of the meat products were further characterized by the API identification system (BioMerieux, Marcy l'Etoile, France) according to the instructions of the manufacturer. Finally, the identification of the selected strains was confirmed by amplification and sequencing of 16S ribosomal RNA as described by Heir et al. (1999).

Results Among the 15 fermented meat products examined, NSLAB dominated in six products and the starter culture was either not detected or occasionally detected but with other strains being dominant. In the other nine products, the starter cultures were considered dominant as the only strain or as co-dominant with other strains. Initially, eleven isolates were selected as the dominant or co-dominant strains of the Swedish fermented meat products, and eleven isolates were selected as dominant or co-dominant strains of the Norwegian fermented meat products. The non-starter strains of the Swedish and Norwegian fermented meat products were identified as Lactobacillus sakei (five strains), Lb.farciminis (five strains), Lb. plantarum/pentosus (five strains) {Lb.plantarum/pentosus species can usually be identified according to their 16S rDNA sequence and/or by multiplex PCR e.g. as in Torriani et al, supra), Lb. alimentarius (four strains), Lb. brevis (two strains) and Lb. versmoldensis (one strain). Using API 50CHL profiles and 16S rRNA sequencing it was not possible to distinguish unequivocally between the species Lb. pentosus and Lb. plantarum. In order to reduce the number of strains for further examinations, heterofermentative strains, strains not growing at 37°C and not lowering pH below 5.1 in the meat model were excluded (results not shown). This initial screening resulted in nine strains (Table 1 , labelled "MF" as strain identity) that were selected for further analyses.

Example 3 - Bacterial strains and growth conditions

Strains to be examined included 9 dominant NSLAB that were homofermentative, growing at 37⁰C and lowering pH below 5.1 in a meat model as shown in Table 1.

Strains used as controls for the adhesion capacity included Lactobacillus rhamnosus GG (Valio Ltd, Helsinki, Finland) and Escherichia coli B44 (VTT Biotechnology, Espoo, Finland) corresponding to high and low adhesive strains (Tuomola and Salminen, 1998), respectively.

Indicator organisms used for determination of the antimicrobial activity included potential pathogenic bacteria which were kindly provided by the Department of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark and by Statens Serum Institute, Copenhagen, Denmark.

All strains were maintained at -4O⁰C in 20% (v/v) glycerol (Merck, Damstadt, Germany). Strains of Lactobacillus and Pediococcus were propagated anaerobically in MRS broth (Oxoid) for 24 h at 37°C. Other strains were propagated aerobically in BHI broth (Oxoid) for 18 h at 37°C.

Example 4 - Acidification properties in a meat model For initial screening, the ability of the dominant NSLAB to decrease pH below 5.1 was investigated. In addition, the rate of acidification was evaluated for strains that were able to decrease pH below 5.1 and fulfilled the probiotic criteria. A meat model system using plastic centrifuge bottles according to Hagen et al. (2000) was applied. A recipe for traditional Norwegian salami was used to prepare the meat batter containing 70% (w/w) beef and pork, 25.5% (w/w) pork back fat, 4.1% (w/w) NaCl and NaNO₂, 0.5% (w/w) glucose, 0.7% (w/w) sodium ascorbate and spices. The frozen meat (-5⁰C) and fat (-18°C) were minced and mixed with the ingredients. The strains were added to the batter at a concentration of 10 -10 CFU/g and the mixture was incubated at 24⁰C for 66 h. In addition, each meat batter was inoculated with a commercial starter culture {Staphylococcus carnosus Mill, Neraal & Co. AS, Oslo, Norway) at a concentration of 10⁶ cfu/g. As a reference, one batch was added the commercial starter culture Lactobacillus curvatus HJ5 (Neraal & Co. AS) in combination with Staphylococcus carnosus Mill. The acidification was recorded by pH measurement each hour as described by Hagen et al. (2000) and duplicate determinations were performed.

Results: For results see Fig.2.

Example 5 - Acid resistance and bile tolerance and ability to grow at 37°C

The acid resistance and the bile tolerance of the strains listed in Table 1 were examined in MRS broth adjusted with hydrochloric acid (HCl) to obtain a final pH of 2.5 and in MRS broth containing 0.3% (w/v) oxgall (Oxoid). The selected strains, propagated in MRS broth for 24 h at 37⁰C, were inoculated at a concentration of 10⁶ cfu/ml and following exposure for 1, 2, 3 and 4 h the survival rates were determined as the number of viable cells enumerated on MRS-agar after anaerobic incubation for 48 h at 37°C. Each determination was conducted in duplicate.

Results The nine NSLAB selected from the Swedish and Norwegian fermented meat products (Table 1) were not able to grow in the media acidified with HCl to a final pH of 2.5. Only 4 strains survived after 1 h of exposure and one of them (MF1300) survived after 4 h of exposure (Table 3). A higher proportion corresponding to 7 strains survived 4 h of exposure to 0.3% (w/v) oxgall (Table 3). All strains except 1 (MF 1295) were able to grow at 37⁰C. The strains were freeze-dried for further experiments resulting in poor growth for one of them (MF1295) and no growth for two other (MF 1296 and MF 1297) as shown in Table 3. These strains were excluded leaving 6 strains for additional examinations.

Example 6 - Adhesion capacity

Adhesion capacity to Caco-2 cells was investigated in vitro using the human colon carcinoma cell line for selected dominant NSLAB as listed in Table 1. The selected dominant NSLAB included the homofermentative strains that lowered pH below 5.1 in a meat model and were able to grow at 37°C following freeze-drying. The Caco-2 cells were purchased from the Deutche Sammlung von Mikroorganism und Zellkulturen (DSMZ), Braunschweig, Germany and cultured in Minimum Essential Medium (Earle's Salt, 25 mM Hepes and GlutaMAX™, Life

Technologies, Gibco, Rockville, MD, USA) supplemented with 16.5% (v/v) fecal bovine serum (FBS, Life Technologies, Gibco) previously heat inactivated (56°C for 30 min), 1% (v/v) non-essential amino acids (Life Technologies, Gibco) and 50 μg ml^"1 gentamicin (Life Technologies, Gibco). The cells were grown at 37⁰C in a humid atmosphere containing 95% air and 5% CO₂. Monolayers of Caco-2 cells were seeded at a concentration of 2x10 cells/ml and dispensed into each 200 mm well of a 24 well tissue culture plate (Nunc, Roskilde, Denmark). The selected strains were resuspended in the Caco-2 growth medium without gentamicin to a final concentration of approximately 10⁶ cfu/ml and 1 ml of this suspension was added to each well of the tissue culture plate. After 1 h of incubation the monolayers were washed three times with phosphate-buffered saline (PBS, pH 7.4) in order to remove non-adherent bacteria. The Caco-2 cells were lysed by addition of 0.1% (v/v) Triton-XlOO (Merck) and the number of viable adherent bacteria were determined by plating serial dilutions onto MRS agar. Colony forming units were enumerated after anaerobic incubation for 48 h at 37°C and the adhesion capacity is described as the percentage of bacteria adhered to Caco-2 cells in relation to the total number of bacteria added. Each adhesion assay was conducted three times (three different passages) with duplicate determinations.

Results

Generally, the adhesion capacity was independent of the bacterial species but showed to be strain specific (Figure 1). Most of the strains adhered moderately compared to the very low adhesive E. coli B44 (4.7 ± 2.8%).

Example 7 - Antimicrobial activity

Strains that showed higher adhesion capacity than the negative control Escherichia coli B44 as well as survived exposure to pH 2.5 and to 0.3% oxgall for at least 1 h were selected and examined for their antimicrobial activity against potential pathogenic bacteria and strains of the human GIT. The agar spot test described by Schillinger and Lϋcke (1989) was applied. In brief, overnight cultures of the selected strains were spotted (2μl) onto agar plates which consisted of MRS broth, 1.2% (w/v) agar and 0.2% (w/v) glucose. The plates were incubated anaerobically for 24 h at 37°C. An overlayer was made of 100 μl indicator strain mixed with 7 ml soft agar (MRS broth containing 0.7% (w/v) agar and 0.2% (w/v) glucose). The plates were incubated anaerobically when lactic acid bacteria were used as indicator strain otherwise they were incubated aerobically. After incubation at 37°C for 48 h, an inhibition zone larger than 1.0 mm was scored as positive. Each determination was carried out four times.

Results

Among the strains examined, four strains showed higher adhesion capacity than Escherichia coli B44 as well as survived exposure to pH 2.5 and to 0.3% oxgall for at least 1 h. These strains were examined for their antimicrobial activity against potential pathogenic bacteria. All four strains (MF1300, MF1298, MF1291 and MF1290) inhibited all the pathogens examined in the present study (Table 4).

Example 8 - Preparation of fermented sausages on an industrial scale

The meat batter was prepared on an industrial scale according to the recipe for Norwegian salami and strains were inoculated at levels described for the meat model. The strains applied included the fastest acid producers that fulfilled the probiotic criteria. The meat batter was stuffed into fibrous casings with a diameter of 94 mm and fermented at 24⁰C for three days followed by drying at 16⁰C for 25 days. The sausages were vacuum-packed and stored at 5⁰C. The viable count was determined during processing (0 and 14 days), at the time of vacuum packaging (28 days) and during storage (50 days) using the procedure as described for the isolation of NSLAB. Each determination was carried out on four individual slices of sausage. Identification of strains in the fermented sausage was assessed by visual examination of uniform colony morphology identical to the strain used as starter culture and finally confirmed by API 50CHL (BioMerieux) profiles for ten colonies from each sausage.

Results

To be used as a starter culture for fermented sausage an important property is the ability to decrease pH of the sausages. The ability to acidify the meat models was investigated for the ten strains selected as described above. The strains MF 1300, MF1298, and MF1291 as well as the commercial starter culture {Lactobacillus curvatus HJ5 ) performed the fastest acidification (Figure 2). Within 25 h these strains acidified the meat model to a pH level of 5.1. A slower acidification was observed for MF 1290 reaching pH 5.1 after 40 h of incubation.

The fastest acid producers (MFl 300, MF1298 and MF1291) were selected for the production of fermented sausages and the viable count during processing and storage was evaluated. All three strains dominated during the fermentation and at the ready-to-sell date (28 days) viable counts of MF1298 and MF1300 reached a high level (2.6x10 cfu/g and 2.9x10 cfu/g, respectively) which remained constant during the storage period (28 - 50 day) i.e. at time of consumption (Figure 3). The viable count of MFl 291 reached 4.7x10⁷ cfu/g at day 28 and decreased slightly during the storage period to a level of 2x 10⁷ cfu/g which is almost identical to the commercial starter culture (Figure 3). The pH of all the sausages decreased from pH 5.8 to pH 4.8-4.9 at day 28 (results not shown).

Example 9 - Sensory analysis

A sensory descriptive profile of sausages which were fermented by strains that fulfilled the probiotic and technological criteria was performed according to the description of Hagen et al. (2000) by 12 trained assessors. After 28 days of processing corresponding to the ready-to-sell date, the following attributes were evaluated: overall flavour intensity, fat flavour, maturity flavour, acidic taste (fruity acid), spicy flavour, garlic flavour, smoke flavour, metallic flavour, sweet taste, sour taste (acetic acid), salt taste, bitter taste, rancid flavour, hardness, tenderness, juiciness, fattiness and graininess. As reference a sausage fermented by the commercial starter cultures Lactobacillus curvatus HJ5 (Neraal & Co. AS) and Staphylococcus carnosus Mill (Neraal & Co. AS) was used.

Results

The flavour profiles of the sausages fermented by the strains that fulfilled the probiotic and technological criteria examined (MF 1291, MF 1298 and MF 1300) were almost identical to the sausage fermented by the commercial starter culture (Figure 4). The differences observed with regard to the acidic taste were not significant and did not influence the overall flavour intensity.

Example 10 - Susceptibility to antibiotics

Antibiotic susceptibility of strains that fulfilled the probiotic, technological and sensory criteria was performed by an E-test according to the instruction of the manufacturer (VIVA Diagnostika GmbH, Kδln, Germany). The strains were plated onto MRS agar with E-test stripes (VIVA Diagnostika GmbH) and the inhibition zones were read after anaerobic incubation for 24 h at 37°C. The minimal inhibitory concentration (MIC, μg/ml) was defined as the lowest concentration of the antibiotics that inhibit the strains. Strains with MIC values higher than the breakpoints proposed by Scientific Committee for Animal Nutrition (SCAN,

European Commission, 2001) or by Danielsen and Wind (2003) were considered to be resistant against the selected antibiotics. Susceptibility to the following antibiotics was evaluated: ampicillin (0.016-256 μg/ml), streptomycin (0.016-256 μg/ml), kanamycin (0.016-256 μg/ml), gentamicin (0.016-256 μg/ml), chloramphenicol (0.016-256 μg/ml), tetracycline (0.016-256 μg/ml), erythromycin (0.016-256 μg/ml), quinupristin/dalfopristin (0.02-32 μg/ml), vancomycin (0.016- 256 μg/ml), trimethoprim (0.02-32 μg/ml), ciprofloxacin (0.002-32 μg/ml), linezolid (0.016-256 μg/ml), rifampicin (0.016-256 μg/ml) and clindamycin (0.016-256 μg/ml).

Results

5

The antibiotic susceptibility of the three strains (MF1291, MF1298 and MF1300) was examined and according to the sensitivity guidelines provided by Danielsen and- Wind (2003) and SCAN (European Commission, 2001) the three strains were sensitive to the majority of the antibiotics. For vancomycin, kanamycin,

10 ciprofloxacin and streptomycin the selected strains (MF 1291 , MF 1298 and MF 1300) were considered to be resistant. Furthermore, MF 1300 were resistant against trimethoprim and gentamicin. Generally, the strains showed the same pattern of sensitivity except for MF 1300 which were less sensitive to ampicillin, gentamycin, trimethoprim, linezolid, chloramphenicol and clindamycin in comparison with

15. MF1291 and MF1298 (Table 5).

Example 11 - Survival and persistence in the human gastrointestinal tract - freeze-dried culture

The aim of this examination is to determine the ability of culture MF 1298 to 20 survive and persist in the human gastro intestinal tract (GIT) when consumed as freeze-dried powder diluted in water. The study was designed as a double blinded cross-over study with 17 healthy test persons who were randomized to consume either a freeze-dried mixture (1) of MF 1298 and MF 1291 or a mixture (2) containing MFl 300 together with two other strains. The study took place during two periods of 25 30 days where one period included a consumption period of 18 days followed by a "wash out period" of 12 days. The test persons consumed the freeze-dried cultures dissolved in water twice a day (6x10⁹ cfu/day). Fecal samples were collected before consumption (day 0), after consumption (day 18) and twice during the "wash out" period (day 24 and 30). The samples were diluted in physiological saline and plated 30 onto MRS -agar (pH 5) added vancomycin. Representative colonies were selected on the basis of colony morphology and microscopy and identified by using internal transcribed spacer PCR (ITS-PCR) for primary selection followed by restriction enzyme analysis (REA) combined with pulsed-field gel electrophoresis (PFGE).

MF 1298 was most frequently re-isolated from 4 out of 17 test persons at day 18 compared to the other strains examined (Lactobacillus plantarum/pentosus MF1291, Lactobacillus plantarum/pentosus MF1300) (Figure 5). These results indicate that MF 1298 survives in the GIT.

Furthermore, MF 1298 was recovered from one test person in the wash out period (day 24) indicating that the strain can persist in the GIT after the intake ended (Figure 5).

Example 12 - Survival and persistence in the human gastrointestinal tract fermented sausage

The aim of this examination is to determine the ability of culture MFl 298 to survive and persist in the human gastro intestinal tract (GIT) when consumed as sausage fermented with the freeze-dried culture. Based on the results of the intervention study using freeze-dried culture, MF 1298 was selected as starter culture to be used in an industrial production of fermented sausage and a new intervention study taking place during 30 days was performed with participation of the same 17 test persons as used for the prior intervention study. The test persons consumed two slices of sausage twice a day for 18 days (corresponding to 6xlO⁹ cfu/day MF1298). The consumption period was followed by a "wash out" period of 12 days. Fecal samples were collected and analyzed as described in example 7.

Results MF 1298 was recovered from 10 out of 17 test persons at day 18. The culture was not recovered during the "wash out" period (Figure 6).

Example 13 - Tight junction barrier - MF 1298

The aim of this examination is to study the influence of MF 1298 on trans- epithelial electrical resistance (TER) and tight junction proteins in Caco-2 cells. Monolayers of Caco-2 cells were seeded at a concentration of IxIO⁵ cells/cm², and grown in filter inserts (pore size: 0.4 μm; diameter: 12 mm) until full polarised monolayers were obtained. The integrity of the polarised monolayers was evaluated by measuring the TER by use of Millicell-ERS Electrical Resistance System (Millipore, Bedford, MA). The net value of electrical resistance was expressed in Ωxcm^" and calculated by subtracting the contribution of the filter and the medium. Defined concentrations of culture MF 1298 were adjusted at OD620 and resuspended in cell growth medium without antibiotics. Approximately 500 μl of the bacteria suspension or bacteria free cell growth media used as control were added onto the polarized monolayers in the Transwell filter inserts (pore size: 0.4 μm; diameter: 12 mm) designed as the inner (apical) chambers. The inner chambers were then placed into the outer (basolateral) chambers (diameter: 22.1 mm) containing 1500 μl bacteria-free cell growth medium and further incubated at 37°C in a humid atmosphere containing 95% air and 5% CO2.

Results MF 1298 was able to increase TER as shown in Figure 7.

Example 14 - Tight junction barrier - MF 1298 and Listeria monocytogenes

The aim of this examination is to study the interaction of MF 1298 and Listeria monocytogenes on the trans-epithelial electrical resistance (TER) of Caco-2 cells. Preparation of Caco-2 mono layers and measurements of TER were carried out as described in Example 13. Defined concentrations of Listeria monocytogenes were adjusted at OD620 and resuspended in cell growth medium without antibiotics. In the studies of the interaction between culture MF1298 and Listeria monocytogenes the mono layers were either pre-incubated with MF 1298 Ih prior to the addition of Listeria monocytogenes or incubated simultaneously.

Results

MFl 298 was able to delay the decrease of TER induced by Listeria monocytogenes as seen in Figure 8. Example 15 - Expression of ZO-I

To analyse tight junction proteins (ZO-I) of Caco-2 cells, lysates of polarized monolayers of Caco-2 cells were prepared after 12 h of exposure to Lb. plantamrn MF1298 (10⁸ CFU/ cm²), L monocytogenes (10⁸CFU/ cm²) or without bacteria (control). The monolayers were washed three times with PBS before lysis which was carried out using Stuart extraction buffer containing 100 mM NaCl (Merck), 1% (v/v) Triton X-IOO (Merck), 0.5% (w/v) deoxycholic acid (Sigma- Aldrich). 0.2% (w/v) SDS (Sigma-Aldrich), 2 mM EDTA (Sigma-Aldrich), 10 mM HEPES (pH 7.5, Sigma-Aldrich), 1 mM sodium orthovanadate (Sigma-Aldrich), 10 mM sodium fluoride (Sigma-Aldrich), 10 mM sodium pyrophosphate (Sigma- Aldrich), 1 mM benzamidine (Sigma-Aldrich), and 1 % (v/v) protease inhibitor cocktail for mammalian tissues (Sigma-Aldrich) for 20 min on ice. The cell lysates were centrifuged at 12000 x g for 30 min at 4°C and pellet was resuspended in 500 μl of Stuart extraction buffer and sonicated (Vibra Cell 72434, Bioblock Scientific, Illkirch. France) at 25 kHz for 20 seconds on ice. The lysate was centrifuged as described above and the supernatant was collected. The total protein concentration of the lysates was measured using BCA protein determination Kit (Pierce Chemicals, Rockford, IL). Each sample of equal amount (25 μg) of total protein was electrophoresed by SDS-PAGE using the NuP AGE^® precast gel system (Invitrogen Life technologies). The electrophoresed proteins were then transferred onto a PVDF membrane (Invitrogen Life Technologies) for 1 h at 30 V and blocked overnight in TTBS (Tris-buffered saline (Bio-Rad, Hercules, CA) supplemented with 0.5% (v/v) Tween 20 (Bio-Rad)) and 6% (w/v) non-fat dry milk (Bio-Rad) to prevent non-specific binding. After TTBS washes, blots were incubated for 1 h at room temperature in TTBS, containing both, monoclonal mouse anti-ZO-1 (Zymed Laboratories, San Francisco, CA) IgGs, at concentrations of 0.1 μg/ml. The blots were then washed 5 times for 10 min with TTBS and incubated for 1 h at room temperature in TTBS containing horseradish peroxidase-conjugated polyclonal goat anti-mouse (Bio-Rad) IgG, at a concentration of 0.04 μg/ml Immunoreactive bands were visualized using Immun-star™ Chemiluminescent Detection Kit (Bio-Rad) and the chemiluminescent signals were detected using autoradiographic film (Kodak BioMax XAR film, Eastman Kodak Company, Rochhester, NY). Quantification was performed using ImageQuant^® 5.2 for Windows NT and the results were expressed as the quantity of each band relative to the control.

Results Figure 9 shows an irnmunoblot of the expression of the protein ZO-I associated with tight junctions of polarized Caco-2 cells following 12 h of exposure to MFl 298 or L. monocytogenes at concentrations of 10 CFU/cm . Compared to polarized monolayers of Caco-2 cells without bacteria added, the addition of MF 1298 resulted in an up-regulation of the protein ZO-I whereas the expression decreased following exposure to L. monocytogenes. The changes of the expression of ZO-I correspond to the alterations observed for TER. The expression of occludin was not affected upon addition of MFl 298 and L. monocytogenes (results not shown).

Example 16 - Pulsed Field Gel Electrophoresis

Bacterial strains, media and growth conditions

L. plantation was grown on MRS agar (De Man et al., 1960) at 30 °C.

PFGE analysis Bacteria were harvested from MRS agar grown in 48 h. Cells were diluted in

LM buffer (Tris 6 mM, NaCl 1000 mM, EDTA 100 niM, N-lauroylsarcosine 1%) containing lysozyme (2 mg ml-1) and mutanolysine (3 U ml-1) to an OD600 of 0.6. The suspension was incubated for 10 min at 37°C and embedded in an equal volume of 2 % low-melting-point agarose in TE buffer (Tris 10 mM, EDTA 1 mM). Bacteria were lysed by incubating plugs over night at 37°C The LM solution was replaced by a PK buffer (Tris 10 mM, EDTA 100 mM, SDS 1%) containing proteinase K (1 mg ml-1), before incubation overnight at 5O⁰C. Plugs were washed once in TE buffer for 30 min at 30°C, once in TE buffer containing PEFABLOC (Roche Diagnostics) (2OmM) for 30 min at 30⁰C, and 5 times for 30 min at 50⁰C in preheated TE buffer. Plugs were kept at 4°C in TE buffer until use. For restriction enzyme digestion the plugs were first washed in sterile water, then equilibrated for 1 h in 200 μl of Ascl specific buffer at 37°C. Ascl (120 U ml-1) (New England Biolabs) was added and the plugs were incubated at 37°C overnight. After digestion, the plugs were submitted to PFGE on a CHEF-DR II apparatus (Bio-Rad). PFGE was performed in 1% GTG SeaKem agarose in 0.5 x TBE buffer (Sambrook et al., 1989). Migration was carried out at 14⁰C for 48 h at 150 V. The switch time was 5 s to 50 s. The molecular mass markers used were yeast chromosomal DNA (2.03-194 kb) and lambda concatemers (48.5-727 kb). After migration, gels were stained in ethidium bromide and photographed under U V light, or analysed on a Typhoon Scanner (Molecular Dynamics, Amersham Pharmacia), with a filter at 500 nm.

Results

Unique fingerprints were obtained for MF1291, MF1298 and MF1300 as can be seen in Figures 10 and 11.

Table 1 Origin and identity of the dominant NSLAB (MF strains). The selected dominant NSLAB included the homofermentative strains tihat lowered pH below 5.1 in a meat model ■

Strain

Group Species Origin identity

Meat isolates Lactobacillus farciminis MF 1288 Swedish salami, Mettwurst-type

Lactobacillus plantarum/pentosus MFl 290 Norwegian salami

Lactobacillus plantarunf MF 1291 Swedish salami

Lactobacillus sakei MFl 295 Swedish salami, Mettwurst-type

Lactobacillus sakei MFl 296 Swedish salami, Mettwurst-type

Lactobacillus alimentarius MF1297 Norwegian blood salami

Lactobacillus plantarum^* MFl 298 Norwegian mutton salami

Lactobacillus plantarum/pentosus MFl 299 Norwegian salami

Lactobacillus pentosus^* MF 1300 Norwegian mutton salami

Abbreviations: MF, Strains isolated fromthe Swedish and Norwegian fermented meat products identified by RAPD, API and sequence analysis of 16S rRNA ^a Differentiation of Lb. plantarum and Lb. pentcsus were carried out using PCR with recA genederived primers (Torriani et aL, 2001)

Table 2 Potential pathogenic bacteria used as indicator strains for the examination of antimicrobial activity.

Group _ In_d-i^;cat ^.or st _rain strain Supplier identity

Potential pathogens Escherichia coli Pl - ΓVT

Bacillus cereus P2 ΓVT

Shigella βexneri P3 SSI

Yersinia enterocolitica P4 ΓVP

Salmonella typhimurium P6 IVP

Listeria monocytogenes P7 IVP Abbreviations: IVP, Department of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark; SSI,Statens Serum Institut, Copenhagen, Denmark Table 3 Survival of the strains following exposureto acid (HCl, pH 2,5) and oxgall (0.3%, w/v) for 1, 2, 3 and 4 has well as the ability of the strains to grow at 37°C following freeze-drying.

Strain identity" Acid^D Bile" Growth at 37⁰C⁰

MF1300

MF1299 - +

MF1298 * * * */ growth_. +

MF1291 +

MFl 290 * +

MF1288 - +

MF1295 - -

MFl 296 - H-

MFl 297 - +/-

³ The name of the species are listed in Table 1 ^b Survival for Oh: -, Ih: *, 2h: **, 3h:***, 4h: ****, growth after 4h:****/growth "DC" strains were used as freeze dried.

⁰ No growth: -, growth: +, poor growth: +/- following freeze drying.

Table 4 Antimicrobial activity of strains against potential pathogenic bacteria measured by agar spot test. The strains examined include strains that survived exposure to pH 2.5 and to 0.3% oxgall for at least 1 h as well as with an adhesion capacity higher than 4.7%. The selected strains are ranked by decreasing antimicrobial activity.

Indicator strain

Strain Escherichia Bacillus Shigella Yersinia Salmonella Listeria coli cereus flexneri enterocolitica typhimurium monocytogenes

MF1300 -H- -H- -H-+ ++ -H- MF1290 -H- ++ +++ + MF1291 + +++

MFl 298 (+) + +++ + ++ -H-

0: 0 mm inhibition, (+): 0.51 mm inhibition zone, +: 1-2 mm inhibition zone, ++: 2-4 mm inhibition zone, : >4 mm inhibition zone. Table 5: The viable count oϊLb. plantarum MP 1291 , Lb. plantarum MF1298and Lb. pentosus MF1300 in the faeces of 17 volunteers when administered asfreeze dried strains or as sausage fermented by MFl 298. The faeces were analyzed before administration (0 day), at the last day of administration (18 day) and during the postadministration period . (days: 19-30). The strains were administered at a concentration of 6 XlO⁹CFU per day of each strain.

Way of Strain Rate of reisolation (CFU/g faeces) from volunteer no. 1-17 administration

O day 18 day 24 day 30 day

Freeze-dried MF1291³ No. 9, (1.2x10⁴) strains

MF1300¹

"included in mixture A (MF129 land MF1298) ^c-, no reisolations in any of the 17 volunteers

Claims

1. An isolated Lactobacillus plantarum or pentosus strain which has the ability to act as a starter culture, has protective activity and which is a probiotic.

2. An isolated Lactobacillus plantarum or pentosus strain of claim 1 wherein said strain can reduce the pH of a sample or acidify a sample.

3. An isolated Lactobacillus plantarum or pentosus strain of claim 1 or claim 2 wherein the pH is reduced to or below 6.5.

4. An isolated Lactobacillus plantarum or pentosus strain of any one of claims 1 to 3 wherein the pH is reduced to or between 4.8 and 5.1

5. An isolated Lactobacillus plantarum or pentosus strain of any one of claims 1 to 4 wherein said strain has the ability to become dominant in a fermented product and/or has the ability to give desirable sensory features in a fermented product.

6. An isolated Lactobacillus plantarum or pentosus strain of any one of claims 1 to 5 wherein said strain has at least one of the abilities selected from the ability to inhabit or survive the gastrointestinal (GI) tract in vivo, the ability to suppress or inhibit growth of microorganisms unwanted in the GI tract, the ability to adhere to the GI tract (GIT) and the ability to strengthen the resistance of the epithelial cell layer to pathogenic microorganisms.

7. An isolated Lactobacillus plantarum or pentosus strain of any one of claims 1 to 6 wherein said strain can survive at a pH of 5.0 or less than 5.0 and/or is bile tolerant.

8. An isolated Lactobacillus plantarum or pentosus strain of any one of claims 1 to 7 wherein said strain has the ability to suppress or inhibit the growth or activity of one or more food deteriorating or pathogenic microorganisms present in a food product.

9. An isolated Lactobacillus plantarum ot pentosus strain of claim 8 wherein said strain suppresses the growth or has an antimicrobial effect against one or more of Bacillus cereus, Shigella flexneri, Yersinia enterocolitica, Salmonella typhimurium, Listeria monocytogenes, Escherichia coli or Campylobacter.

10. An isolated Lactobacillus plantarum or pentosus strain of any one of claims 1 to 9 wherein said strain is sensitive to one or more antibiotics.

11. An isolated Lactobacillus plantarum or pentosus strain of any one of claims 1 to 10 wherein said strain is MF1298 (DSM 16997, 22/12/04 in DSMZ), MF1291 (DSM 17320, 17/5/05, DSMZ) or MF1300 (DSM 17321, 17/5/05 in DSMZ) or a strain derived therefrom, or a mutant or variant thereof.

12. An isolated Lactobacillus plantarum or pentosus strain of claim 11 wherein said mutant or variant is a strain with a closely related genetic finger print or a closely related Restriction Enzyme Analysis pattern.

13. A culture comprising the strain of any one of claims 1 to 12.

14. A strain or culture of any one of claims 1 to 13 which is freeze dried.

15. A food product comprising one or more of the strain or culture of any one of claims 1 to 14 wherein the strain or culture has been added to the food product.

16. A food product of claim 15 wherein said food product comprises more than 10⁷ CFU/g of one or more of the strains or cultures as defined in any one of claims 1 to 14.

17. A food product of claim 15 or 16 wherein said food product is a meat product.

18. A food product of claim 17 wherein said food product is a sausage.

19. The food product of any one of claims 15 to 18 which is fermented.

20. Use of the strain or culture of any one of claims 1 to 14 as a starter culture for the fermentation of a food product, and/or as a probiotic culture and/or as a protective culture.

21. A method for producing a fermented food product comprising the steps of inoculating, mixing or combining a food product with a strain or culture of any one of claims 1 to 14 and incubating the food product for a time and under conditions necessary for fermentation to occur.

22. A method for producing a food product with probiotic and/or probiotic properties comprising the steps of inoculating, mixing or combining a food product with a strain or culture of any one of claims 1 to 14.

23. A composition comprising a strain or culture of any one of claims 1 to 14.

24. The composition of claim 23, wherein said composition is a pharmaceutical composition.

25. The strain, culture or food product of any one of claims 1 to 19 or the composition of claim 24 for use in therapy.

26. Use of a strain, culture or food product of any one of claims 1 to 19 or the composition of claim 24 in the manufacture of a composition for use in therapy.

27. Method of treating a disease or condition in a mammal which method comprises administering to said mammal an effective amount of a strain, culture or food product of any one of claims 1 to 19 or the composition of claim 24.

28. Use or method of claim 26 or claim 27 to treat intestinal infections, any disease associated with increased permeability of the intestinal epithelium or reduced epithelial barrier function, gastrointestinal disorders, any disease involving the presence of pathogenic bacteria in the GIT or atopic eczema or allergies or to reduce serum cholesterol or the risk of sepsis with bacterial infections or to suppress or prevent cancer.

29. A kit comprising at least one strain or culture of any one of claims 1 to 14.

30. A kit of claim 29 wherein said kit is for use as a starter culture for fermentation and/or as a probiotic culture and/or as a protective culture.