ITMI20130792A1 - METHOD FOR THE PREVENTION AND / OR REDUCTION OF THE DEVELOPMENT OF LISTERY IN THE CHEESE INDUSTRY AND PREVENTION OF LYSTERIOSIS IN MAN. - Google Patents

Description

"Method for the prevention and / or reduction of the development of Listeria in the dairy industry and prevention of listeriosis in humans."

The present invention relates to a method for preventing and / or reducing the development of listeria in the dairy industry. Dairy products can be chosen from hard cheeses, semi-hard cheeses, stretched curd cheeses, soft cheeses, blue cheeses and in particular gorgonzola cheese. In particular, the present invention relates to a method for contrasting and / or reducing the development of listeria in cheeses which provides for the use of selected bacterial cultures with inhibitory activity for listeria spp. and / or producers of nisin and / or bacteriocins and / or metabolites active against Listeria spp. Finally, the present invention refers to a composition comprising said bacterial cultures for human use in the preventive and / or curative treatment of listeriosis.

Listeria is a genus of bacteria that includes six species, all Gram-positive, which primarily attack the immune system. Listeria monocytogen.es is an asporigenous Gram-positive bacterium, anaerobic facultative mobile at 28 ° C due to the presence of peritrichous flagella and is responsible for bacterial Listeriosis. Listeriosis is an infectious disease, generally transmitted with food, which despite having a low incidence still represents a serious problem to be countered and solved due to the serious consequences it can have on human health, since it can also cause forms severe meningitis.

Infection can occur by ingestion of certain contaminated dairy products, but also by direct contact with the food during its production phases. Some cheeses such as gorgonzola have a rind that is considered inedible precisely because of the possible presence of Listeria which could give rise to a listeriosis.

Cheese is a very popular food that is produced using the techniques and equipment known to experts in the dairy sector. Cheese is the product obtained from the acid or rennet coagulation of whole milk, partially or totally skimmed. Gorgonzola is a blue cheese produced in Italy from whole cow's milk. It is a soft, fat, raw, pale yellow cheese, whose green streaks are due to the marbling process, that is to the formation of mold (Penicillium). The method of obtaining (EC Regulation no. 510/2006 -GU L 93 of 31.03.2006, page 12) provides for the following operational sequence:

Production:

- whole cow's milk from the production area is pasteurized,

coagulation: once pasteurized whole cow's milk is inseminated with lactic ferments with a suspension of Penicillium spores and selected yeasts, added with calf rennet at a temperature of 28-36 ° C,

the curd is placed in the role bands and the original mark containing the number of the dairy is affixed to both flat faces,

the form obtained is subjected to dry salting, which is continued for a few days with temperatures of 18-24 ° C,

- during ripening, varieties and strains of Penicillium, characteristic of Gorgonzola and determining the blue-greenish color (natural herb), develop.

Seasoning: the minimum duration is 50 days and it is possible to obtain large wheels (minimum seasoning 50 days), medium (minimum seasoning 80 days) and small wheels (minimum seasoning 60 days).

For all types, seasoning is carried out in environments with a temperature of 2-7 ° C and with a humidity of 85-99%. During the maturation the paste is pierced several times to favor the development of the characteristic varieties and strains of Penìcillìum. At the end of the maturing period the product is checked and placed on the market.

The consumption of dairy products containing listeria (internal contamination) or listeria on the outside because they came into contact with the pathogen during their processing / production (external contamination) in a quantity of listeria above a certain limit value generally tolerated, it can cause listeriosis, in particular towards certain categories of consumers such as, for example, towards subjects undergoing drug treatment or with ongoing infections.

Listeria is sensitive to acid / medium pH values found in subjects not treated with IPP drugs (Inhibitors of the Protionic Pump) also known with the English abbreviation PPI, while it shows a good survival at pH above 5. Strains of listeria spp . have been isolated from the faeces of subjects treated with H2 receptor inhibitor drugs, which have effects similar to PPIs. Therefore, listeria can "in its own right be included among the pathogens that can survive the stomach unscathed in subjects taking IPP drugs, to the detriment of their health.

Therefore, the need is strongly felt to have a composition and a method to prevent and / or reduce the development of listeria in dairy products and for the preventive and / or curative treatment of listeriosis, in particular in the case of subjects under treatment. with H2 receptor inhibitor drugs or IPP drugs.

Therefore, it is desirable to be able to produce dairy products with an insignificant quantity / content of listeria so that their consumption by all categories of consumers (including subjects treated with H2 receptor inhibitor drugs or PPI drugs) is not harmful to human health and does not lead to the development of listeriosis.

For this reason, the need for operators in the dairy and pharmaceutical sector is strongly felt to have a valid and effective composition and method to prevent and / or reduce the development of listeria in dairy products and for preventive treatment. and / or curative of listeriosis, in particular for which subjects being treated with H2 receptor inhibitor drugs or PPI drugs.

The Applicant has responded to the aforementioned needs by developing a valid and effective composition and treatment method that are able to prevent and / or reduce the development of Listeria spp. in dairy products and for the preventive and / or curative treatment of listeriosis, in particular for those subjects being treated with H2 receptor inhibitor drugs or IPP drugs.

The proposed composition and method are inexpensive, easy to apply, effective and safe for processed food products and effective and safe for human health.

The object of the present invention is a composition for use in the preventive and / or curative treatment of listeriosis, having the characteristics as reported in the attached independent claim.

The object of the present invention is a first treatment method conceived to prevent and / or reduce the development of listeria on the outside of dairy food products once they have been prepared, having the characteristics as reported in the accompanying claim (external treatment method ).

The object of the present invention is a second treatment method conceived to counter and / or reduce the development of listeria within dairy food products which acts during their preparation (internal treatment method - Barrier effect).

The object of the present invention is a third treatment method conceived to counter and / or reduce the development of listeria both outside and inside the food products selected from meat and dairy products in accordance with said first and said second treatment method ( combined treatment method).

Said second and third methods are suitable for treating all types of cheeses that are not subject to a Protected Designation of Origin (PDO) specification. The present invention relates to the use of a composition comprising or, alternatively, consisting of at least one strain selected from Lactobacillus plantarum LMG P-21021 -LP01 and Lactobacillus lactis DSM 19072 -NS01 to prevent and / or reduce the development of listeria spp. in food products, as claimed in the accompanying claim.

The Lactobacillus plantarum LMG P-21021 -LP01 strain of bacteria was deposited by the Mofin S.r.l. on 16.10.2001 at the BCCM LMG center.

The Lactobacillus lactis DSM 19072 -NS01 strain was deposited by the Anidral Society (now Probiotical SpA) on 02.21.2007 at the DSMZ.

Preferred forms of the present invention are described below in the detailed description which follows and form the subject of the present invention.

The Applicant has developed a method for the prevention and / or reduction of the development of a pathogen belonging to the genus Listeria, in particular belonging to the species of Listeria monocytogenes in dairy products. Dairy products can be chosen from hard cheeses, semi-hard cheeses, stretched curd cheeses, soft cheeses, blue cheeses and in particular gorgonzola cheese.

The outer surface of a cheese wheel comprises a portion called the plate and a portion called the side. Once a form of cheese has been produced, for example a form of gorgonzola, this is subjected to a subsequent phase of maturation which can last from a minimum of 50 days to a maximum of 80 days, for the gorgonzola cheese.

Maturation includes a salting phase, a salting phase, a drilling phase and a seasoning phase.

For example, a form of gorgonzola cheese once prepared is left to rest for 12-24 hours (t = 0). Then after it goes, for example, into the dressing room for about 3 days (tl = 3) at a temperature of about 20-21 ° C where the dry salting of the cheese is done. Subsequently, the wheel is put, for example, in a seasoning cell at a temperature of about 4-5 ° C and after about 10 days (t2 = 13) the salting phase is carried out using a water solution. and salt, usually using a soaked sponge. This salting phase is, for example, usually repeated at least 2-3 times after about 10 days (one from the other treatment), for example at t3 = 23 days and t4 = 33 days.

The treatment method of the present invention (external method) was designed to prevent and / or reduce the development of a pathogen belonging to the genus of Listeria, in particular belonging to the species of Listeria monocytogenes.

The method involves at least a partial or total treatment of the external surface of a dairy product, in particular gorgonzola cheese, once said dairy product has been prepared.

Said at least a partial or total treatment, of the external surface of said dairy product, in particular gorgonzola cheese, is carried out by means of a manual or mechanical method which provides for the use of a solution comprising or, alternatively, consisting of at least one strain of selected bacteria from the group comprising or, alternatively, consisting of bacteriocin producing strains active against listeria and / or bacterial strains active in the production of nisin.

In a preferred embodiment, said solution comprises or, alternatively, consists of at least one bacteriocin-producing bacteria strain belonging to the Lactobacillus plantarum species and / or at least one nisin-producing bacteria strain belonging to the Lactobacillus lactis species.

The external surface of a cheese wheel is placed in direct contact with a liquid solution comprising or, alternatively, consisting of at least one strain of bacteria selected from the group of lactic bacteria producing bacteriocins active against listeria such as Lactobacillus plantarum LMG P-21021 - LP01 and / or at least one strain of bacteria selected from the group of lactic bacteria active in the production of nisin such as the strain Lactobacillus lactis DSM 19072 -NS01.

The strains of bacteria object of the present invention have been deposited in accordance with the provisions of the Budapest Treaty.

The Lactobacillus plantarum LP01 bacterium strain produces bacteriocins and has a marked inhibition activity of the pathogen Listeria monocytogenes as shown by the halos reproduced on the plates (see experimental part and Figure G).

The bacterium strain Lactobacillus lactis DSM 19072 -NS01 produces nisin as evidenced by the experimental tests.

Said liquid solution is applied by means of a mechanical device capable of spraying or nebulising or spraying said solution on the external surface of said dairy product, in particular gorgonzola cheese. The solution is applied for a time which depends on the device used and the pressure used, but as a rule it is applied until the external surface of said dairy product, in particular gorgonzola cheese, is completely covered or wet.

It is important that the liquid solution is distributed evenly and homogeneously until the entire external surface of the cheese wheel is wet. Once the liquid solution has been applied to the external surface of the cheese wheel, it is left to dry.

In an embodiment that concerns the treatment of a cheese wheel, preferably gorgonzola cheese, the treatment is carried out on the external surface (bare or flat and bare) and is carried out by brushing, using the same machine as the salting, and / or by nebulization. manual or mechanical.

Said first treatment method comprises from 1 to 4, preferably from 2 to 3, partial or total treatments of the external surface of a dairy product. The method provides a first partial or total treatment which is performed after 7 days, preferably within 10 days, starting from when the wheel enters the seasoning cell, preferably in place of the first salting. The method can "provide for a second partial or total treatment that is performed after 7 days, preferably within 10 days, starting from said first treatment, preferably in place of the second salting. The method can" provide for a third partial or total treatment that is performed after 7 days, preferably within 10 days, starting from said second treatment, preferably in place of the third salting. The method can "foresee a fourth partial or total treatment which is performed after 7 days, preferably within 10 days, starting from said third treatment, preferably in place of the fourth salting. A period of 7-10 days between one treatment and the next it is necessary to avoid that the pathogen of listeria has the time to replicate and develop in an uncontrolled manner and, therefore, to prevail over the bacteria object of the present invention.

Said liquid solution comprising or, alternatively, consisting of a mixture of bacteria as described above, is placed in contact only on the external surface of the cheese wheel (side or flat and side). Said solution is applied during the maturation phase, in particular during salting or salting, or in place of salting or salting.

Said liquid solution can be applied by brushing the external surface using the same machine used for salting or salting, in particular during salting or preferably in place of it.

Said liquid solution is applied in the integral version which includes all the metabolites and bioactive peptides present in the cultural growth substrate of the respective strains of bacteria used.

For example, the L. plantarum LP01 strain was prepared in MRS broth as follows: 1 kg glucose, 0.5 kg meat extract, 0.5 kg casein peptone, 0.25 kg yeast extract, 0.25 Kg sodium acetate, 0.10 Kg dibasic potassium phosphate, 0.10 Kg ammonium citrate, 5 grams magnesium phosphate and 2 grams manganese sulphate is pasteurized at 90 ° C for 15 seconds. Once cooled, 10 grams of the lyophilized L. plantarum LP01 strain are added to 50 liters of broth. The resulting product has a pH of about 3.80 and an SH value of 35.0 / 36.0.

For example, the L. lactis NS01 strain was prepared on a milk / whey basis as follows: 2.5 kg of milk and 2.5 kg of whey are pasteurized at 90 ° C for 15 seconds. Once cooled, 2 grams of the L. lactis NS01 strain are added and proceeded with an incubation at 32 ° C for 16 hours. The resulting product has a pH of 4.30 and an SH value of 14.0 / 15.0.

The Lactobacillus plantarum LP01 strain of bacteria is used in the form of an MRS broth, at a concentration ranging from 1x10 <s> to lxlO <9>. The Lactobacillus lactis NS01 strain of bacteria is used once prepared on a milk / whey basis, at a concentration ranging from lxlO <6> to lxlO <9>.

In a preferred embodiment, the liquid solution is a mixture comprising the Lactobacillus plantarum LP01 strain and the Lactobacillus lactis NS01 strain in a weight ratio ranging from 3: 1 to 1: 3, preferably from 2: 1 to 1: 2, even more preferably 1: 1.

Another object of the present invention is a method which can be defined as "combined" which provides a double level of safety against the development of Listeria monocytogenes in the production of cheeses.

A first level of safety (first treatment) against the development of Listeria monocytogenes is obtained by acting on the outside of the wheel by using an aqueous liquid solution on the external surface of a cheese wheel comprising a mixture of bacteria comprising or, alternatively, consisting of Lactobacillus plantarum LP01 and / or Lactobacillus lactìs NS01, under the conditions indicated above.

Advantageously, the first treatment is able to inhibit and reduce from the outside (flat and barefoot) the development of the pathogen Listeria monocytogenes, thanks to the use of said mixture of bacteria.

While a second level of safety (second treatment) against the development of Listeria monocytogenes is obtained by acting inside the form through the use in milk of a mixture of bacteria comprising or, alternatively, consisting of Lactobacillus plantarum LP01 and / or Lactobacillus lactis NS01, during the processing of the milk that leads to the obtaining of the cheese wheel.

Advantageously, the second treatment is able to exert a barrier effect inside the paste (which will lead to the cheese) which inhibits and reduces the development of the pathogen Listeria monocytogenes, especially thanks to the use of the Lactobacillus lactis NS01 bacterium strain.

The mixture of bacteria comprising or alternatively consisting of Lactobacillus plantarum LP01 and / or Lactobacillus lactis NS01 is used in association with the starter bacteria strains in a variable quantity with respect to the processed milk and the starter culture used.

The combined method that provides for said first and said second treatment is able to implement a complete inhibition of the development of the pathogen Listeria monocytogenes ensuring a high level of food safety.

EXPERIMENTAL PART

1. Gorgonzola cheese-making tests to prevent and control the development of Listeria spp with cultures with inhibitory activity - External treatment.

The tests were carried out on forms of gorgonzola cheese whose external surfaces (bare or flat and bare) were treated using a liquid solution of the culture in the integral version which includes all the metabolites and bioactive peptides present in the cultural substrate of growth of the respective strains of bacteria used such as the L. plantarum LP01 strain (prepared in MRS broth) and the L. lactis NS01 strain (prepared on a milk / whey basis).

For example, the L. plantarum LP01 strain was prepared in MRS broth as follows: 1 kg glucose, 0.5 kg meat extract, 0.5 kg casein peptone, 0.25 kg yeast extract, 0.25 Kg sodium acetate, 0.10 Kg dibasic potassium phosphate, 0.10 Kg ammonium citrate, 5 grams magnesium phosphate and 2 grams manganese sulphate is pasteurized at 90 ° C for 15 seconds. Once cooled, 10 grams of the lyophilized L. plantarum LP01 strain are added to 50 liters of broth. The resulting product has a pH of about 3.80 and an SH value of 35.0 / 36.0.

For example, the L. lactis NS01 strain was prepared on a milk / whey basis as follows: 2.5 kg of milk and 2.5 kg of whey are pasteurized at 90 ° C for 15 seconds. Once cooled, 2 grams of the L. lactis NS01 strain are added and proceeded with an incubation at 32 ° C for 16 hours. The resulting product has a pH of 4.30 and an SH value of 14.0 / 15.0.

Starting from a 1: 1 mixture of the two strains, a liquid solution was prepared having a bacterial load of: L. lactis (NS01) 3.5xl0 <9>; method: ISO 27205: 2010 IDF 149: 2010 ISO 7889 IDF 117: 2003 (lactococcus count); L. plantarum (LP01) 5.8xl0 <9>; method: ISO 27205: 2010 IDF 149: 2010 ISO 7889 IDF 117: 2003 (lactobacilli count).

The treatments concerned only the external surface (bare or flat and bare) and were carried out by brushing (with the same salting machine) and by manual nebulization, as reported below. The treatments concerned n.

56 shapes for test (total 224 = 56 shapes for test 1; 56 shapes for test 2; 56 shapes for test 3 and 56 shapes for standard) divided into two subgroups identified "right" and "left" (left indicated with: Standard X, Test 1, Test 2 X, Test 3 X) based on the physical position on the curing pallet. The efficacy of a single, double and triple treatment was verified (see Table 1). As a reference, 56 untreated forms were considered, also divided into two subgroups identified "right" and "left" based on the physical position on the maturing pallet (28 Standard forms and 28 Standard X forms).

The 56 forms of Test 1, Test 2 and Test 3 were treated by as indicated in Table. The brushing was performed with the same machine as the salting.

The search for Listeria spp. and of Listeria monocytogenes was carried out by taking samples of the crust from the bare feet of all 28 forms constituting the two subgroups identified "right" and "left". The overall quantity of each sampling was subjected to manual homogenization before taking the sample to be analyzed. The search for Listeria {spp. and monocytogenes) was carried out using the following official methods: (a) Qualitative Research: AFNOR method with enrichment of 25 g of the crust mix in 250 ml of broth at 30 ° C for 24 hours and subsequent smear in L Rapid Mono a 37 ° C for 48 hours; (b) Quantitative Research: ISO 11290-2 method with pre-activation of 10 g in 100 ml of peptone water for 1 hour at 30 ° C and treatment with a spatula in PALCAM agar of 1 ml of the appropriate dilution divided into n. 3 plates. The results obtained are reported in Table 2A and 2B.

The results show the effectiveness of the treatment already starting with a first treatment. Advantageously, a first treatment after 10 days from the cheese-making followed by another two / three treatments at a distance of 10-15 days from each other improve even more the results obtained with said first treatment. On the contrary, it has been found that late treatments, when the number of Listeria spp. has become important, they have no effect.

Similar results were obtained with the internal treatment and with the combined treatment.

Nisin producing strain: h. lactis DSM19072 -NS01 The L. lactis NS01 strain was isolated from cheeses from Monte Veronese. Nisin belongs to the class of bacteriocins called antibiotics (such as lactacin 481, lactococcin S and carnicine U149): they are peptides active on cell membranes, containing non-traditional amino acids such as lantionine and b-methyl lanthionine as well as dehydrated residues.

The points of the experimentation are listed below:

1. Development of a method for the verification of nisin producing strains.

Inhibition tests were performed on a test strain L. plantarum DSMZ 20174, reported in the bibliography to be sensitive to nisin, using the Lc strain. Lactis subsp. lactis from international collection DSMZ 20729, declared producer of this bacteriocin. A broth test was conducted in which the test organism was grown in contact with the supernatant of the producer strain:

a) the two strains were both inoculated in medium M17 + lact .0.5% and incubated o.n. at 30 ° C. At the same time, the broth culture of the L. plantarum DSMZ 20174 strain was set up in MRS medium;

b) the broth cultures in M17 were used, after incubation, as a source of supernatant, removing the cells by centrifugation and filtering at 0.45pm. The pH value of the two filtered supernatants was 6.15 per Lc respectively. Lactis and 6.13 for L. plantarum, demonstrating that the M17 medium, containing diglycerol-phosphate, has a certain buffering power on the pH variations that normally occur during bacterial growth. In this case, it was not necessary to intervene by neutralizing the supernatants in order to bring them to the same pH value before carrying out the broth test;

c) a series of test tubes were prepared in which 2ml of the solutions listed in the order in the table were added to 2ml of the broth culture in MRS of the test microorganism L. plantarum, standardized to 0.4 of OD560 with fresh MRS. The test tubes, performed in duplicate, were incubated at 30 ° C for 6 hours, after which the OD was read at 560nm, subtracting from each its own blank, stored at 4 ° C:

OD50 samples after 6 hours pH

added

H20 1,680 4, 37

Supernatant 1,700 4, 63

L.plantarum TQ

Supernatant -0 .115 6, 00

L.plantarum TQ

lmg / ml nisin

1:10 in water -0 .193 5.90

1: 100 in water 0.843 5.00

1: 1000 in water 1.625 4, 37

Supernatant -0 .157 6.08

Le.lactis

DSM20729 TQ

1:10 in water -0 .175 5.89

1: 100 in water 1,581 4.46

The results obtained show that the producer strain Lc. lactis DSMZ 20729 determined a real inhibition of the test strain sensitive to nisin and this inhibition can be correctable to that induced by a solution in which the bacteriocin (nisin SIGMA N5764) has been specially resuspended as a control (data reported in lines 3 and 7 ).

From the absorption data shown in the table it is also possible to quantify the concentration of nisin produced and present in the supernatant, on the basis of a standard curve that relates the OD560 value of the broth culture to the known concentration of nisin. Substituting in the equation of the curve, in place of x, the OD value found with the supernatant of Le. Lactis (dii. 1: 100), ie 1.581 (see previous table), the concentration value 1.2pg / ml is obtained, which multiplied by the dilution factor 100, determines the unknown concentration value of nisin in the supernatant of Lc. lactis, equal to about 12OÎ1⁄4g / ml.

2. Application of a molecular method (POR) for the identification of producer strains.

On the log Lc. lactis DSMZ 20279 the presence of the gene coding for nisin was verified using a specific primer (Y. Cait at all, 1997), to correlate the demonstrated production of nisin in broth (point 1) with the actual presence of the coding gene for it. The photo of the gel confirms the presence of the aforementioned gene: specific PCR for the gene encoding nisin. Expected amplified: 311bps

1) PCR Marker (Sigma), 2) White, 3) ID 1615 Lc. lactis sub lactis DSMZ 20729 (producer of nisin), 4) ID 1618 Lc. lactis sub cremoris DSMZ 20384, 5) ID 1619 Le. lactis sub lactis DSMZ 20069, 6) ID 922 L. helveticus (from internal collection anti-listeria strain), 7) ID 923 L. helveticus (from internal collection anti-listeria strain), 8) ID 1400 L. plantarum DSMZ 20174 (susceptible strain with nisin).

Following the negative result of the amplification with a specific primer for nisin on two strains of L. helveticus (internal collection ID 922 and 923), which were declared as producing strains of an anti-listeria bacteriocin, it was however decided to apply the broth protocol, developed with the producer strain Le. lactis (point 1), as confirmation of the negative PCR result, i.e. the real absence of nisin production by the two strains in question, excluding the possibility that the primer could give a false negative result as it is specific for the nisin gene of lactococcus strains. The following are the new results of the broth test:

OD 56o samples after 6 hours pH

added

H20 1,566 4.38

Supernatural L. 1.651 4.62

plantarum TQ

Supernal L. -0.152 5.92

plantarum TQ

100g / ml nisin

[10g / ml] 0.341 5.29

[5g / ml] 1.278 4.61

[lg / ml] 1,625 4.35

Supernatural Le lactis -0.141 6.05

DSMZ 20729 TQ

1: 100 in H20 1.581 4.40

Supernatural L. 1.763 4.55

helveticus ID

922 TQ

Supernatural L. 1.730 4.56

helveticus ID

923 TQ

The data confirm the absence of inhibitory activity on the L. plantarum DSMZ strain, sensitive to nisin, by the two strains of L. helveticus ID 922 and 923, which therefore do not produce this bacteriocin. PCR was then applied with a specific primer for nisin on a series of strains belonging to the species Le. lactis subsp. lactis and cremoris (verification by PCR speciespecifica), of different origins, available from the internal collection, in order to allow a more immediate identification of those possessing the gene encoding bacteriocin.

The gel shows an intense amplification band, corresponding to a Lc strain. lactis (ID 1254 from Anidral collection). A less intense amplification band is then visible in correspondence with two other strains ID 1612 and 1618.

3. Microbiological verification of producer strains identified by PCR

The broth test was carried out primarily on the Le strain. lactis ID 1254, which has an intense amplification band following PCR with a specific primer for the nisin gene (point 2, photo gel above). The method referred to in point 1 was followed. The absorption values after incubation of the broth cultures are shown below:

OD56 Samples After 6 hours

added

H20 1.617

L. plantarum surname 1.733

TQ

L. plantarum surname -0.167

TQ 100g / ml nisin

[10g / ml] 0.209

[5g / ml] 1.366

[lg / ml] 1,639

L lactis DSMZ -0.157

20729 TQ

1: 100 in H20 1.551

L. lactis ID -0.148

1254 TQ

1: 100 in H20 1.526

L. lactis ID -0.142

1254 TQ 1000g / ml

nisin

[lg / ml] 1,567

As can be seen, the producer strain identified L. lactis ID 1254 induces a true inhibition on the nisin-sensitive test strain (data shown lines 7 and 9) with an efficacy comparable to that of the control strain L. lactis DSMZ 20729. To confirm that the effect observed with the supernatant of the new strain, positive for the nisin gene, Lc. lactis ID 1254 is specific to the strain and due to the actual expression of the aforementioned gene, the broth test was repeated including as a negative control the supernatant of another strain of the same species ID 1252 Le. Lactis sub cremoris (internal collection), which does not amplify with specific primers for the nisin gene. To then exclude any inhibition attributable to lactic acid produced by dairy strains ID 1254 and 1252, another control was set up, consisting of only fresh M17 medium brought to a pH of about 4.50, corresponding to that of the supernatants obtained directly after incubation. of the strains Lc. lactis ID 1252 and 1254, using lactic acid; after which the medium was neutralized up to a value of about 6.20, to which the supernatants included in the test were found or corrected, using 7.5N NaOH. The absorption and pH values are shown below:

OD560 samples after 6 hours pH

added

H20 1.602 4.34

Supernal L. 1 .714 4.60

plantarum TQ

Supernatural Le lactis -0.111 6.09

DSMZ 20729 TQ

L. lactis surname 1.633 4.70

ID 1252 TQ

L. lactis surname -0.087 6.02

ID 1254 TQ

Land only M17 1.681 4.64

acidified with

B.C. lactic e

then reported to

pH 6.20 with

soda*

<â ™ ¦> land MI7 -0.059 6.01

+ 1000g / ml nisin

[10g / ml] 0.422 5.19

The above data confirm that the activity observed for the Lc. Lactis ID 1254 is specific and correctable to the production of nisin by the strain itself.

4. Characterization of the Lactobacillus plantarum strain LMG P-21021 LP01 (ID 1171)

Origin: Lactobacillus plantarum LP01 strain was isolated from feces of healthy individuals. The strain was not genetically engineered.

1) Biochemical characterization - Table A

2) Enzymatic profile: API Zym Bio MÃ © rieux - Int Met 015 - Table B

3) Total protein profile PAGE (PolyAcrylamide Gel Electrophoresis) Int. Med 003 (Biavati B., Scardovi V. and Moore W.E.C. "Electrophoretic patterns of proteins in thà ̈ genus Bifidobacterium and proposai of four new species"; Int J Syst Bacteriol. 1982 ; 32: 358-373) FIGURE C.

FIGURE C: 1. Negative reference: Lactohacillus rhamnosus ATCC 53103.2. Sample Strain: LP 01 (ID 1171) LMG P-21021 - Working Celi Bank, 3. Sample Strain: LP 01 (ID 1171) LMG P-21021 - Freeze-dried product, 4. Positive Reference: Lactohacillus plantarum DSM 9843 Stroke 2 -3: the profiles of the strain belonging to the Working Cell Bank (2) and of the lyophilized culture (3) are superimposable (characterizing bands indicated in green);

Strokes 4 and 2-3: the profile of the strains (2,3,4) are characteristic of the Lactohacillus plantarum species (arrows in blue)

Runs 1 and 2-3: the profiles of the strain belonging to the Working Cell Bank (2) and of the lyophilized culture (3) are different from that characterizing different species (1) (see red arrows).

4. Molecular characterization

1) Classification at the species level:

- PCR (Polymerase Chain Reaction) with primers: LFPR / PLAN II - Int. Met. 049 (Walter J. et al. "Detection and Identification of gastrointestinal Lactohacillus Species by using denaturing gel electrophoresis and species specific PCR primer". Appi Environ Microbiol. 2000; 66 (1): 297-303; -FIGURE D.

In FIGURE D there is a positive reaction for L. plantarum: 1. PCR marker: Sigma 50-2,000 bp; 2. Experimental Blank: No DNA; 3. Positive Reference: L. plantarum ID 091; 4. Positive Reference: L. plantarum ID094; 5. Postive Reference: L. plantarum ID126; 6. Sample strain: L. plantarum LP 01 (ID1171) LMG P-21021; 7. Positive reference: L. plantarum ID1283; 8. Positive reference: L. plantarum DSM20174; 9. Negative reference: L. casei sub. casei DSM 20011.

2) Fingerprinting Profile:

PFGE (Pulsed Field Gel Electrophoresis) with Not I (FIGURE E) and Sfi I (FIGURE F) - Int. Met. 161 (Tynkkynen S. et al. "Comparison of Ribotyping, randomly ampli fied polymorphic DNA analysis and pulsed- field gel electrophoresis in typing of Lactobacillus rhamnosus and L. casei strains"; Appi Environ Microb. 1999; 65 (9): 3908- 3914.

In Figure E and Figure F we have: 1. Electrophoretic marker: Sigma 50-1,000 kb; 2. Sample strain: L. plantarum LP 01 (ID1171) LMG P-21021; 3. Comparison strain: L. plantarum ID091; 4. Comparison strain: L. plantarum ID1393; 5. Comparison strain: L. plantarum ID 1521

5. Inhibition of Listeria monocytogenes

The ability of the L. plantarum LP01 strain to inhibit the ATCC 19112 strain of listeria monocytogenes was tested.

A broth culture was set up which was incubated for 24 hours in MRS broth. The cells were then washed and resuspended in fresh MRS broth medium. On the surface of plates containing the agar medium, specific for the pathogenic species to be inhibited, this pathogenic bacterium is sown starting from a fresh culture broth at the rate of 100 Î1⁄4Î per plate of the first decimal dilution. The cells thus treated are adsorbed on a paper disk at the rate of 100 Î1⁄4Î per disk. After incubation at 37 ° C for 24 hours, the inhibition halo is measured, represented by the space between the edge of the disk and the growth margin of the tested pathogen. Below are listed and represented in photos the evidence of inhibition by six strains of probiotic bacteria against the pathogen Listeria monocytogenes ATCC 19112.

Halos of inhibition expressed in millimeters (mm):

1.L. reuteri mm 0

2.L. reuteri ID 1774 mm 0

3.L. reuteri Biogaia (reference) mm 0

4.L. plantarum LP01 mm 5

5.L. delbr.susp. bulgaricus LDD01 mm 2

6.L. pentosus PCB 101 mm 4

Claims (8)

CLAIMS 1. A composition comprises or, alternatively, consists of at least one bacteriocin-producing bacteria strain belonging to the Lactobacillus plantarum species with activity against the pathogen Listeria monocytogenes and at least one nisin-producing bacteria strain belonging to the Lactobacillus lactis species. 2. The composition according to claim 1, wherein said at least one bacteriocin producing bacteria strain belonging to the Lactobacillus plantarum species is the Lactobacillus plantarum LMG P-21021 -LP01 strain of bacteria, while said at least one nisin producing bacteria strain belonging to the Lactobacillus lactis species is the Lactobacillus lactis strain DSM 19072 -NS01. The composition according to claims 1 and 2, for use in the preventive and / or curative treatment of bacterial listeriosis. The composition for use according to claim 3, wherein said treatment is aimed at subjects taking drugs inhibiting the H2 receptor or drugs inhibiting the protion pump. 5. A method to prevent and / or reduce the development of the pathogen belonging to the genus Listeria in dairy products which includes the following steps: to. preparing a liquid solution comprising the bacterial strains of claim 2 together with the metabolites and bioactive peptides present in the culture substrate for their growth, b. apply at least once said solution of step (a) on the external part of said dairy product in a homogeneous way. 6. The method according to claim 5, in which the pathogen is Listeria monocytogenes, the dairy product is a cheese, preferably a blue cheese or gorgonzola cheese, and step b) is applied from 1 to 4 times, preferably from 2 to 3 times, after a period of time ranging from 7 to 15 days, preferably 10 days from each other. The method according to one of claims 5 or 6, in which after said at least one step (b) follows a drying step during which the applied solution is allowed to dry on the external part of the dairy product. The method according to one of claims 5-7, wherein said at least one step (b) is applied during the maturation step of the dairy product; preferably instead of salting.