DESCRIPTION

"METHOD FOR PREPARING A MILK FOR MILK-DAIRY APPLICATIONS, THE MILK OBTAINED BY SAID METHOD AND THE USES THEREOF" * * * * *

The present invention relates to a method, called Mofinazione process, for preparing a milk intended for milk-dairy applications, the milk obtained with said method and the uses thereof. Said method includes an opportune heat treatment of the milk, followed by a pre-maturation concerning the addition and the development within said milk of opportune bacterial strains selected for this purpose, in particular strains with a probiotic valence. It is known that the clotting is the base of the caseation process which leads to the formation of cheese, yoghurt and other dairy products. The clotting phenomenon consists of a structural modification of the casein micelles which join together to form aggregates due to the action of the heat, the acidification and, as a consequence, of an enzymatic action.

The milk clotting due to the thermal heating is mainly due to the denaturation of the serum proteins which aggregate together and successively complex with the

casein forming co-precipitates at temperatures above 7O ⁰ C.

On the contrary, the acid clotting is due to the aggregation of the casein micelles because of the loss of calcium phosphate from the micelles themselves; the lowering of the pH for the concentration increase of acids within the milk causes the protonation/neutralization of the negatively charged casein functions. This causes a decrease of the zeta potential (a deter- minant parameter for the stability of the dispersed systems and defined as the electric potential value recordable at the surface of the double electric layer which usually is around any particles dispersed in a liquid) which, in turn, increases the solubilization of the calcium salts.

Such phenomenon induces a progressive passage of calcium from the calcium phosphocaseinate of the casein micelle to the aqueous matrix of the milk. At pH values between 5.7 and 5.8, 50% of the colloidal calcium is passed in solution, while at a pH = 4.6 (isoelectric point of the casein) the demineralisation of the casein is complete and therefore the destabili- zation of the casein micelles, which aggregate leading to the clot formation, is the greatest. The enzymatic coagulation of the milk takes place

through the addition of substances, generally defined as "milk coagulants" capable of exerting a hydrolytic action on the casein k, with a relative destabiliza- tion of the casein micelles, which promotes the aggre- gation of the micelles themselves to give the formation of a gel defined "curd" . The curd maturation is operated by:

- coagulating enzymes;

- enzymes produced by lactic bacteria used during the processing;

- residual enzymes typical of the fresh milk;

- enzymes produced by microorganisms contaminating the milk.

Said maturation generally determines the structural and organoleptic characteristics of the different cheeses ready to use.

These latter represent an uncontrolled variable and, so far, an uncontrollable variable both for the number and the typology. Such enzymatic complement resulting from bacterial flora contaminating the milk can affect in an absolutely negative way the organoleptic characteristics of the different cheeses produced.

The enzymatic clotting can also be defined as "rennet" clotting, as from time immemorial in the caseation

process it is used the "rennet" or curd, which is an enzymatic preparation of animal origin constituted by the natural extract of the bovine, ovine and goat abo- masum, prepared according to traditional known meth- ods . The main coagulating enzymes existing in the rennet are rennin and pepsin.

The rennet clotting certainly is the more used typology for the manufacturing of cheeses all over the world. The coagulating effect of the enzymes can schematically be divided in three successive steps: the first step consists of the enzyme attack on the micellar casein with hydrolysis of the phenylalanine-methionine bond (in the 105-106 position of the primary structure of the casein k) and leads to the detachment of a strongly hydrophilic casein-glycopeptide; the second step consists of the formation of hydrophobic bonds and calcium-phosphate salt bridges between the destabilized casein micelles. In fact, the casein molecules no more protected by the glycopeptide mutually hurt themselves and, thanks to the calcium existing in a ionic form within the milk, they start to bind to each other by producing the flocculation phenomenon; the third step follows the flocculation and consists of a reinforcement of the casein network through the forma-

tion of an always increasing number of bonds of a different nature.

Within the casein matrix, which constitutes the support structure of the caseous gel, the serous part re- mains entrapped.

During the third step, the gel becomes always thicker following to the increase of intermicellar bonds; micelles approach to each other and the clot is contracted causing the expulsion of the serum. This phe- nomenon, also known as draining or syneresis, is accelerated by the cut of the curd, the increase of the temperature and the increase of the acidity (with a relative lowering of the pH) produced by lactic bacteria which, by developing themselves, rapidly transform the lactose in lactic acid. Only the first two steps above described determine the real clotting, namely the passage of the casein from the colloidal suspension state to the gel state, while the third step essentially consists of the gelation of all the mass of milk and the start of proteolytic phenomena not specific in other sites of the casein k and on caseins α _s and β.

The rate and the course of the flocculation and the following gelation affect in a determining extent the rheological characteristics of the curd with reference

to elasticity, texture, permeability and contractility of the clot and consequently to the syneresis capability of the serum.

Several factors affect the steps above described, in particular the first two steps.

The length of the first step (also called "floccula- tion time") depends on the temperature, which must be, within reasonable limits, close to the optimal one for the catalytic effectiveness of the enzyme; the concen- tration of the total enzyme, calcium and phosphorous; the free acidity values (pH) ; the tertiary and quaternary structure of the casein (which can facilitate or block the enzyme access to the attack sites) . The features of the second step (gelation) mainly de- pend on the protein and casein concentration, the concentration of the free calcium and phosphate ions,- the free acidity (namely, the pH) and the temperature which increases the reactions rate. The development of these two steps can be followed and evaluated by means of an equipment called lactody- namograph, thanks to which the flocculation (or "setting") time corresponding to the first step and the extent of the gelation corresponding to the second step can be measured. It is therefore possible to establish in advance if

the characteristics of the milk under examination are such to make it suitable to the caseation. Said lacto- dynamograph thus allows to determine the clotting time and the consistency of the clot of milk. Information re- lating to such technique are provided, for example, in the "Trattato di tecnologia casearia" by Ottavio SaI- vadori del Prato, Ed. Agricole, 1998, pages 203-205. The aptitude of the milk to the rennet clotting, that is its reactivity towards the rennet, therefore con- stitutes, together with its fermentative aptitude, namely the tendency to the growth of lactic bacteria, a fundamental parameter for a correct and optimal dairy transformation. The bonification heat treatment to which the milk must necessarily be subjected before its industrial use, is a particularly delicate factor. In fact, if conducted at too high temperatures and/or for excessively long times, said treatment remarkably reduces the aptitude to the milk clotting and, consequently, the quality of dairy products resulting thereof.

Said bonification heat treatment of the milk is usually carried out through pasteurization or sterilization. According to what is usually known, the pasteurization is a thermal bonification treatment with the main pur-

pose of eliminating the pathogenic microorganisms existing in the milk, as well as greatly reducing a great part of the remaining microbial flora, such as yeasts, coliforms and generally so-called "anti -dairy" microorganisms, as they are responsible of structural and/or organoleptic defects of the milk-dairy products obtained from said milk.

Actually, in the last years it has been found that some pathogenic microbial species, such as for example certain strains of the genus Listeria, are capable of surviving to said treatment .

In some cases, milk-dairy derivatives strongly contaminated by Listeria monocytogenes, still existing after traditional pasteurization of the milk, have de- termined mortality events in the consumers.

Usually, the pasteurization consists of heating the milk at a temperature below its boiling point for an opportune time. The pasteurized milk can be intended both for the human diet and the dairy transformation; the one for a food use usually has a shelf life of 6 days under refrigerated conditions.

Typically, said pasteurization includes the following steps : - pre-heating step, at a temperature between 4O ⁰ C and 45°C; ^"

- homogenization step, wherein the milk is exited from a high-pressure nozzle (150-200 bar) thus breaking fat clots and eliminating the tendency to the surface cream formation; - degassing step, wherein the air bubbles are removed bringing the milk at 45° under a partial vacuum;

- real pasteurization step, which can be of two kinds: low and slow, or high and quick; in the first case, the milk is brought at a temperature of 63 ⁰ C for about 30 min, in the second case at a temperature between 72°C to 75°C for 10-20 seconds,-

- cooling step, until a temperature depending on the technological process.

The sterilization, on the contrary, is a process capa- ble of destroying any microbial forms, vegetative or in form of spore, as well as the viral forms, among which the bacteriophages.

Said process is used when it is necessary to store the milk for 3-6 months at room temperature. The most commonly used technological process is the

UHT (Ultra High Temperature) , conducted at 140-144 ⁰ C for 2-4 seconds.

The sterilization, because of the high temperatures reached by the milk during the process, causes the al- teration of , some components thereof, in particular

proteins and sugars, by compromising, in each case, its natural clotting aptitude.

In conclusion, the purification of milks intended for dairy transformation is now exclusively carried out through pasteurization of a traditional type (at a temperature between 72 ⁰ C and 75 ⁰ C for 10-20 seconds) , the only treatment suitable for the maintenance of the natural clotting aptitude of the fresh milk. However, the same is not able to reduce the load (quantity) of the bacterial sporing strains (for example some species of the genus Clostridium and/or Bacillus) at acceptable residual levels, as well as the non- sporing thermoduric microorganisms (for example, some species of the genus Micrococcus, Mycobacterium, Enterococcus, Streptococcus and Pediococcus) originally existing within the milk.

The substantial persistence of the "antidairy" strains above mentioned in the milk intended for miIk-dairy applications (also after purification through tradi- tional pasteurization) gives rise to a reduced quality of the end dairy products (for example, due to swelling phenomena of the cheese and, generally, to qualitative-quantitative alterations of the fermentation/maturation kinetics which compromise the or- ganoleptic peculiarities of the products themselves) .

Furthermore, under the above pasteurization conditions it is also noted the non-elimination of the phages (with a particular reference to those specific of the lactic bacteria) . Said problem can compromise the growth of the microbial cultures used within the dairy processing (selected starters, natural lacto- or serum-grafts) , with serious technological problems and unacceptable economic losses. Furthermore, as above mentioned, the non-complete elimination of the pathogenic agents, such as some species of Listeria, above all the species Listeria monocytogenes, can represent a serious sanitary problem, making the product no more suitable for the consumption. Concerning this, it is worthwhile to point out that said pathogenic species have improved, in the course of the years, the own capability of withstanding to the traditional pasteurization conditions usually employed (72°C-75°C, for 10-20 seconds) . Because of this phenomenon, due to the natural and evolutionary selection of the species, with the above traditional pasteurization it is no more possible to ensure the desired elimination (or reduction below an acceptable threshold) of said pathogens. Moreover, it is known that, in addition to the pro-

gressive worsening of the hygienic-microbiological qualities of the milk and the products resulting therefrom because of the development of potentially pathogenic microbial species above described, an addi- tional problem is represented by the thermoresistant toxins produced from pathogenic microorganisms possibly contaminating the milk, and by the presence of enzymes still active after the traditional pasteurization step. Said enzymes can be both of an endogenous nature, intrinsic of the milk composition, and of which one of the most representative is the alkaline phosphatase, and of a bacterial nature. The typical time/temperature combination of a tradi- tional pasteurization is not capable of bringing the residual total enzymatic activity below an acceptable threshold for the purposes of a good dairy transformation and, therefore, of the quality of the milk-dairy products obtained. Therefore, it would be useful being able to provide a milk intended for dairy applications which is substantially free of undesired and disadvantageous or harmful contaminating substances. In particular, there remains the need of providing a milk intended for dairy applications, in which the re- sidual quantity of phages, unfavourable enzymatic ac-

tivities, thermoduric microorganisms, pathogenic agents and their toxins, often present within the raw milk, is substantially null (or at least below a value considered acceptable from the sanitary and industrial point of views) , without qualitatively-quantitatively altering (in a negative sense) the tendency of said milk to the clotting.

It is an object of the present invention to provide an adequate answer to the need above pointed out . This and other aims, which will result apparent from the following detailed description, have been attained by the Applicant, which has unexpectedly found that, by subjecting the milk intended for dairy applications to a heat treatment at a particular, proper tempera- ture, followed by the addition and successive development (pre-maturation) , within said treated milk, of an effective quantity of at least one properly selected bacterial strain, it is possible to obtain a milk substantially free of the undesired contaminating sub- stances above described and which preserves the original tendency to the clotting.

Another object of the present invention is a method for producing a milk suitable for dairy applications, as reported in the appended independent claim. A further object of the present invention is the milk

obtainable through the method of the present invention, as reported in the appended independent claim. Another object of the present invention is then the use of said milk above mentioned in the dairy sector, as reported in the appended independent claim.

Preferred embodiments of the present invention are reported in the appended dependent claims. Features and advantages of the present invention are pointed out in detail in the following description; furthermore, they are also shown, by way of example, in the enclosed Table 1, in which: Table 1 shows the thromboelastogram of:

1- a reference milk, thermally treated at 72°C for 40 seconds (that is, under conditions of a traditional pasteurization) ;

2- the same milk thermally treated at 82.5°C for 40 seconds, without pre-maturation,-

3- the same milk thermally treated at 82.5°C for 40 seconds and, successively, pre-matured with the bacte- rial strain LMG-P-21385 through incubation at a temperature of 9 ⁰ C for 18 hours;

4- the same milk, previously maintained under refrigerated conditions at 4 ⁰ C for 4 days, thermally treated at 72 ⁰ C for 40 seconds; 5- the same milk, previously maintained under refrig-

erated conditions at 4 ⁰ C for 4 days, thermally treated at 82.5 ⁰ C for 40 seconds, without pre-maturation; 6- the same milk, previously maintained under refrigerated conditions at 4°C for 4 days, thermally treated at 87.5°C for 40 seconds and, successively, pre- matured with the bacterial strain LMG-P-21385 through incubation at a temperature of 9°C for 18 hours. The Applicant has completely unexpectedly found that, by subjecting a milk intended for dairy applications for an opportune time interval to a heat treatment at a higher temperature than 75 ⁰ C, followed by the addition and the development (pre-maturation) , within said thermally treated milk, of an effective quantity of at least a proper physiologically compatible bacterial strain, it is possible to obtain a milk which maintains unaltered its original tendency to clotting and that, in the meantime, it is substantially free of un- desired contaminating agents (above described) . Said pollutants, on the contrary, are still present, in a different extent, in the milk pasteurized according to conventional techniques known to the skilled in the art.

The method according to the present invention for the preparation of a milk intended for dairy applications, includes at least a step a) (called heat treatment) ,

in which the starting milk is subjected to a heat treatment at a temperature above 75 ⁰ C for a time above 1 second.

Preferably, the temperature of said heat treatment is > 80 ⁰ C.

Advantageously, said temperature is between 80 ⁰ C and 95°C; still more preferably, from 83°C to 90 ⁰ C. In a particularly preferred embodiment, said temperature is about 85°C. Preferably, the time of said heat treatment is above 3 seconds .

Preferably, said time is between 5 seconds and 10 minutes; more preferably, it is between 15 seconds and 5 minutes; still more preferably, it is between 20 sec- onds and 1 minute.

In a particularly preferred embodiment, the time of the heat treatment is about 40 seconds.

In a preferred embodiment of the invention, said step a) includes: - heating said milk until a temperature preferably between 83°C and 90 ⁰ C (more preferably of about 85°C) , for a time preferably between 20 seconds and 1 minute (more preferably, about 40 seconds) ; - cooling the milk thus thermally treated at a tem- perature between 1°C and 15 ⁰ C, preferably between 3

and 12 ⁰ C, however as a function of the treatment conditions of the subsequent step.

The method of the present invention further includes at least a step b) (called pre-maturation step) , in which the thermally treated milk resulting from the step a) is additioned with an effective quantity of at least a physiologically compatible microbial strain, followed by the development, in opportune conditions within said milk, of the additioned microbial strain. Advantageously, said development of at least one microbial strain allows to restore the original tendency to the clotting of the milk itself thanks to the production and the catalytic activity of specific enzymes produced by the additioned strains. Preferably, said at least one microbial/bacterial strain is selected from the group including the genera: Lactobacillus, Bifidobacterium, Lactococcus, Leu- conostoc, Pediococcus, Streptococcus, Propionibacte- rium and mixtures thereof. For example, of the genus Lactobacillus the species: L. pentosus, L. plantarum, L. casei, L. casei ssp. paracasei , L. rhamnosus, Lactobacilli belonging to the group acidophilus, L. delbrueckii ssp. bulgaricus, L. fermentum are preferred. For example, of the genus Bifidobacterium the species:

B. longum, B. breve, B. lactis, B. adolescentis, B. pseudocatenulatum, B. catenulatum, B. infantis are preferred.

For example, of the genus Lactococcus the species: L. lactis and L. lactis ssp. lactis are preferred.

For example, of the genus Streptococcus the species S. thermophilics is preferred.

In a preferred embodiment of the invention, said at least one strain is selected from the group including the species: Lactobacillus plantarum, Lactobacillus casei, Lactococcus lactis, Lactobacillus acidophilus, Bifidobacterium lactis and mixtures thereof. In a particularly preferred embodiment of the invention, said at least one strain is selected from the group including the species: Lactobacillus plantarum and Lactococcus lactis and mixtures thereof. In a particularly preferred embodiment, said strain is selected from the group consisting of: Lactobacillus plantarum LMG-P-21385 deposited on 01.31.2002, Lacto- coccus lactis subsp. lactis LMG-P-21387 deposited on 03.15.2002, Lactococcus lactis subsp. lactis LMG-P- 21388 deposited on 01.31.2002 and Lactobacillus plantarum LMG-P-21389 deposited on 03.15.2002 (all c/o the BCCM/LMG Bacteria Collection of Gent, Belgium) and mixtures thereof.

The acronyms relating to the strains above shown refer to the access number of the relative deposits carried out by the Firm MOFIN S.r.l., Via Pietro Cu-stodi, 12, Novara, in accordance with the Budapest Treaty on the international acknowledgement of the microorganisms deposit of the 28.04.1977.

The strains belonging to the genus Lactobacillus, species plantarum (with the deposit numbers LMG-P-21385 and LMG-P-21389) are characterized by: isolation: from human fecal samples with methods known to the skilled in the art; growth in a MRS culture broth (DIFCO, ref . 288130) at 30 ⁰ C; they are in form of single, short-chained, short rods; they grow well at 30 ⁰ C; they do not generate spores; gram-positive,- facultative heterofermentings ; at temperatures higher than 70 ⁰ C, said strains are inactivated; therefore, when subjected to traditional pasteurization conditions, their complete degradation occurs.

Strains belonging to the genus Lactococcus, species lactis (having deposit numbers LMG-P-21387 and LMG-P- 21388) , are characterized by: isolation: from samples of cow's milk pre-heated at 25 ⁰ C for 15 minutes, with following isolations accord-

ing to what usually foreseen by the know technique of the sector; growth in milk at 30 ⁰ C overnight (possibility of growing also in culture broth M17 at 3O ⁰ C) ; they are in form of elongated ovoidal cells with a diameter between 0.5 and 1 μm; doublet or short-chained growth; they do not generate spores; gram-positive,- microaerophilic ; final pH, after growth in broth having glucose as a carbon source, between 4.0 and 4.5; obliged omofermentings ; they form lactic acid starting from glucose, galactose, maltose and lactose and, in a lower extent, also from other sugars; at temperatures higher than 70 ⁰ C they are inactivated, therefore, when subjected to the conditions of a tra- ditional pasteurization, their complete degradation occurs .

Such strains can be used alone or in admixture therebetween in a varying mutual ratio as a function of the strain combinations . In one of the preferred embodiments of the present in- Vention, the mixture consists of the following strains: LMG-P-21385 in a quantity between 10% and 40%; LMG-P-21387 in a quantity between 10% and 40%; LMG-P-21388 in a quantity between 10% and 40%; LMG-P- 21389 in a quantity between 10% and 40%.

In the step b) , said at least one bacterial strain (or mixture of bacterial strains) is added to the milk resulting from step a) in the more opportune physical form, selected from liquid, anhydrous or frozen, de- pending on the type of milk and/or the type of micro- organism/s employed.

Preferably, it is sufficient to add very low quantities of the above strains to the thermally treated milk resulting from a) . Independently of the physical form of the culture used, the quantity added to the milk is such to obtain a concentration between 10 ⁴ and 10 ⁹ CFU/ml of milk. Preferably, said concentration is between 10 ⁵ and 10 ⁸ CFU/ml of milk; particularly preferred, between 10 ⁶ and 10 ⁷ CFU/ml of milk.

Usually, after the addition of at least one microbial strain according to the invention to a milk resulting from a) , there is the development of said strain in the milk in opportune conditions. Preferably, said development takes place at temperatures between 1 and 15°C, preferably from 6 to 12 ⁰ C, for time > 1 hour, preferably between 4 and 48 hours, more preferably from 8 to 30 hours,- particularly preferred, between 12 and 24 hours. For example, said development is conducted at a milk

temperature of 9 ⁰ C for a time of 18 hours. In a particularly preferred embodiment of the invention, the preparation method of the milk for a dairy use according to the present invention includes, therefore, the following steps: a) subjecting the milk to a heat treatment at the temperature and for the time above shown; b) adding and developing in suitable conditions, within the milk resulting from step a) , a quantity, between those above mentioned, of at least one bacterial strain selected from those above mentioned. The milk obtained with the method according to the present invention above described has proved to have the same typical original tendency to the clotting. Accordingly, with the addition and the following development of at least one bacterial strain, selected from those above described, in the milk after the heat treatment of the same, according to step a) , it was unexpectedly possible to completely restore the normal tendency to the clotting of the milk itself, without altering the normal clotting parameters of the initial milk.

Therefore, an object of the present invention is also the use of said strains above mentioned for restoring the coagulative tendency of a milk thermally treated

under the conditions above described.

Furthermore, said milk has proved to be substantially free of the undesired contaminating agents above shown. In particular, it has been shown that the milk obtained with the method of the present invention has a significant lowering of the Listeria monocytogenes in its thermoresistant variant, with respect to what usually occurs by means of a traditional pasteurization. As for the phages problem, it has also been shown that said heat treatment completely eliminates the same, thus avoiding the serious technological problems during the dairy transformation. A further and unexpected advantageous aspect of the milk obtained according to the method of the present invention is the remarkable activity reduction of the enzymes naturally existing within said milk and/or liberated from the lactic flora cells after its degradation during the thermization. The heat treatment of a milk according to the present invention, in fact, is able to irreversibly denature most of the enzymes and toxins initially existing within said milk. The milk, when the heat treatment and pre-maturation steps (Mofinazione process) are ended, can be simply heated at the temperature of the

dairy processing, thus maintaining the probiotic valences, if any, or being subjected to a mild heat treatment, such to inactivate the bacterial forms used in the pre-maturation process. The quality of the dairy food products obtained from the same results considerably improved, being said milk substantially free of the undesired residual contaminating agents which, on the contrary, are still existing within the milk pasteurized with traditional methods.

The Mofinazione process, besides the advantages above described, further ensures a better yield in the caseation as it allows to combine within the curd the serum proteins denatured by the heat effect. In other words, the present invention ensures a greater yield of the cheese, with consequent incontrovertible advantages of economic nature.

Therefore, an object of the present invention is also the milk intended for milk-dairy applications obtain- able with the method of the invention above described. Advantageously, also the food products resulting from the caseation of said milk are substantially free of the undesired contaminating and/or pathogenic residual agents above mentioned. Consequently, said products are differentiated from the known ones because of the

lack of defects and for the best sanitary characteristics .

Another extremely advantageous aspect is that two of the particularly preferred strains of the invention (Lactobacillus plantarum LMG-P-21385 and Lactobacillus plantarum LMG-P-21389) belong to the species Lactobacillus plantarum, a typology with remarkable probiotic properties . The pre-maturation operated with said microorganisms imparts, in this way, a probiotic valence to the milk itself and, accordingly, also to the cheese produced therefrom.

Analytical investigations carried out by the Applicant have pointed out the presence of specific peptides both within the pre-matured milk having the two aforesaid strains and within the cheeses obtained therefrom; waiting for additional investigations, it is believed that such peptides are bioactive and that part of the probiotic activities of the two strains of L. plantarum may be due to the same.

Therefore, also the dairy food products, in particular yoghurt and/or cheeses, obtainable from the milk intended for milk-dairy applications according to the present invention form another object of the present invention.

The following experimental part shows, by way of absolutely not limiting example, the clotting tendency of milk samples obtained with the method of the present invention, in comparison with that of a milk treated in a traditional way, and the reduction of the catalytic activity of an enzyme naturally existing in the milk in high concentrations, the alkaline phosphatase, following to different heat treatment conditions of the milk itself. Example 1- Evaluation of the clotting tendency of the milk.

In order to check the clotting tendency of the milk obtained according to the present invention, clotting tests on milk samples pasteurized at 72 ⁰ C with a tra- ditional pasteurization method and on milk samples thermally treated according to the method of the present invention, respectively at 82.5°C and 87.5°C, have been carried out . Said samples have not been ad- ditioned with any microorganisms before the determina- tion of their clotting tendency.

In parallel, the same clotting tests have been carried out on samples similar to those above mentioned, but pre-matured with the bacterial strain LMG-P-21385 before being subjected to the evaluation of the clotting tendency.

The thromboelastograms recording of the samples above mentioned has been carried out with a lactodynamograph FOSS Italia under the following experimental conditions : - temperature 32 ⁰ C;

- substrate of cow's milk having pH = 6.75;

- calf liquid rennet having a titer equal to 1:4000 (80% rennin and 20% pepsin), additioned in an extent of 23 μl per 10 ml of milk (0.23%, v/v); - within the samples additioned with the bacterial strain above shown, said addition has been carried out starting from a liquid culture in an extent of the 0.5% (volume/volume, v/v) and the resulting mixture has been maintained for 18 hours at 9°C for the devel- opment of said strain before the thromboelastogram recording.

The adopted procedure was the following: to a volume of milk of the samples above mentioned (both those without addition of the microorganism and those previously additioned with the mentioned microorganism) , heated at 32°C, an effective quantity of rennet (23 μl per 10 ml of milk, that is 0.23% in v/v) has been added for inducing the clotting thereof. The milk-containing wells were supported on a movable base, which performs a very slow circular movement.

The nib dipped within the milk does not encounter, at the beginning, a large friction and it remains still, then, after the progress of the clotting, it brings the nib, which follows in this way the movement of the movable base .

Resulting thromboelastograms are those reported in the enclosed Table 1. The most important parameter for evaluating an optimal tendency to the clotting of a milk, for the purposes of a correct caseation, is the one identified as K20, which shows the required time, starting from the beginning of the clotting process, for obtaining such a mechanical resistance of the clot to induce a total displacement of the nib of 20 mm. The parameter K20 is therefore strictly connected with the rheological characteristics of the rennet.

High values of K20 are indicative of a less thick clot, namely a poor tendency of the milk to clotting in the times required for obtaining a milk-dairy product of a good quality. Values of pH and parameter K20 of six milk samples, whose thromboelastograms are reported in the table 1, are presented in the following Table 1. In particular, the first three thromboelastograms have been obtained from fresh milk samples, while the last

three have been recorded starting from milk samples stored under refrigerated conditions for 4 days.

TABLE 1

As it is apparent from table 1, milk samples after heat treatment at 82.5°C or 87.5°C show K20 values at least 2-2.5 times longer than the values typical of the milk pasteurized according to the conventional process.

On the contrary, milk samples thermized under the same conditions, and successively pre-matured with the

strain LMG-P-21385, according to the invention, have shown to have absolutely comparable K20 values with those of the milk pasteurized according to the conventional procedure. Therefore, it is unexpectedly shown that the addition and the development of said microorganism in milk thermized according to the method of the present invention has allowed to restore the original tendency to the milk clotting. Example 2 - Evaluation of the residual catalytic activity of the alkaline phosphatase in the milk. The alkaline phosphatase is an enzyme naturally existing within the raw milk; moreover, it is liberated following to the thermal degradation of almost all the microbial species existing in said milk.

Said enzyme, like all the protein molecules, is denatured under high temperatures conditions, therefore it is a valid indicator both of the entity of the thermal treatment undergone by a milk (time/temperature combi- nation) and of the level of residual enzymatic activities in the milk after the heat treatment itself. In order to quantify the residual activity of the enzyme, an international method (FIL IDF 155A:199) has been carried out, based on a continuous fluorometric procedure which uses, as a substrate, a non-fluo-

rescent aromatic monophosphoric ester which, in the presence of the alkaline phosphatase, undergoes a hydrolysis reaction by producing a highly fluorescent molecule . In the table 2 below there are reported the values of a residual phosphatase activity (in milliunits of enzyme/liter, mU/1) existing in a milk pasteurized according to the traditional procedure and in a milk thermized at 85°C for 40 seconds. TABLE 2

As shown by data of table 2, the residual phosphatase activity in a thermally treated milk according to the method of the present invention is equal to about a tenth of the typical residual activity of a milk pas- teurized at 72°C for 20 seconds.

The data shows how the method of the present invention is capable of remarkably reducing the concentration of active enzymes, also including those anti-dairy, if present, which originate defective and poor quality productions of the milk-dairy products resulting therefrom.