WO2008065491A2 - Method for preparing freezed bacterial cultures for use in the food, dietary, nutraceutic and pharmaceutical field - Google Patents

Method for preparing freezed bacterial cultures for use in the food, dietary, nutraceutic and pharmaceutical field Download PDF

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Publication number
WO2008065491A2
WO2008065491A2 PCT/IB2007/003485 IB2007003485W WO2008065491A2 WO 2008065491 A2 WO2008065491 A2 WO 2008065491A2 IB 2007003485 W IB2007003485 W IB 2007003485W WO 2008065491 A2 WO2008065491 A2 WO 2008065491A2
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Prior art keywords
lactobacillus
bifidobacterium
ssp
culture
frozen
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PCT/IB2007/003485
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French (fr)
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WO2008065491A3 (en
Inventor
Giovanni Mogna
Federico Bruno
Original Assignee
Anidral S.R.L.
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Priority to EP07825662A priority Critical patent/EP2094829A2/en
Publication of WO2008065491A2 publication Critical patent/WO2008065491A2/en
Publication of WO2008065491A3 publication Critical patent/WO2008065491A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/02—Making cheese curd
    • A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G1/00—Preparation of wine or sparkling wine
    • C12G1/02—Preparation of must from grapes; Must treatment and fermentation
    • C12G1/0203—Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms

Definitions

  • the present invention relates to a method for preparing frozen bacterial cultures for use in the food, dietary, nutraceutics and pharmaceutical fields.
  • Said method includes the packaging of the bacterial culture under aseptic conditions, followed by the freezing process, thus avoiding all those handlings which are a potential contamination source.
  • starter cultures which are added to the starting raw materials in order to obtain the desired end products in the dairy-, baked products-, fermented sausages- and vinification sectors .
  • Said starter cultures are selected grafts, consisting of known microbial strains (or mixtures thereof) , with well defined biochemical properties and which therefore allow to obtain repeatable and enduring results.
  • One of the physical forms in which starter cultures are marketed is the frozen form, which shows the advantage of combining a good shelf life of the same with an absolutely acceptable cost, if compared with the one of cultures in a freeze-dried form.
  • the freezing process is carried out by subjecting the bacterial culture to a quick falling of the temperature of the product itself (for example up to temperatures ⁇ -40 0 C) , followed by the packaging of the same .
  • the most currently used methods foresee the rapid freezing of the same usually through a contact with refrigerated surfaces or through air flows at temperatures generally between -40 and -50 0 C, as well as through the direct use of a coolant, such as liquid nitrogen or carbon dioxide at the solid state, which is contacted with the food.
  • a coolant such as liquid nitrogen or carbon dioxide
  • the freezing process is, at present, generally performed through direct percolation in liquid nitrogen, with a consequent immediate change of the existing water from the liquid form to the solid one.
  • a disadvantage resulting from such process is that the starter culture, once frozen as above described, must then be stored at very low temperatures, which are on average between -70 and -55°C, with the obvious need of making use of particularly exacting and expensive cooling environments and devices.
  • a further substantial drawback connected with the freezing processes commonly used is due to the fact that, during the execution of such processes, there is a direct, more or less extensive, contact between the freezing agent, or the device, and the product intended for the freezing, with consequent possible microbial contamination phenomena of different origin and nature. Said type of contamination persists in the frozen end product also after its packaging, although this can occur in controlled hygienic conditions.
  • liquid cultures In the specific case of liquid cultures, a possible contamination source can result from the contacting surfaces of the freezing plant itself and the air existing therein.
  • the liquid nitrogen allows the preservation of the existing biological pollutants, if any, in a frozen state but viable and capable of reproducing themselves when permitted by the environmental conditions.
  • the presence of biological pollutants in a frozen starter culture intended for dairy applications is particularly harmful, as the inoculum temperature of the milk in the boiler and its following processing is on average suitable to the growth of most of said pollutants .
  • the sanitary situation of the food or the product for a food use is particularly serious, as the spores are generally able to withstand most of the heat treatments to which foods, before their consumption, are generally subjected. Therefore, there remains thus the .need of being able to provide frozen bacterial cultures for the use in ⁇ food, dietary, nutraceutics and pharmaceutical fields which are free of each kind of contamination.
  • bacterial cultures must be free of contaminations due to the plants and means used for the freezing (especially when the freezing operation is carried out by percolation in liquid nitrogen) .
  • the object of the present invention is to provide an adequate answer to the need above pointed out .
  • an object of the present invention is a method for preparing frozen bacterial cultures for the use in food, dietary, nutraceutics and pharmaceutical fields which are free of contaminations, having the features described in the appended independent claim.
  • Another object of the present invention is a bacterial culture which can be obtained with the above method, having the features described in the appended independent claim.
  • Another aspect of the present invention is the use of said bacterial culture in a food, dietary, nutraceutics and pharmaceutical fields, having the features described in the appended independent claim.
  • Preferred embodiments of the present invention are re- ported in the appended dependent claims .
  • the method subject of the present invention, relates to the preparation of a bacterial culture including: a) at least a first step wherein the starting culture is packaged under completely sterile conditions; b) at least a second step wherein said culture resulting from a) is subjected to freezing.
  • the first step a) is performed on the product under complete sterile conditions by using known equipments and modes and usually employed in the sector. For example, one works in a sterile environment at a temperature on average between 1°C and 20 0 C, preferably from 3°C to 1O 0 C, and having an air quality at least equal to the class 100, obtained through opportune filtering systems of the HEPA (High Efficiency Particulate Air) type.
  • HEPA High Efficiency Particulate Air
  • the environment ⁇ in which the packaging takes place is kept in overpressure conditions of sterile air (with a quality at least of class 100) equal to at least 1.01 atmospheres.
  • the method according to the present invention is characterized in that the frozen microbial culture obtained with said method is substantially free of pollutants .
  • the method allows to eliminate/reduce the contamina- tion sources in a production line of frozen microbial cultures; in particular, in the food and pharmaceutical industries .
  • said process for use in the food field is a starter micro- bial culture in a liquid form.
  • Said starter culture in a liquid form is prepared, for example, by adopting the following steps: - development of the microbial biomass in an industrial fermenter using a proper fermentation medium and opportune development conditions of the microbial bio- mass according to the microorganism to be reproduced;
  • Cryoprotectants are substances commonly used in the freezing sector. Said substances are usually carefully selected, as a function of the microorganism of interest, among physiologically compatible substances capa- ble of protecting the microbial cells from the osmotic and mechanical stresses caused by the freezing process.
  • said substances are selected from the group including; amino acids and derivatives thereof; monosaccharides and disacch.arid.es; polymers presenting alcoholic functions within their structure; polyethers; other substances known to a skilled in the art.
  • cryoprotectants are selected from the group including: sodium glutamate, mannitol, lactitol, sucrose, fructose, starch, glycerol, propanediol, polyvinyl pyrrolidone (PVP) , dimethyl sulfoxide
  • DMSO dimethyl methacrylate
  • PEG polyethylene glycol
  • Said cryoprotectants are additioned to the water volume used for re-suspending the washed microbial biomass in a quantity on average between 10% and 65% (weight/volume or volume/volume) based on the total weight of the water; preferably, from 25% to 50% (weight/volume or volume/volume) .
  • the used quantity of the cryoprotectant is, however, varying as a function of the physical-chemical features of the cryoprotectant and the biomass cell con- centration after its re-suspension in the cryoprotec- tive solution.
  • the starter culture in a liquid form includes at least a microorganism physiologically compatible with the human and/or animal organism.
  • said at least one microorganism is se- lected from the group including the following genera: Lactobacillus, Bifidobacterium, Lactococcus, Leu- conostoc, Pediococcus, Streptococcus, Bacillus, Propi- onibacterium, Saccharomyces, Enterococcus, Staphylo- coccus .
  • Lactobacillus are preferred the species: L. pentosus, L. plantarum, L. casei, L. casei ssp. paracasei , L. casei ssp. rhamnosus, L. acidophilus, L. delbrueckii ssp. bulgaricus, L. fermen- turn, L. gasseri.
  • B. longum B. breve
  • B. lactis B. adolescentis
  • B. pseudocatenulatum B. catenulatum
  • L. lactis L. lactis ssp. lactis.
  • Streptococcus are preferred the species: S. thermophilus.
  • the starter culture includes a mixture of two or more bacterial strains selected among those above mentioned. It is understood to mean that the freezing method according to the present invention is, in any case, applicable to any type of starter microbial culture in a liquid form, independently from the type of process with which the same is prepared.
  • Starter cultures in liquid form once ready for being frozen, are packaged (preferably under a laminar flow of sterile air at a temperature between 1 and 10 0 C) , in sterile primary packaging materials, preferably selected from: polyethylenes , aluminium and polyesters and/or opportune multilayered combinations thereof .
  • the same are preferably tightly sealed under the same aseptic conditions above described.
  • the following freezing (relative to the step b) above described) is carried out using known techniques in the art of the refrigeration; preferably, through the use of liquid nitrogen or an opportune refrigerator system at a temperature ⁇ -18 0 C for a sufficient time to obtain the freezing of the water existing in the packaged product .
  • the freezing time of a total volume between 10 1 and 100 1 is on average between 5 seconds and 2 minutes .
  • a starter culture in liquid form resulting from the step a) is frozen by using liquid nitrogen at -200 0 C for times between 3 seconds and 1 minute.
  • the viability of the frozen product obtained with the method of the present invention is equivalent to the one typical of the traditional frozen starter cultures.
  • the Applicant has found that, in the starter liquid cultures in which the filtration and the washing of the microbial biomass have been carried out, there can be found a sensible decrease, or a total ab- sence, of the lactic acid organic salts (such as ammonium lactates and/or calcium lactates, usual metabolites resulting from the fermentation and development steps of the biomass within the fermenter) .
  • This working mode allows to obtain a stability increase of the culture itself after the freezing.
  • frozen starter cultures according to the method of the present invention have shown to have an excellent shelf life at temperatures between -24 0 C and -18 0 C, with consequent much more limited storage costs.
  • refrigerator devices at lower temperatures usually between, in the general use routine procedure, -70 0 C and -55 0 C) are not needed anymore, while, on the contrary, they are still needed in case of traditional frozen cultures .
  • the shelf-life of a starter culture frozen according to the method of the present invention has been evaluated based on the maintenance, over time, of the ability of exact volumes of said culture, inoculated in a proper medium, for example milk, of lowering the pH until precise values after the same time intervals.
  • the shelf-life of said frozen starter culture, stored at a temperature between -24 0 C and -18 0 C, has proved to be > 4 months, preferably between 4 and 12 months; more preferably, > 6 months.
  • the frozen starter culture obtained with the method of the present invention, is used as starter in the food sector, for example in the dairy industry; dietary sector, for example in the supplements; nutraceu- tic and pharmaceutical sectors.
  • the cheese production process to be followed with the frozen starter culture obtained with the method of the present invention is equal to the one which is followed with the traditional frozen cultures .
  • Products which originate therefrom have the same organoleptic characteristics, but show high guarantees from a sanitary point of view.

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Abstract

The present invention relates to a method for preparing frozen bacterial cultures for use in food, dietary, nutraceutic and pharmaceutical fields. Said method includes the packaging of the bacterial culture in aseptic conditions, followed by the freezing process, thus avoiding all those handlings which are a potential contamination source.

Description

DESCRIPTION
"METHOD FOR PREPARING FREEZED BACTERIAL CULTURES FOR USE IN THE FOOD, DIETARY, NUTRACEUTIC AND PHARMACEUTICAL FIELD" * * * * *
The present invention relates to a method for preparing frozen bacterial cultures for use in the food, dietary, nutraceutics and pharmaceutical fields. Said method includes the packaging of the bacterial culture under aseptic conditions, followed by the freezing process, thus avoiding all those handlings which are a potential contamination source.
It is known the use of microbial cultures, so-called starter cultures, which are added to the starting raw materials in order to obtain the desired end products in the dairy-, baked products-, fermented sausages- and vinification sectors .
Said starter cultures are selected grafts, consisting of known microbial strains (or mixtures thereof) , with well defined biochemical properties and which therefore allow to obtain repeatable and enduring results. One of the physical forms in which starter cultures are marketed is the frozen form, which shows the advantage of combining a good shelf life of the same with an absolutely acceptable cost, if compared with the one of cultures in a freeze-dried form. Generally, the freezing process is carried out by subjecting the bacterial culture to a quick falling of the temperature of the product itself (for example up to temperatures < -400C) , followed by the packaging of the same .
In case of the food products, the most currently used methods foresee the rapid freezing of the same, usually through a contact with refrigerated surfaces or through air flows at temperatures generally between -40 and -500C, as well as through the direct use of a coolant, such as liquid nitrogen or carbon dioxide at the solid state, which is contacted with the food. On the contrary, in case of the starter microbial cul- tures in a liquid form, the freezing process is, at present, generally performed through direct percolation in liquid nitrogen, with a consequent immediate change of the existing water from the liquid form to the solid one. A disadvantage resulting from such process is that the starter culture, once frozen as above described, must then be stored at very low temperatures, which are on average between -70 and -55°C, with the obvious need of making use of particularly exacting and expensive cooling environments and devices. A further substantial drawback connected with the freezing processes commonly used is due to the fact that, during the execution of such processes, there is a direct, more or less extensive, contact between the freezing agent, or the device, and the product intended for the freezing, with consequent possible microbial contamination phenomena of different origin and nature. Said type of contamination persists in the frozen end product also after its packaging, although this can occur in controlled hygienic conditions.
In the specific case of liquid cultures, a possible contamination source can result from the contacting surfaces of the freezing plant itself and the air existing therein. The same applies with reference to the freezing of the liquid cultures through percolation in liquid nitrogen, as this medium does not absolutely ensure the complete sterility. On the contrary, the liquid nitrogen allows the preservation of the existing biological pollutants, if any, in a frozen state but viable and capable of reproducing themselves when permitted by the environmental conditions. For example, the presence of biological pollutants in a frozen starter culture intended for dairy applications is particularly harmful, as the inoculum temperature of the milk in the boiler and its following processing is on average suitable to the growth of most of said pollutants . In the particular case of contaminations due to the presence of sporogenic microorganisms, the sanitary situation of the food or the product for a food use is particularly serious, as the spores are generally able to withstand most of the heat treatments to which foods, before their consumption, are generally subjected. Therefore, there remains thus the .need of being able to provide frozen bacterial cultures for the use in ■ food, dietary, nutraceutics and pharmaceutical fields which are free of each kind of contamination. In particular, bacterial cultures must be free of contaminations due to the plants and means used for the freezing (especially when the freezing operation is carried out by percolation in liquid nitrogen) . The object of the present invention is to provide an adequate answer to the need above pointed out . These and other aims, which will result apparent from the following detailed description, have been attained by the Applicant, which has unexpectedly found that, by packaging the bacterial cultures under completely aseptic (i.e. sterile) conditions and successively freezing the same, an end culture free of each kind of pollutant is obtained. Therefore, an object of the present invention is a method for preparing frozen bacterial cultures for the use in food, dietary, nutraceutics and pharmaceutical fields which are free of contaminations, having the features described in the appended independent claim. Another object of the present invention is a bacterial culture which can be obtained with the above method, having the features described in the appended independent claim. Another aspect of the present invention is the use of said bacterial culture in a food, dietary, nutraceutics and pharmaceutical fields, having the features described in the appended independent claim. Preferred embodiments of the present invention are re- ported in the appended dependent claims .
The method, subject of the present invention, relates to the preparation of a bacterial culture including: a) at least a first step wherein the starting culture is packaged under completely sterile conditions; b) at least a second step wherein said culture resulting from a) is subjected to freezing.
The first step a) is performed on the product under complete sterile conditions by using known equipments and modes and usually employed in the sector. For example, one works in a sterile environment at a temperature on average between 1°C and 200C, preferably from 3°C to 1O0C, and having an air quality at least equal to the class 100, obtained through opportune filtering systems of the HEPA (High Efficiency Particulate Air) type.
More preferably, the environment ■ in which the packaging takes place is kept in overpressure conditions of sterile air (with a quality at least of class 100) equal to at least 1.01 atmospheres. The method according to the present invention is characterized in that the frozen microbial culture obtained with said method is substantially free of pollutants . The method allows to eliminate/reduce the contamina- tion sources in a production line of frozen microbial cultures; in particular, in the food and pharmaceutical industries .
In another preferred embodiment of the invention, said process for use in the food field is a starter micro- bial culture in a liquid form.
Said starter culture in a liquid form is prepared, for example, by adopting the following steps: - development of the microbial biomass in an industrial fermenter using a proper fermentation medium and opportune development conditions of the microbial bio- mass according to the microorganism to be reproduced;
- separation, for example through filtration or cen- trifugation, of the microbial biomass developed by the fermentation medium;
- washing of the microbial biomass thus separated with an isotonic liquid;
- separation, for example through filtration or cen- trifugation, of the microbial biomass from the washing liquid;
- re-suspension of the obtained biomass in an opportune volume of an aqueous solution including an effective quantity of one or more cryoprotectants to obtain the desired starter microbial culture in a liquid form ready for the freezing step.
Cryoprotectants are substances commonly used in the freezing sector. Said substances are usually carefully selected, as a function of the microorganism of interest, among physiologically compatible substances capa- ble of protecting the microbial cells from the osmotic and mechanical stresses caused by the freezing process.
By mere way of absolutely not limiting example, said substances are selected from the group including; amino acids and derivatives thereof; monosaccharides and disacch.arid.es; polymers presenting alcoholic functions within their structure; polyethers; other substances known to a skilled in the art.
Preferably, cryoprotectants are selected from the group including: sodium glutamate, mannitol, lactitol, sucrose, fructose, starch, glycerol, propanediol, polyvinyl pyrrolidone (PVP) , dimethyl sulfoxide
(DMSO) , polyethylene glycol (PEG) and other molecules, having the same characteristics compatible with the microbial biomass.
Said cryoprotectants are additioned to the water volume used for re-suspending the washed microbial biomass in a quantity on average between 10% and 65% (weight/volume or volume/volume) based on the total weight of the water; preferably, from 25% to 50% (weight/volume or volume/volume) .
The used quantity of the cryoprotectant is, however, varying as a function of the physical-chemical features of the cryoprotectant and the biomass cell con- centration after its re-suspension in the cryoprotec- tive solution.
Usually, the starter culture in a liquid form includes at least a microorganism physiologically compatible with the human and/or animal organism. Preferably, said at least one microorganism is se- lected from the group including the following genera: Lactobacillus, Bifidobacterium, Lactococcus, Leu- conostoc, Pediococcus, Streptococcus, Bacillus, Propi- onibacterium, Saccharomyces, Enterococcus, Staphylo- coccus .
For example, of the genus Lactobacillus are preferred the species: L. pentosus, L. plantarum, L. casei, L. casei ssp. paracasei , L. casei ssp. rhamnosus, L. acidophilus, L. delbrueckii ssp. bulgaricus, L. fermen- turn, L. gasseri.
For example, of the genus Bifidobacterium are preferred the species: B. longum, B. breve, B. lactis, B. adolescentis, B. pseudocatenulatum, B. catenulatum. For example, of the genus Lactococcus are preferred the species: L. lactis and L. lactis ssp. lactis.
For example, of the genus Streptococcus are preferred the species: S. thermophilus.
For example, of the genus Staphylococcus the species S. xylosus is preferred. Not limiting examples of strains which can be used in the context of the present invention are given below in the table.
TABLE
Publication Publication ..
Name . . . Applxcant. number date Streptococcus hMQ p_18383 5.05.1998 ANIDRAL S.R. L. thermophilus Streptococcus LMG P-18384 5.05.1998 AMIDRAL S.R. L. thermophilus
Lactobacillus
LMG P-21019 10.16.2001 MOFIN S. R. L. pentosus
Lactobacillus
LMG P-21020 10.16.2001 MOFIN S. R. L. plantarum
Lactobacillus
LMG P-21021 10.16.2001 MOFIN S. R. L. plantarum
Lactobacillus
LMG P-21022 10.16.2001 MOFIN S. R. L. plantarum
Lactobacillus
LMG P-21023 10.16.2001 MOFIN S. R. L. plantarum
Lactobacillus casei
LMG P-21380 01.31.2002 ANIDRAL S. R. L. ssp. paracasei
Lactobacillus belonging to the aciLMG P-21381 01.31.2002 ANIDRAL S. R. L. dophilus group Bifidobacterium
LMG P-21382 01.31.2002 ANIDRAL S.R. L. longum Bifidobacterium
LMG P-21383 01.31.2002 ANIDRAL S.R. L. breve Bifidobacterium
LMG P-21384 01.31.2002 ANIDRAL S. R. L. lactis Lactobacillus
LMG P-21385 01.31.2002 MOFIN S. R. L. plantarum Lactococcus lactis
LMG P-21387 03.15.2002 MOFIN S. R. L. ssp. lactis Lactococcus lactis
LMG P-21388 01.31.2002 MOFIN S. R. L. ssp. lactis Lactobacillus
LMG P-21389 03.15.2002 MOFIN S. R. L. plantarum Streptococcus
DSM 16506 06.18.2004 ANIDRAL S. R. L. thermophilus Streptococcus
DSM 16507 06.18.2004 ANIDRAL S.R. L. thermophilus Bifidobacterium
DSM 16603 07.20.2004 ANIDRAL S.R.L. longum Bifidobacterium
DSM 16604 07.20.2004 ANIDRAL S. R. L. breve Lactobacillus casei
DSM 16605 07.20.2004 ANIDRAL S.R. L. ssp. rhamnosus Lactobacillus del'- brueckii ssp. bulga- DSM 16606 07.20.2004 ANIDRAL S.R. L. ricus Lactobacillus del- brueckii ssp. bulga- DSM 16607 07.20.2004 ANIDRAL S. R. L. ricus Streptococcus
DSM 16590 07.20.2004 ANIDRAL S. R. L. thermophilus Streptococcus
DSM 16591 07.20.2004 ANIDRAL S.R.L. thermophilus Streptococcus
DSM 16592 07.20.2004 ANIDRAL S. R. L. thermophilus
Streptococcus
DSM 16593 07.20.2004 ANIDRAL S. R. L. thermophilus
Bifidobacterium
DSM 16594 07.21.2004 ANIDRAL S.R. L. adolescentis
Bifidobacterium
DSM 16595 07.21.2004 ANIDRAL S.R. L. adolescentis
Bifidobacterium
DSM 16596 07.21.2004 ANIDRAL S. R. L. breve
Bifidobacterium
DSM 16597 07.21.2004 ANIDRAL S. R. L. pseudocatenulatum
Bifidobacterium
DSM 16598 07.21.2004 ANIDRAL S.R. L. pseudocatenulatum
Staphylococcus
DSM 17102 02.01.2005 ANIDRAL S.R. L. xylosus
Bifidobacterium
DSM 17103 02.01.2005 ANIDRAL S. R. L. adolescentis
Lactobacillus
DSM 17104 02.01.2005 ANIDRAL S. R. L. plantarum
Streptococcus
DSM 17843 12.21.2005 ANIDRAL S.R. L. thermophilus
Streptococcus
DSM 17844 12.21.2005 ANIDRAL S.R. L. thermophilus
Streptococcus
DSM 17845 . 12.21.2005 ANIDRAL S. R. L. thermophilus
Lactobacillus
DSM 18295 05.24.2006 ANIDRAL S. R. L. fermentum
Lactobacillus
DSM 18296 05.24.2006 ANIDRAL S. R. L. fermentum
Lactobacillus
DSM 18297 05.24.2006 ANIDRAL S.R. L. fermentum
Lactobacillus
DSM 18298 05.24.2006 ANIDRAL S.R. L. fermentum
Lactobacillus
DSM 18299 05.24.2006 ANIDRAL S. R. L. gasseri
Lactobacillus
DSM 18300 05.24.2006 ANIDRAL S. R. L. gasseri
Lactobacillus
DSM 18301 05.24.2006 ANIDRAL S. R. L. gasseri
Lactobacillus
DSM 18302 05.24.2006 ANIDRAL S.R. L. gasseri
Bifidobacterium
DSM 18350 06.15.2006 ANIDRAL S.R. L. adolescentis
Bifidobacterium
DSM 18351 06.15.2006 ANIDRAL S.R. L. adolescentis
Bifidobacterium
DSM 18352 06.15.2006 ANIDRAL S. R. L. adolescentis Bifidobacterium DSM 18353 06.15.2006 ANIDRAL S. R. L. catenulatum
Streptococcus
DSM 18613 09.13.2006 MOFIN S.R. L. thermophilics
Streptococcus DSM 18614 09.13.2006 MOFIN S. R. L. thermophilus
Streptococcus DSM 18615 09.13.2006 MOFIN S. R. L. thermophilus
Streptococcus DSM 18616 09.13.2006 MOFIN S.R. L. thermophilus
Streptococcus DSM 18617 09.13.2006 MOFIN S.R. L. thermophilus
Streptococcus DSM 18618 09.13.2006 MOFIN S. R. L. thermophilus
Streptococcus DSM 18619 09.13.2006 MOFIN S. R. L. thermophilus
Streptococcus DSM 18620 09.13.2006 MOFIN S. R. L. thermophilus
Streptococcus DSM 18621 09.13.2006 MOFIN S.R. L. thermophilus
Streptococcus DSM 18622 09.13.2006 MOFIN S. R. L. thermophilus
Streptococcus DSM 18623 09.13.2006 MOFIN S. R. L. thermophilus
Streptococcus DSM 18624 09.13.2006 MOFIN S. R. L. thermophilus Streptococcus DSM 18625 09.13.2006 MOFIN S. R. L. thermophilus
In a preferred embodiment of the invention, the starter culture includes a mixture of two or more bacterial strains selected among those above mentioned. It is understood to mean that the freezing method according to the present invention is, in any case, applicable to any type of starter microbial culture in a liquid form, independently from the type of process with which the same is prepared. Starter cultures in liquid form, once ready for being frozen, are packaged (preferably under a laminar flow of sterile air at a temperature between 1 and 100C) , in sterile primary packaging materials, preferably selected from: polyethylenes , aluminium and polyesters and/or opportune multilayered combinations thereof . At the end of the packaging within said packaging ma- terials, the same are preferably tightly sealed under the same aseptic conditions above described. The following freezing (relative to the step b) above described) is carried out using known techniques in the art of the refrigeration; preferably, through the use of liquid nitrogen or an opportune refrigerator system at a temperature < -180C for a sufficient time to obtain the freezing of the water existing in the packaged product . In case of using liquid nitrogen, the freezing time of a total volume between 10 1 and 100 1 is on average between 5 seconds and 2 minutes .
In a particularly preferred embodiment of the invention, a starter culture in liquid form resulting from the step a) is frozen by using liquid nitrogen at -2000C for times between 3 seconds and 1 minute.
In the specific case of the starter microbial cultures, it has been ascertained that the viability of the frozen product obtained with the method of the present invention is equivalent to the one typical of the traditional frozen starter cultures. Moreover, the Applicant has found that, in the starter liquid cultures in which the filtration and the washing of the microbial biomass have been carried out, there can be found a sensible decrease, or a total ab- sence, of the lactic acid organic salts (such as ammonium lactates and/or calcium lactates, usual metabolites resulting from the fermentation and development steps of the biomass within the fermenter) . This working mode allows to obtain a stability increase of the culture itself after the freezing.
Such stability increase can also be found when the frozen culture is stored at higher temperatures than those usually employed today for frozen cultures prepared with traditional methods . In fact, frozen starter cultures according to the method of the present invention have shown to have an excellent shelf life at temperatures between -240C and -180C, with consequent much more limited storage costs. In fact, in order to obtain such shelf-life, refrigerator devices at lower temperatures (usually between, in the general use routine procedure, -700C and -550C) are not needed anymore, while, on the contrary, they are still needed in case of traditional frozen cultures . The shelf-life of a starter culture frozen according to the method of the present invention has been evaluated based on the maintenance, over time, of the ability of exact volumes of said culture, inoculated in a proper medium, for example milk, of lowering the pH until precise values after the same time intervals. The shelf-life of said frozen starter culture, stored at a temperature between -240C and -180C, has proved to be > 4 months, preferably between 4 and 12 months; more preferably, > 6 months.
The frozen starter culture, obtained with the method of the present invention, is used as starter in the food sector, for example in the dairy industry; dietary sector, for example in the supplements; nutraceu- tic and pharmaceutical sectors.
The cheese production process to be followed with the frozen starter culture obtained with the method of the present invention is equal to the one which is followed with the traditional frozen cultures . Products which originate therefrom have the same organoleptic characteristics, but show high guarantees from a sanitary point of view.

Claims

1. Method for the preparation of a frozen microbial culture including at least the following steps : a) packaging a microbial culture in a container under aseptic conditions; and b) subjecting said container to a freezing step.
2. Method according to claim 1, wherein said step b) directly follows said step a) .
3. Method according to claim 1 or 2 , wherein said step a) is carried out in a sterile environment at a temperature on average between 1°C and 200C, preferably from 3°C to 1O0C, and having an air quality at least equal to the class 100.
4. Method according to one or more of claims 1-3, wherein said step b) is carried out through the use of liquid nitrogen or a cryogenic system working at a temperature lower or equal to minus 18°C (-180C) for a sufficient time to obtain the freezing of the water present in the packaged bacterial culture .
5. Method according to claim 4, wherein the freezing time by the use of liquid nitrogen of a total volume of bacterial culture between 10 1 and 100 1 is between 5 seconds and 2 minutes .
6. Method according to one or more of the preceding claims, wherein the microbial culture includes bacte- ria and yeasts having a probiotic or symbiotic valence .
7. Method according to claim 6, wherein said microorganism is selected from the group including the gen- era: Bifidobacterium, Lactobacillus, Leuconostoc, Lac- tococcus, Streptococcus, Pediococcus, Propionibacte- rium, Bacillus, Enterococcus, Saccharomyces, Staphylococcus.
8. Method according to claim 7, wherein said microor- ganism is selected from the group including the species: Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus casei ssp. casei, Lactobacillus easel ssp. paracasei, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus delbrueckii ssp. bulgari- cus, Lactobacillus delbrueckii ssp. lactis, Lactobacillus fermentum, Lactobacillus gasseri; Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium animalis ssp. lactis, Bifidobacterium adolescentis, Bifidobacterium pseudocatenulatum, Bifidobacterium catenulatum, Bifidobacterium infantis, Lactococcus lactis e Lactococcus lactis ssp. Lactis ; Streptococcus thermophilus; Staphylococcus xylosus .
9. Method according to one or more of the preceding claims, wherein said microbial culture is in a liquid form.
10. Frozen microbial culture obtainable by the method according to one or more of the claims 1-9.
11. Method for eliminating or reducing the contamina- tion sources in a production line of frozen microbial cultures including one or more of claims 1-9.
12. Use of the frozen microbial cultures prepared according to one or more of the claims 1-9 in the food and pharmaceutical industries .
13. Use of a starter microbial culture according to any one of the claims 1-9, for the preparation of products in the food industry, such as cheese, yogurts, fermented milks, bread, baked products, charcu- teries and sausages, alcoholic beverages,- preferably, for the preparation of products of the dairy industry.
14. Dairy product obtainable through a microbial culture according to any one of the claims 1-9.
PCT/IB2007/003485 2006-11-28 2007-11-12 Method for preparing freezed bacterial cultures for use in the food, dietary, nutraceutic and pharmaceutical field WO2008065491A2 (en)

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