PROCESS FOR DIRECT INOCULATION FROM CONCENTRATED FERMENTS AND ASSOCIATED DEVICE

Process for direct inoculation from concentrated ferments and associated device

The present invention relates to an equipment and a process for continuous inoculation from concentrated ferments requiring neither incubation or preculture which have a potential health risk, nor interruption o f the inoculation process during production.

Inoculation in the food-processing industry and in the dairy industry in particular is o f essential importance for producing a product. Indeed, the industrial and qualitative performance levels o f the final products depend on the nature and the efficiency o f the ferments used and on their method of addition.

The obtaining o f precultures, also known as starter cultures, i.e . prior to activation o f the culture in order to reduce the lag phase, for the inoculation o f milk is known from documents WO 200 170935 and EP688864. Patent application WO 99/09838 describes a method for preparing a fresh product in which the starter culture can be in frozen form.

The fermentation o f liquid medium to be inoculated with frozen concentrated ferments means that the manufacturer using them has to work in batchwise mode for the inoculation and fermentation phases . Indeed, since the form and type o f packaging is generally as bags or tins, the microorganisms must necessarily be added directly to the fermentation tank.

Other systems using concentrated ferments require the presence of a supplemental container for intermediate thawing of the ferments in case o f frozen ferments or for dilution in case o f dried ferments, which increases the risk of contaminations.

These reactivation and/or dilution systems have the drawback of making it necessary to handle the concentrated ferments upstream of the inoculation phase, thereby risking contaminations.

The applicant has discovered surprisingly that the introduction of frozen concentrated ferments can be carried out by direct ino culation. Alternatively, the freeze-dried concentrated ferments can be introduced by direct inoculation.

This allows continuous inoculation without having to interrupt the fermentation process for the production of the final product. It thus becomes possible to substantially increase fermented product production rates.

The subj ect of the invention is thus a process for continuous ino culation o f a food product, in particular a dairy product, with concentrated ferments.

According to one general characteristic, the process comprises the fo llowing steps :

- either frozen concentrated ferments are thawed in a temperature controlled chamber, such as a refrigerator, operating on a container containing frozen concentrated ferments, or the freeze-dried ferments are rehydrated.

- the concentrated ferments obtained are continuously inj ected, from the container, into a flow of liquid to be inoculated.

The subj ect of the invention is also an equipment for continuous ino culation o f ferments into a liquid to be inoculated, wherein the ferments originate from either frozen or freeze-dried concentrated ferments, said equipment comprising either a temperature controlled transforming chamber for thawing a container comprising frozen concentrated ferments, or a transforming chamber in which the freeze-dried ferments are rehydrated in the container, an ino culation chamber provided with support means for installing at least two containers o f ready-to-use ferments and with at least one weighing device capable o f continuously determining the remaining vo lume in the container being emptied, the equipment further comprising an inj ection circuit connecting the containers to a circuit for continuous feeding o f the liquid to be inoculated, the inj ection circuit comprising a valve allowing switch from one container to another container and means for regulating the flow rate of the ferments in liquid form. In one embo diment, the container containing the frozen concentrated ferments is stored at a temperature of -20 to -70°C prior to the thawing thereof.

In an other embodiment, the container containing the freeze- dried concentrated ferments is stored at a temperature of -20 to ambient temperature prior to rehydratation.

The container may be rigid, deformable, soft. Preferentially, the container is soft.

Advantageously, once placed in the ino culation chamber, the container containing the ready-to-use concentrated ferments is continuously weighed in order to determine, during emptying, the remaining volume of liquid ferments in the container weighed.

The inj ection o f the ferments is carried out via means o f connection to a circuit for continuous feeding o f liquid to be ino culated. These connection means may be pipes o f an inj ection circuit, which can be cleaned and sterilized after each passage o f the liquid to be inoculated in the line, or more or less flexible tubing provided with means o f temporary connection, for example via clip- fastening or snap-fastening.

Microbial contamination o f surfaces constitutes a danger to health through the possible contamination o f foods during transformation thereof. This is, for example, the case when bacterial spores occur in bio films, i. e. multicellular communities o f microorganisms adhering to one another and to a surface. Indeed, bacterial spores exhibit remarkable resistance characteristics and contaminate the surfaces o f the equipment and o f connecting piping. For industrial manufacturers, the removal o f bio films in mo st cases requires the use of excessive hygiene procedures in order to ensure good preservation of the transformed foods, and to avoid food contaminations .

Thus, alternatively, the means o f connection to a circuit for continuous feeding of liquid to be inoculated are disposable in order to ensure perfect sterility and easy use. These means o f connection may also be changed according to the ferments used. Preferably, the container after thawing or after rehydrating is placed in an ino culation chamber at a pressure above atmospheric pressure.

Thus, in one embo diment of the invention, the ino culation chamber containing the concentrated ferments is pressurized by means of a neutral sterile gas in order to maintain as far as possible in said chamber a constant pressure which thus facilitates the accuracy o f the flow of the concentrated ferments. Furthermore, an overpressure in the container limits the possibilities o f contamination by outside air. An overpressure typically of 100 g/cm² allows a more even metering.

In one embodiment of the invention, several containers are placed in parallel arrangement in the inoculation chamber so that, when one of them is in the process of being emptied, at least one other container containing ready-to-use concentrated ferments is on standby.

Preferably, by means of this process, a metered amount o f ready-to-use concentrated ferments is continuously introduced into a flow o f liquid to be inoculated. This inoculated liquid will then be put in a fermentor, a tank for producing fermented products or a fermentation device, directly in the container intended to be marketed. In the case o f a dairy product, for examp le, the fermentation unit may be a pot of dairy product.

This continuous inoculation has the effect of improving the regularity o f the quality o f the final products . The invention thus allows direct use, from their container, of the previously frozen or freeze-dried concentrated ferments directly in the line o f liquid to be ino culated without invo lving a risky intermediate phase. Any intermediate handling phase indeed inevitably leads to risks o f accidental contamination which are detrimental to the who le o f the subsequent process for producing the fermented product. Furthermore , directly inoculating into the line o f liquid just before renneting makes it possible to limit any proliferation o f phages and the creation o f bio films on the maturation zone. Preferably, means for regulating the flow rate of the ferments in liquid form are placed upstream o f the circuit for continuous feeding o f the liquid to be inoculated. These means may be a pump .

The thawing time for the frozen concentrated ferments in the container is variable depending on the amounts o f products present in the container.

The thawing time for the frozen concentrated ferments in a temperature controlled chamber is from 5 to 30 hours, and preferably around 12 hours.

In order to ensure a homogeneous melting of the concentrated ferments without creating any large thermal shock which would be detrimental to the correct course of the subsequent steps o f the production process, the temperature in the transforming chamber is regulated.

Preferably, the temperature o f the atmosphere in the temperature controlled chamber is from 0 to 15 °C and preferably 4°C .

Preferably, the frozen concentrated ferments are stirred during the thawing in order to homogenize them and to avoid incompletely melted aggregates.

The rehydrating time for the freeze-dried concentrated ferments in the container is variable depending on the amounts of products present in the container. It is generally from 30 minutes to 2 hours, and preferably around 1 hour.

Preferably, the freeze-dried concentrated ferments are stirred during the rehydrating in order to homogenize them and to avoid incompletely rehydrated aggregates.

For this purpose, in one embodiment of the equipment, the temperature controlled transforming chamber may comprise means for stirring the container.

Once placed in the inoculation chamber, the ready-to-use liquid ferments are maintained at a relatively low temperature which may be from 2 to 12°C, or any other temperature compatible with maintaining the functionalities of the ferments. This makes it possible to limit as much as possible the resumption o f the bacterial metabo lism and to guarantee a quality of ino culation which is constant over time.

In one embo diment of the equipment, the inoculation chamber may comprise refrigeration means and means for maintaining the pressure above atmospheric pressure.

The ino culation chamber o f the equipment may advantageously comprise means o f homogenization o f at least one container during emptying.

Thus, homogenization o f the mixture o f concentrated ferments during emptying makes it possible to ensure the homogeneity o f the mixture of bacterial cultures constituting the ferments.

Preferably, the homogenization step comprises blending.

The frozen or freeze-dried concentrated ferments can be packaged and stored in packaging with a more or less large capacity ranging from 200 g to several kilo s. The transfer must be carried out under strict hygiene conditions in order to avoid any contamination detrimental to the whole of the subsequent fermentation process.

The concentrated ferments used are composed of bacteria which are used for producing cheeses such as, for example, so ft cheeses, cooked pressed cheeses, uncooked pressed cheeses, spun-curd cheeses, and fermented milks such as, for example, stirred or set, flavoured or natural yoghurts, drinking yoghurts, creme fraiche and fromages frais and also for producing other fermented products such as, for example, wine.

The bacteria used may be mesophilic microorganisms, the optimum growth temperature of which is from 25 to 35°C . Among the mesophilic microorganisms typically used, mention may in particular be made of, for example, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Leuconostoc cremoris, Lactoccus lactis biovar. diacetylactis, Lactobacillus casei, Streptococcus durans, Streptococcus faecalis.

Use may also be made o f thermophilic microorganisms, i. e . organisms o f which the growth temperature may be from 35 to 45 °C . Mention may in particular be made o f, for examp le, Streptococcus thermophilus, Lactobacillus lactis, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus or any other appropriate microorganism.

Likewise, strictly anaerobic microorganisms o f the bifidobacteria type, including Bifidus bifidum and Bifidobacterium longum (animalis) can be used.

Use may also be made o f propionic bacteria such as Lactobacillus helveticus, Propionibacteirium freudenreichii, Propionibacterium freudenreichii subsp shermanii, etc.

The bacteria used may be wine bacteria, for example

Oernococcus oeni (Leuconostoc oenos), Lactobacillus plantarum or Pedicoccus sp.

Use may also be made o f yeasts of the family Saccharomycetaceae or moulds such as Penicillium or Geotrichum .

The level o f ready-to-use concentrated ferment or concentrated bacterial culture inoculation varies according to the technologies and the products under consideration. Generally, this proportion is from 0.005 % to 0.025 % based on the total weight of the medium to be ino culated.

Generally, upon being produced, the ferments are frozen using liquid nitrogen, then stored at a temperature from -20 to -70°C .

Depending on their freezing temperature, the frozen ferments can be stored for some time before use : up to 1 month in case o f storing at -20°C, up to 6 months in case of storing at -40°C , and up to 12 months in case of storing at -45 °C .

Alternatively, the freeze-dried ferments are dried by sublimation o f the frozen water from a frozen culture by reducing pressure in the surrounding allowing the water to evaporate directly to gas without going by liquid phase. For the purpose of the invention, the expressions freeze-drying, lyoph i i isat io n a nd cryodesiccation hav e the same signification.

Freeze-dried fements are generally stored at -20°C . In this case, the shelf life can be up to 24 month. It can also be stored at +5 ° C but in this case its shelf life is about 6 weeks. Other purposes, characteristics and advantages will appear upon reading the fo llowing description of an embodiment and o f different modes o f implementation o f the invention, given only as nonlimiting examples, and given with reference to the attached drawings in which:

- Figure 1 illustrates schematically a flowchart of the various steps o f a process according to one mo de o f imp lementation o f the invention,

- Figure 2 illustrates schematically a flowchart of the various steps of a process according to a second mode of imp lementation o f the invention,

- Figure 3 illustrates schematically a first embodiment according to the invention,

- Figure 4 illustrates schematically a second embodiment according to the invention,

- Figure 5 represents monitoring curves for the acidification of the culture medium at 40°CC o f the S SC l OO culture after thawing, as a function of the thawing time Oh, 24h, 48h 72h or 96h in the refrigerator,

- Figure 6 represents monitoring curves for the acidification of the culture medium at 37°CC o f the STI06 culture after thawing, as a function of the thawing time Oh, 24h, 48h 72h or 96h in the refrigerator,

Represented schematically in Figure 1 is a flowchart of the various steps o f an ino culation process according to one embodiment of the invention.

Prior to the inoculation, concentrated ferments are frozen. Then they can be blended and packed in containers.

For this, in a first step E01 , a container is sterilely filled with concentrated frozen ferments. The containers may be packagings o f more or less large capacity ranging from 200 g to several kilograms that are capable o f maintaining concentrated ferments composed o f bacteria that are used for producing cheeses, fermented milks and other fermented products. Then, in a step E02, the orifice o f the container is sealed, still while maintaining sterility, so as to obtain a hermetically sealed container filled with concentrated ferments.

In a subsequent step E03 , these frozen ferments are stored at a temperature from -20 to -70°C for a relatively long time o f a few days to several months.

It is possible to repeat steps E01 to E03 with different containers so as to obtain a plurality of containers comprising the same frozen concentrated ferments.

For the ino culation, in step E04, frozen ferments are thawed in situ in one of the containers previously kept frozen. By "in situ", it is meant that the ferments stored in the container are transformed in the same container into liquid concentrated ferments without transfer. This thawing step is carried out via refrigeration means acting on the container and more particularly on the frozen ferments contained in the container. In the embodiment presented, the frozen concentrated ferments are stirred during the thawing, in order to distribute the heat evenly and to avoid incompletely melted aggregates.

In a subsequent step E05 , the container which has undergone thawing is connected to a disposable inj ection circuit.

In a subsequent step E06, the container connected to the inj ection circuit is installed in an inoculation chamber and the container is opened.

The thawed container is then emptied in a step E07. During the emptying, the inoculation chamber is pressurized with a neutral sterile gas in order to maintain a constant pressure therein as much as possible and thus to facilitate the accuracy o f the flow o f the concentrated ferments . The thawed liquid ferments are also maintained at a temperature from 2 to 12°C, so as to limit as much as possible the resumption o f the bacterial metabolism and to guarantee an inoculation quality which is constant over time.

While being emptied, the container is regularly weighed, in a step E08 , so as to determine the amount of ferments remaining in the container. Next, in a step E09, the weight measured in the preceding step is compared to a thresho ld value corresponding to the weight o f the empty or almost empty container. In addition, depending on the weight of the container, and therefore depending on the amount of ferments remaining in said container, the container-emptying operation is continued by resuming it in step E07 via a loop BCL l , or the virtually empty container is exchanged with a full thawed container in a step E 10. The thawing o f the full container may have been initiated during the emptying o f the previous container, or before the beginning o f the emptying o f said previous container, for examp le after the beginning of the thawing o f said previous container using another thawing chamber.

These steps o f emptying a container, weighing, and optionally changing container according to the vo lume o f remaining ferments are carried out via a loop BCL2.

The parallel arranging of several containers in an inoculation chamber and step E 10 of exchanging a container to be emptied make it possible to obtain a continuous inoculation process wherein a metered amount of thawed concentrated ferments is continuously introduced into a flow of liquid to be inoculated, wherein the inoculated liquid can then be introduced in a fermentor, a tank for producing fermented products or a device for fermentation, directly in the container intended to be marketed.

This continuous ino culation results in improving the regularity of the quality of the final products.

Represented schematically in Figure 2 is a flowchart of the various steps o f an inoculation process according to a second embodiment of the invention.

Prior to the inoculation, concentrated ferments are freeze- dried. Then they can be blended and packed in containers.

For this, in a first step E010, a container is sterilely filled with concentrated freeze-dried ferments. The containers may be packagings of more or less large capacity ranging from 200 g to several kilograms that are capable o f maintaining concentrated ferments composed o f bacteria that are used for producing cheeses, fermented milks and other fermented products.

Then, in a step E020, the orifice of the container is sealed, still while maintaining sterility, so as to obtain a hermetically sealed container filled with concentrated freeze-dried ferments.

In a subsequent E030, these ferments are stored at a temperature o f -20°C for a relatively long time o f a few days to 24 months.

It is possible to repeat steps E010 to E030 with different containers so as to obtain a plurality of containers comprising the same freeze-dried concentrated ferments.

For the ino culation, in step E040, freeze-dried ferments are rehydrated in situ in the container previously stocked. By "in situ", it is meant that the ferments stored in the container are transformed in the same container into liquid concentrated ferments. In this case, the rehydrating step is carried out in the container itself without transfer of the ferments from another container.

In a preferred embodiment, the container containing the ferments comprises a packaging forming a two-compartment container, the first compartment comprising the freeze-dried ferments separated by a breakable membrane from the second compartment comprising the sterile liquid in which the ferments are rehydrated once the breakable membrane between the two chambers is broken.

In the embodiment presented, the concentrated ferments are stirred during the rehydrating, in order to avoid incompletely disso lved aggregates.

In a subsequent step E050, the container which has undergone rehydrating is connected to a disposable inj ection circuit.

Steps E060 to E 100 are the same as the steps of the inoculation process of Figure 1 .

Represented schematically in Figure 3 is an inoculation equipment 1 according to a first embo diment o f the invention. The equipment 1 comprises a thawing chamber 2 comprising a refrigerator capable o f thawing a container o f frozen concentrated ferments Cfc according to step E04 of the process illustrated in Figure 1 . The thawing chamber 2 comprises means for stirring the ferments during the thawing, which are not represented in the figure, for homogenization o f the ferments.

The equipment 1 also comprises an inoculation chamber 3. The ino culation chamber illustrated in this figure comprises two support means 4 each capable of supporting a container of thawed concentrated ferments Cfcl and Cfc2, for example a vertical attachment device or a device for gripping the container, comprising a set of plates for ho lding the container in place and/or a hook. It is possible to store certain types o f concentrated ferments once thawed in the ino culation chamber 3 for several hours and up to 24 hours, but preferably between 4 and 8 hours without particular effect on the resumption o f the bacterial metabolism or on the activity o f the bacteria constituting the concentrated ferments.

The inoculation chamber 3 o f the equipment 1 comprises, moreover, means 5 for weighing the container in order to deduce the vo lume of the remaining ferments during emptying (steps E07 to E09) . The ino culation chamber 3 also comprises homogenization means 6 for homogenizing the ferments located in the container. By way o f nonlimiting example, use may be made of a plurality o f plates applying a different pressure per plate which varies with passing time . The homogenization can be carried out continuously or intermittently as required.

In addition, the inoculation chamber 3 may comprise air- conditioning means not represented in Figure 2. Thus, the inoculation chamber 3 can be refrigerated at a temperature of from 2 to 12°C throughout the duration of the inoculation.

The inoculation chamber 3 may comprise a plurality o f means for supporting the container o f thawed concentrated ferments Cfc, the Cfc containers being connected via an inj ection circuit 7 to a circuit for continuous feeding 10 of the liquid to be inoculated. In the embodiment illustrated in Figure 2, the inj ection circuit 7 comprises a valve 8 connected to a first container Cfcl via a first circuit portion 12, to a second container Cfcl via a second circuit portion 13 and to the feeding circuit 10 via a third circuit portion 14. The valve 8 thus makes it possible to change container Cfcl or Cfcl without interrupting the inj ection process.

The injection circuit 7 also comprises a pump 9 installed on the third circuit portion 14, consequently downstream o f the valve 8. The pump 9 serves to regulate the flow rate of afferent liquid concentrated ferments o f the container Cfcl or Cfcl in place in the inoculation chamber 3. The regulating pumps used, such as pump 9, can be proportioned according to the flow rate of the main circuit o f the medium inoculated; typically in the dairy industry, the pump flow rates range from 0. 1 1/hour to 4 1/hour, for equipment of 2 to 10 000 1/hour, up to 0.75 1/hour to 12 1/hour for equipment o f 1 5 000 to 30 000 1/hour.

The inj ection circuit 7 may also comprise connecting means 1 5 at the level o f the container(s) Cfcl and Cfcl in the inoculation chamber 3 , and at the level o f the junction between the circuit portion 14 and the feeding circuit 1 0.

These connecting means 1 5 make it possible to sterilize and clean the inj ection circuit 7 more easily. In another embodiment, these connecting means make it possible to change the portions 12, 13 and 14 of the inj ection circuit 7 in order to replace them with others which are sterile, during, for example, the changing in the composition of the ferments being used to inoculate the pipe 10 for feeding o f the liquid to be inoculated.

The inoculation chamber 3 may also comprise means, not represented in the figure, for checking the pressure inside the ino culation chamber 3.

The equipment 1 also comprises a fermentation unit 1 1 connected to the circuit 1 0 for feeding the liquid to be inoculated. The ino culation o f said liquid is carried out by means o f a tapping on the pipe o f the feeding circuit 1 0, making it possible to connect the third circuit portion 14 of the inj ection circuit 7. The fermentation unit 1 1 is in this case reproduced in the form of a fermentor. Of course, it is also possible to envisage that the fermentation unit 1 1 is a tank for producing fermented products or a device for fermentation directly in the container intended to be marketed, for example a pot of dairy product.

The quantification of the thawed ferments is an essential part of the fermentation unit ino culation process.

Represented schematically in Figure 4 is an inoculation equipment 1 according to a second embodiment of the invention.

The equipment 1 comprises a transforming chamber 2 comprising the container containing the concentrated ferments Cfd rehydrated according to step E040 of the process illustrated in Figure 2. The transforming chamber 2 comprises means for stirring the ferments during the rehydrating, which are not represented in the figure, for homogenization of the ferments .

The equipment 1 also comprises an inoculation chamber 3. The ino culation chamber illustrated in this figure comprises four support means 4, capable o f supporting the containers o f rehydrated concentrated ferments Cfdl and Cfd2, for example a vertical attachment device or a device for gripping the container, comprising a set of plates for ho lding the container in place and/or a hook. It is possible to store certain types of concentrated ferments once rehydrated in the inoculation chamber 3 for several hours and up to 24 hours, but preferably between 4 and 8 hours without particular effect on the resumption o f the bacterial metabo lism or on the activity of the bacteria constituting the concentrated ferments.

Same parts as in Figure 3 are assigned the same reference numbers.

Whatever the embodiment of the invention, the inoculation equipment makes it possible to obtain a continuous and accurate on line flow o f a small amount of concentrated ferments from concentrated ferments for inoculating a fermentation unit. The invention thus allows to directly use from their container the concentrated ferments, directly in the line of liquid to be inoculated without a risky intermediate phase being invo lved. Any intermediate handling phase in fact inevitably leads to risks o f accidental contamination which are detrimental to the who le o f the subsequent process for producing the fermented product. Furthermore, directly ino culating into the line o f liquid just before renneting makes it possible to limit any possible phage proliferation.

Example 1 : Monitoring the acidification of the culture medium fo llowing thawing using a refrigerator device

The ferments S SC- 100 {Streptococcus thermophilics with a slow acidification) and STI06 {Streptococcus thermophilus with a rapid acidification) are packaged in sterile pouches o f 5 litres, i.e . 2.5 kg o f ferments in a form o f frozen granules stored at a temperature of either -40°C or -20°C .

The pouches are placed in a refrigerator.

The ferments previously stored at -40°C were subj ected to refrigeration for 12 hours to achieve complete melting.

The pouches are placed on a stirrer throughout the thawing in order to ensure homogeneous melting of the concentrated ferments.

The tests for acidification of the culture medium were carried out on milk reconstituted at 9.5 % so lids content from skimmed milk powder, heated at 99°C for 30 min. The inoculation dose is, 0.01 % for S SC- 100 with a maturation temperature of 40°C and 0.01 % for STI06 with a maturation temperature of 37°C .

The results o f the monitoring o f the acidifying activity o f each of the strains tested are given below as curves of variation in pH of the ino culated medium as a function o f time, the test strains having been previously thawed in a refrigerator device (Figures 5 and 6) .

In particular, Figure 5 represents monitoring curves for the acidification o f the culture medium o f the SCC- 100 culture, Figure 6 represents monitoring curves for the acidification o f the culture medium o f the STI06 culture, after thawing in a refrigerator as a function o f the time in the refrigerator. In each o f the figures, the first curve referenced C I corresponds to the control for culture of the ferments without previous thawing, and curves C2 to C5 represent the curves obtained after thawing during thawing time of respectively of 24, 48 , 72 or 96 hours .

The acidification monitorings for the various strains tested allow one to deduce that there is no significant effect of the thawing time in a refrigerator of the ferments on the acidifying activity performance levels.

Thus, it was demonstrated that it is possible to thaw various types o f frozen concentrated bacterial cultures for several hours at a temperature of 2 to 12°C without particular effect on the resumption of the bacterial metabo lism and on the activity, in particular acidifying activity, of the ferments under consideration.