DRY COMPOSITION, NEWBORN POWDER, FORMULA, MILK, FOOD PRODUCT OR DIETARY SUPPLEMENT

专利摘要:
dry composition, newborn powder, formula, milk, food product or dietary supplement. The present invention relates to a dry composition for lactic acid bacteria and in particular to a dry composition comprising from 10(9) to 10(13) cfu/g of the lactic acid bacteria cell composition, wherein the composition is characterized by the fact that it also comprises the following amounts of protective agents (all amounts of protective agents below are given in relation to 1 g of lactic acid bacteria cells in the composition): from 6 to 9 g of trehalose, from 0.1 to 1 g of inulin and from 0.5 to 3 g of hydrolyzed casein, and by the fact that it does not comprise an alginic acid salt. the composition has an improved storage stability of the cell of interest. Comparison experiments were done between compositions with and without alginate and it was found that there is substantially no difference between compositions with and without alginate with regard to stability. further, the invention relates to a method for preparing a dry lactic acid bacteria composition.
公开号:BR112013033565B1
申请号:R112013033565-3
申请日:2012-06-30
公开日:2021-06-29
发明作者:Birgitte Yde；Jakob Blenker Svendsen
申请人:Chr. Hansen A/S；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[001] The present invention relates to the field of dry compositions for lactic acid bacteria, a method for preparing dry compositions of lactic acid bacteria and compositions that can be prepared by said method. FUNDAMENTALS OF THE INVENTION
[002] Cells such as microorganisms are involved in numerous industrially relevant processes. For example, bacterial cultures, in particular bacterial cultures which are generally classified as lactic acid bacteria (LAB) are essential in the manufacture of all fermented dairy products, cheese and butter. Cultures of such bacteria can be referred to as starter cultures and they communicate specific characteristics to various dairy products performing various functions.
[003] Many lactic acid bacteria are known to have probiotic properties (ie they have a beneficial health effect on humans when ingested). In most cases, it is critical that microorganisms remain viable after prolonged storage so that they communicate their beneficial effect on ingestion. Attempts have been made, in which freeze-dried bacteria are mixed with additives that act as wet barriers, or as protectors needed to freeze cells (so called cryoprotectants). Various types of additives have been added to microorganisms in attempts to make them more stable.
[004] For some uses - it can be said that one should preferably have a very storage stable lactic acid bacteria composition/formulation.
[005] For example - if the LAB composition is mixed with powdered milk to make a suitable newborn powder, it generally needs a very storage stable LAB composition - essentially because of this a powdered product for newborn as such is normally very stable in storage and can be supplied to newborns long after its actual date of manufacture. Consequently, if newborn powder is provided to newborns eg 30 weeks (or later) after their actual manufacturing date - it is evident that the composition of LAB incorporated in the newborn powder must be quite stable on storage in order to maintain the viability of the LAB cells.
[006] WO2010/138522A2 (Advanced Bionutrition Corporation) describes a LAB cell culture composition which is said to be useful to be incorporated into a powdered newborn product.
A preferred composition comprises alginate, inulin, trehalose and hydrolyzed protein (see table 1, paragraph [0094]).
[008] It can be said that the LAB compositions of table 1 of WO2010/138522A2 comprise a relatively high amount of what may be termed protective agents - i.e. agent which can help to improve the storage stability of acid bacteria cells lactic.
[009] For example - it can be said that the LAB compositions of table 1 of WO2010/138522A2 comprise a relatively high amount of trehalose.
[0010] Paragraph [0097] of WO2010/138522A2 reads:
[0011] "Lactobacillus Acidophilus (100 g of frozen concentrate from a laboratory fermentation product) was thawed at 37°C. Protein hydrolyzate premix (100 g, Table 1)."
[0012] As known to the skilled person, a LAB cell concentrate, as described in this paragraph [0097], can often comprise around 10% cell dry matter. Under this assumption - it can be said that the dry LAB composition described in this paragraph [0097] is a LAB composition comprising around 10 times more protective agents than LAB cells as such - according to the technique, this can be said to be a composition of LAB with a relatively high amount of protective agents.
A problem with such LAB compositions with a relatively high amount of protective agents may be that they can often be quite difficult to dry properly as such - for example, without significant inactivation of the relevant LAB cells.
[0014] WO2010/138522A2 describes processes for drying for example, in paragraph [0097] described the LAB composition, which as discussed above can be said to be a LAB composition with a relatively high amount of protective agents.
[0015] Paragraph [0081] of WO2010/138522A2 says (emphasis added):
[0016] "Typical processes for the conservation of bioactive microorganisms such as live or attenuated organisms include a combination of freezing and vacuum conditions that can result in damage to the membrane and denaturation of cell constituents. The prior art shows the use of pressures higher vacuum (eg less than 100 Torr), addition of specific cryoprotective agents, concentration steps to obtain thick solutions (syrup), and/or higher starting temperatures to prevent freezing.”
[0017] This paragraph [0087] of WO2010/138522A2 can be said to provide an overall summary that the prior art generally shows with respect to suitable drying processes relevant herein.
[0018] Here it can be said to be relevant to note that the direct drying method and unambiguously described in WO2010/138522A2 for example is not involving a freezing step to form solid frozen particles/pellets.
[0019] WO2010/138522A2 (Advanced Bionutrition Corporation) describes a cell culture composition comprising alginate, inulin, trehalose and hydrolyzed protein (see table 1, paragraph [0094]).
[0020] However, there is still a need for improved compositions that are able to withstand high moisture/high water activity. SUMMARY OF THE INVENTION
[0021] The problem to be solved by the present invention relates to providing a new method for preparing a dry powder composition comprising lactic acid bacteria cells (LAB) and what can be said to be a relatively high amount of agents to provide compositions that are stable on prolonged storage under moist conditions.
[0022] In a preferred embodiment, the invention relates to a dry composition comprising from 109 to 1013 cfu/g of the composition of lactic acid bacteria cells, wherein the composition is characterized by the fact that it also comprises the following amounts of protective agents (all amounts of protective agents below are given in relation to 1 g of lactic acid bacteria cells in the composition):
[0023] (i): from 6 to 9 g of trehalose,
[0024] from 0.1 to 1 g of inulin and
[0025] from 0.5 to 3 g of hydrolyzed casein, and
[0026] by the fact that it does not comprise an alginic acid salt.
[0027] Reference is made to working examples here, in which it is demonstrated that the storage stability of the LAB Lactobacillus rhamnosus LGG® cell, which is commercially available from Chr. Hansen A/S, Hoersholm, Denmark, is significantly improved in a composition as described here. The present inventors also experimented with the LAB L. casei 431® cell (Lactobacillus paracasei subsp. paracasei) - and good storage stability was also demonstrated for dry compositions as described herein.
[0028] As illustrated in the working examples here, the novel compositions as described here result in an improved storage stability of the cells. In particular, the compositions are more stable at high humidity/high water activity. The present invention provides compositions comprising infant LAB powder with an aqueous activity of 0.3 where the log loss of active cells is <2.5 when stored in 30% RH at 35°C and tested after 13 weeks. Preferably, the log loss of active cell compositions is <2.5 after 17 weeks.
[0029] As demonstrated in the examples, substantially no difference is observed between compositions with or without sodium alginate with reference to stability. However, for compositions with sodium alginate that have been heat treated, heat treatment has a negative impact on stability as opposed to compositions without sodium alginate.
[0030] Accordingly, one aspect of the invention relates to a dry composition comprising from 109 to 1013 cfu/g of the composition of lactic acid bacteria cells, wherein the composition is characterized in that it also comprises an amount relatively high amount of protective agents as described in more detail in the following. Preferably, the composition does not comprise an alginic acid salt such as sodium alginate for reasons explained in more detail later.
[0031] As discussed here - a dry composition as described here can be used in eg newborn powder with high aqueous activity (aw = 0.3) or in other high aw applications such as cereals, musli bars or chocolate.
[0032] The solution is grounded in that the present inventors have worked intensively with numerous different parameters to dry such compositions and have identified that by using the drying method as described herein a person is able to properly dry such compositions in an efficient manner that it can be applied on an industrial scale with relatively large amounts of the LAB composition - i.e. it can be manufactured at relatively low costs and drying can be carried out within a relatively short period (eg within 15 to 30 hours).
[0033] As discussed above - the direct drying method and unambiguously described in WO2010/138522A2 for example is not involving a freezing step to form solid frozen particles/pellets. The drying method as described here also comprises other method steps which are different from what is directly and unambiguously described in WO2010/138522A2.
[0034] For example - in the primary drying step (e) of the method of the first aspect of the invention a vacuum pressure of 0.7 to 2 millibar (mbar) is used (corresponding to 525 mTORR to 1500 mTORR). As discussed here - the use of this vacuum pressure range of 0.7 to 2 mbar can be seen as an essential element here of the method of the invention. As discussed here - it is only by working within this vacuum pressure range of 0.7 to 2 mbar in step (e) that one obtains a satisfactory method here for drying a LAB relevant here.
[0035] The use of this range of 0.7 to 2 mbar as done in step (e) herein is not described or suggested in WO2010/138522A2 or any other relevant herein to inventors of the known prior art.
[0036] In summary, it is claimed that the drying method of the present invention represents a significant improvement over the direct and unambiguously described drying methods eg in WO2010/138522A2.
Accordingly, a first aspect of the invention relates to a method for preparing a dry powder composition comprising: (i): from 108 to 1014 cfu/g of the lactic acid bacteria cell (LAB) composition; and (ii) an amount of protective agent(s) from 2 to 40 g - wherein the amount of protective agent(s) is given in relation to 1 g of lactic acid bacteria cells in the composition dry, wherein the method for preparing a dry powder composition comprises the following steps: (a): fermenting the LAB cell and harvesting the cells to obtain a LAB cell concentrate comprising the LAB cells and water - wherein the concentrate comprises from 108 to 1014 cfu/g dry matter of lactic acid bacteria cell concentrate (LAB); (b): mixing a suitable amount of protective agent(s) with the LAB cell concentrate to form a slurry - wherein the slurry comprises an amount of protective agent(s) of 2 g 40 g - where the amount of protective agent(s) is given in relation to 1 g of lactic acid bacteria cells in the slurry and both the amount of protective agent(s) and cells of lactic acid bacteria are measured as dry matter in a slurry; (c): freezing the slurry to form solid frozen particles/pellets; (d): load a tray with 2 kg/m2 to 50 kg/m2 of the frozen particles/pellets to obtain the material here relevant in the tray; (e): primarily dry the material in the tray under a vacuum pressure of 0.7 to 2 millibar (mbar), at a temperature where the temperature of the material is not so high that more than 75% of the LAB cells are inactivated and for a period of time until at least 90% of the water in the slurry from step (b) has been removed; and (f): secondarily drying the material from step (e) under a vacuum pressure of 0.01 to 0.6 millibar (mbar), at a temperature where the temperature of the material is not so high that more than 75 % of the LAB cells are inactivated and for a period of time sufficient to reduce the aqueous activity (aw) to less than 0.30 and thereby obtain the dry powder composition comprising: (i): from 108 to 1014 cfu/ g of the composition of lactic acid bacteria (LAB) cells; and (11) an amount of protective agent(s) from 2 to 40 g - wherein the amount of protective agent(s) is given in relation to 1 g of lactic acid bacteria cells in the composition dry.
[0038] The term "protective agent(s)" is to be understood herein as any agent that can help to improve the storage stability of lactic acid bacteria cells of interest. In relation to a dry powder composition and the method of drying such a dry powder composition as described herein - the term "protective agent(s)" may also be noted as any agents present in the dry powder composition as such, which is not the acid bacteria cells lactic acid (LAB) as such.
[0039] As understood by the person versed in the present context - the term "in which the amount of protective agent(s) is given in relation to 1 g of lactic acid bacteria cells in the dry composition" - means that if the dry powder composition of the first aspect for example comprises 2 g of lactic acid bacteria cells so should the dry powder composition also comprise from 4 g to 80 g of protective agent(s) - since the method of The first aspect says that there should be 2 g to 40 g of protective agent(s) per 1 g of lactic acid bacteria cells.
[0040] Similarly, as understood by the person versed in the present context - the term "all amounts of protective agents below are given in relation to 1 g of active lactic acid bacteria cells in the composition" means if the composition for example, comprises 2 g of lactic acid bacteria cells then should the composition for example also comprise from 4 to 8 g of sucrose.
[0041] Since the composition is a dry powder composition - it is evident to the skilled person that the amounts given for the individual components (eg lactic acid bacteria cells and protective agents) of the composition are measured as dry matter.
[0042] A composition as described herein may be included in suitable packaging - for example, a bottle, box, vial, capsule etc. As would be understood by the person skilled in the present context - when referred to herein as the weight of the composition (e.g. called "g of the composition") then it is referred to the weight of the composition as such - i.e. not including the possible weight of a package proper.
[0043] Embodiments of the present invention are described below, by way of examples only.
[0044] Other dry compositions relevant herein are described below - each of which can be said to be characterized by specific preferred amounts of protective agents.
[0045] One aspect of the invention relates to a dry composition comprising from 109 to 1013 cfu/g of the composition of lactic acid bacteria cells, wherein the composition is characterized in that it also comprises the following amounts of protective agents (all amounts of protective agents below are given in relation to 1 g of lactic acid bacteria cells in the composition): (i): from 2 to 5 g of sucrose, 1 to 3 g of maltodextrin and 0 .75 to 2 g of Na ascorbate.
[0046] A second aspect of the invention relates to a dry composition comprising from 109 to 1013 cfu/g of the composition of lactic acid bacteria cells, wherein the composition is characterized in that it also comprises the following amounts of protective agents (all amounts of protective agents below are given in relation to 1 g of lactic acid bacteria cells in the composition): (i): from 5 to 9 g of sucrose, from 1 to 3 g of maltodextrin and 0.75 to 2 g of Na ascorbate.
[0047] A third aspect of the invention relates to a dry composition comprising from 109 to 1013 cfu/g of the composition of lactic acid bacteria cells, wherein the composition is characterized in that it also comprises the following amounts of protective agents (all amounts of protective agents below are given in relation to 1 g of lactic acid bacteria cells in the composition): (i): from 3 to 6 g of sucrose, from 4 to 8 g of trehalose and 0.0 (preferably 0.1) to 0.5 g of Na ascorbate.
[0048] A fourth aspect of the invention relates to a dry composition comprising from 109 to 1013 cfu/g of the composition of lactic acid bacteria cells, wherein the composition is characterized in that it also comprises the following amounts of protective agents (all amounts of protective agents below are given in relation to 1 g of lactic acid bacteria cells in the composition): (i): from 0.5 to 3.5 g of maltodextrin, from 0.0 ( preferably 0.1) to 0.5 g Na-ascorbate, 6 to 9 g trehalose and 0.1 to 0.5 g modified starch.
[0049] The term "modified starch" is well known to the knowledgeable person and the knowledgeable person knows whether an agent of specific interest is modified starch or not. As known to the knowledgeable person - modified starch, also called starch derivatives, are prepared by treating The native starch is physically, enzymatically, or chemically.A suitable commercially available modified starch herein is the commercially available modified starch product Remy HC-P.
[0050] A fifth aspect of the invention relates to a dry composition comprising from 109 to 1013 cfu/g of the composition of lactic acid bacteria cells, wherein the composition is characterized in that it also comprises the following amounts of protective agents (all amounts of protective agents below are given in relation to 1 g of lactic acid bacteria cells in the composition): (i): from 6 to 9 g of trehalose, from 0.1 to 1 g of inulin and from 0.5 to 3 g of hydrolyzed casein.
[0051] A sixth aspect of the invention relates to a dry composition comprising from 109 to 1013 cfu/g of the composition of lactic acid bacteria cells, wherein the composition is characterized in that it also comprises the following amounts of protective agents (all amounts of protective agents below are given in relation to 1 g of lactic acid bacteria cells in the composition): (i): from 0.5 to 3.5 g of maltodextrin, from 0.0 ( preferably 0.1) to 0.5 g Na-ascorbate and 2 to 5 g trehalose.
[0052] A seventh aspect of the invention relates to a dry composition comprising from 109 to 1013 cfu/g of the composition of lactic acid bacteria cells, wherein the composition is characterized in that it also comprises the following amounts of protective agents (all amounts of protective agents below are given in relation to 1 g of lactic acid bacteria cells in the composition): (i): from 2 to 5 g of sucrose, from 1.5 to 3.5 g of maltodextrin and from 0.0 (preferably 0.1) to 0.5 g of Na ascorbate.
[0053] An eighth aspect of the invention relates to a dry composition comprising from 109 to 1013 cfu/g of the composition of lactic acid bacteria cells, wherein the composition is characterized in that it also comprises the following amounts of protective agents (all amounts of protective agents below are given in relation to 1 g of lactic acid bacteria cells in the composition): (i): from 4.5 to 7.5 g of sucrose, from 2.5 to 5.5 g of maltodextrin and from 0.0 (preferably 0.1) to 0.5 g of Na ascorbate.
[0054] A ninth aspect of the invention relates to a dry composition comprising from 109 to 1013 cfu/g of the composition of lactic acid bacteria cells, wherein the composition is characterized in that it also comprises the following amounts of protective agents (all amounts of protective agents below are given in relation to 1 g of lactic acid bacteria cells in the composition): (i): from 2.5 to 5.5 g of maltodextrin and from 0.0 ( preferably 0.1) to 0.5 g of Na-ascorbate and from 4.5 to 7.5 g of trehalose.
[0055] A tenth aspect of the invention relates to a dry composition comprising from 109 to 1013 cfu/g of the composition of lactic acid bacteria cells, wherein the composition is characterized in that it also comprises the following amounts of protective agents (all amounts of protective agents below are given in relation to 1 g of lactic acid bacteria cells in the composition): (i): from 1.5 to 4.5 g of maltodextrin and from 0.0 ( preferably 0.1) to 0.5 g of Na-ascorbate and from 3 to 6 g of trehalose.
[0056] An eleventh aspect of the invention relates to a dry composition comprising from 109 to 1013 cfu/g of the composition of lactic acid bacteria cells, wherein the composition is characterized in that it also comprises the amounts following of protective agents (all amounts of protective agents below are given in relation to 1 g of lactic acid bacteria cells in the composition): (i): from 0.5 to 3 g of maltodextrin and from 0.0 (preferably 0.1) to 0.5 g of Na ascorbate and from 1 to 4 g of trehalose.
[0057] Experimental results have shown that all dry compositions described above have very good storage stability.
[0058] Generally the specific preferred industrial use of a lactic acid bacteria cell (LAB) containing composition as described herein would normally depend on the specific characteristics of the cell in question.
[0059] The composition can be given to a human, an animal or a fish for health promotion purposes. This is generally more relevant if the cell has probiotic properties and is particularly relevant when the cell is a probiotic LAB cell.
[0060] Accordingly, another aspect of the invention relates to a method for delivering lactic acid bacteria (LAB) cells to a human, an animal or a fish, comprising administering at least one dry composition of any of the separate dry composition aspects as described herein. DETAILED DESCRIPTION OF THE INVENTION Dry powder composition
[0061] The knowledgeable person understands what a dry composition is in the present context. To describe this quantitatively - the aqueous activity (aw) of the dry powder composition as described here is less than 0.30. More preferably - the aqueous activity (aw) of the dry powder composition as described herein is less than 0.25, even more preferably less than 0.20 and most preferably the aqueous activity (aw) of the dry powder composition as described here is less than 0.15. The person skilled in the art knows how to determine the aqueous activity (aw) of the dry composition as described here.
[0062] The skilled person knows how to make a dry composition as described here. Manufacturing the dry composition as described herein involves, for example, mixing a cell culture with a protective agent. The second step involves drying said mixture. Drying can be done by freeze drying, spray drying, modified spray drying and/or vacuum drying. Other means for drying may be possible.
[0063] In the case of freeze or vacuum drying, the mixture is preferably formed into pellets by methods that are known in the art. One method might be to let droplets of the mixture fall into liquid nitrogen. Another method to form pellets can be by extrusion. Said pellets can be subsequently dried using the above drying methods. Preferably, the composition is dried using the method for preparing a dry powder composition described herein.
[0064] The dry composition may be in a powdered form.
[0065] The weight of the dry composition (for example, termed "g of the composition") will generally depend on different factors such as the use of the composition (for example, to manufacture a powdered newborn product as discussed below).
[0066] The weight of the dry composition as described here for example can be from 1 g to 1000 kg.
[0067] For example - if the dry composition is to be used as a newborn product - then it is the dry composition usually mixed with powdered milk and other supplements to obtain a powdered newborn product comprising bacterial cells of the lactic acid.
[0068] As known to the skilled person - production of powdered newborn products can be done on a very large scale - for example, mixing 1 to 10 kg of a dry composition as described here with an adequate amount of powdered milk and other supplements.
Therefore, it may be preferred that the weight of the dry composition as described herein is from 50 g to 10000 kg, such as for example from 100 g to 1000 kg or from 1 kg to 5000 kg or from 100 kg to 1000 kg.
[0070] As is evident to the person skilled in the present context - in order to obtain, in step (f) of the method of the first aspect, a dry powder composition with a weight of, for example, 100 kg - one needs to use relatively high amounts corresponding LAB cell concentrate in step (b) of the first aspect and protective agent(s) in step (c) of the first aspect.
[0071] The dry powder composition of the invention can be encapsulated, for example, in a gelatin capsule, or formulated in, for example, tablets, or sachets. This aspect is particularly relevant if the composition is to be used in a dietary supplement. Lactic acid bacteria cells
It may be preferred that the dry powder composition as described herein comprises from 109 to 1013 cfu/g of the lactic acid bacteria cell composition (LAB).
[0073] The lactic acid bacteria (LAB) cell in principle can be any suitable LAB cell of interest.
[0074] Preferably, the LAB cell is a probiotic cell.
[0075] The term "probiotic cell" designates a class of cells (eg, microorganisms) that is defined as a microbial food supplement that beneficially affects the human or animal host by improving its gastrointestinal microbial balance. Known beneficial effects include improved colonization resistance against harmful microflora due to oxygen consumption and acid production of probiotic organisms. An example of the effectiveness of probiotically active organisms in preventing the overgrowth of potential pathogens and thus diarrhea is shown in a study where administration of capsules containing probiotically active organisms viable to tourists traveling in Egypt resulted in a 39.4% production rate against traveler's diarrhea (Black et al. 1989). A review of probiotics and their effects on humans and animals can be found in Fuller, 1989 and 1994.
[0076] In the present context, the expression "lactic acid bacteria" designates a group of Gram positive, catalase negative, non-motile, microaerophilic or anaerobic bacteria that ferment sugar (including lactose) with the production of acids including lactic acid such as Predominantly produced acid, acetic acid, formic acid and propionic acid.Below described are preferred LABs.
[0077] The most industrially useful lactic acid bacteria are found among Lactococcus species, Streptococcus species, Enterococcus species, Lactobacillus species, Leuconostoc species, Bifidobacterium species, Propioni and Pediococcus species. Therefore, in a preferred embodiment the lactic acid bacteria are selected from the group consisting of these lactic acid bacteria.
[0078] In a preferred embodiment the lactic acid bacteria are lactic acid bacteria selected from the group consisting of Lactobacillus rhamnosus, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Leuconostoc lactis, Leuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactobacillus casei subsp. casei, Streptococcus thermophilus, Enterococcus, such as Enterococcus faecum, Bifidobacterium animalis, Bifidobacterium lactis, Bifidobacterium longum, Lactobacillus lactis, Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus. Within this group, the most preferred lactic acid bacteria is Lactobacillus rhamnosus.
The composition may comprise one or more strains of a lactic acid bacterium which may be selected from the group comprising: BB-12® (Bifidobacterium animalis subsp lactis BB-12®), DSM 15954; ATCC 29682, ATCC 27536, DSM 13692, and DSM 10140, LA-5® (Lactobacillus acidophilus LA-5®), DSM 13241, LGG® (Lactobacillus rhamnosus LGG®), ATCC 53103, GR-1® (Lactobacillus rhamnosus GR- 1®), ATCC 55826, RC-14® (Lactobacillus reuteri RC-14®), ATCC 55845, L. casei 431® (Lactobacillus paracasei subsp. paracasei L. casei 431®), ATCC 55544, F19® (Lactobacillus paracasei F19 ®), LMG-17806, TH-4® (Streptococcus thermophilus TH-4®), DSM 15957, PCC® (Lactobacillus fermentum PCC®), NM02/31074, LP-33® (Lactobacillus paracasei subsp. paracasei LP-33®) ), CCTCC M204012.
[0080] The LAB culture can be a "mixed lactic acid bacteria (LAB) culture" or a "pure lactic acid bacteria (LAB) culture". The term "mixed lactic acid bacteria (LAB) culture", or "LAB" culture, denotes a mixed culture comprising two or more different LAB species. The term a "pure lactic acid bacteria (LAB) culture" denotes a pure culture comprising only a single LAB species. Accordingly, in a preferred embodiment the LAB culture is a LAB culture selected from the group consisting of these cultures.
[0081] The LAB culture can be washed, or not washed, before mixing with the protective agents.
[0082] If the composition comprises an alginic acid salt such as sodium alginate, it is often necessary to wash the cells with demineralized water before adding the protective agents to prevent the formation of calcium alginate. Protective agent(s)
[0083] The term "protective agent(s)" is to be understood herein as any agent that can help to improve the storage stability of lactic acid bacteria cells of interest. In relation to a dry powder composition and the method of drying such a dry powder composition as described herein - the term "protective agent(s)" may also be observed as any agents present in the dry powder composition as such, other than the bacterial cells of the lactic acid (LAB) as such.
[0084] It may be preferred that the amount of protective agent(s) of point (ii) of the first aspect is an amount of protective agent(s) from 4 g to 20 g, such as from 5 g to 15 g or from 6 g to 12 g.
[0085] In a preferred embodiment - at least 30% (more preferably at least 50%, even more preferably at least 70% (such as for example at least 80% or at least 90%) of the agent(s) ) protectant(s) of point (ii) of the first aspect are carbohydrates.
[0086] Preferably the carbohydrates are saccharides and preferred saccharides are for example sucrose, maltodextrin, trehalose and/or inulin.
[0087] In working examples here the drying of a LAB composition is described, in which more than 80% of the protective agent(s) are saccharides, since the composition comprises 75.3 g of trehalose + 5.0 g of inulin per 100 g of all protective agents present in the composition.
[0088] In addition, other preferred dry powdered LAB compositions herein, wherein greater than 70% of the protective agent(s) are saccharides, are discussed below. Without being bound by theory - it is believed that all of these LAB compositions described here, "more than 70% of the protective agent(s) are saccharides" have very good storage stability - said in others words, using a (as described here) relatively high amount of carbohydrates (such as eg saccharides) as protective agents one can obtain a dry powdered composition of LAB with a very good commercially relevant storage stability (eg , for use as a component in a newborn powder).
[0089] As used herein, by the term "newborn" is meant a human being from about birth to 12 months of age. In the present context, the term "newborn formula" refers to a composition in form liquid or powder that meets the nutritional needs of a newborn by being a substitute for human milk. These formulations are regulated by EU and US regulations that define macronutrient, vitamin, mineral and other ingredient levels in an effort to simulate the nutritional and other properties of human breast milk. Of course, the formula must not contain any potentially allergenic substances. Thus, when hydrolyzed casein is used, it preferably should be hydrolyzed so that more than 90% of the peptides have a molecular weight of less than 1,000 Daltons, with more than 97% having a molecular weight of less than 2,000 Daltons.
[0090] As used herein, "infants" are defined as human beings over the age of about 12 months to about 12 years. The infant powder compositions of the present invention can be used for infant formula, follow-on formula, growth milk and special formula as well as a nutritional product for newborns and children to improve their intestinal microflora while simultaneously providing nutrition to the newborn or child.
[0091] A protective agent suitable herein is a protective agent selected from the group consisting of: Na ascorbate, modified starch, hydrolyzed casein and alginate (eg sodium alginate).
[0092] Alginic acid, also called algin or alginate, is an anionic polysaccharide distributed widely in the cell walls of brown algae. Alginate is present in the cell walls of brown algae as the calcium, magnesium and sodium salts of alginic acid. Alginic acid forms water-soluble salts with monovalent cations but is precipitated on acidification.
[0093] Alginates of many divalent cations, particularly of Ca2+, Sr2+ and Ba2+, are insoluble in water and can be prepared when sodium ions of NaAlg are replaced by bi- and trivalent cations. This property is used in the isolation of alginic acid from algae. The purpose of the extraction process is to obtain dry powdered sodium alginate. Calcium and magnesium salts do not dissolve in water; the sodium salt does.
[0094] Due to their physical and chemical properties the monovalent salts of alginic acid such as hydrogen alginate (HAlg), potassium alginate (KaAlg) and sodium alginate (NaAlg), have been widely used in food processing, medical industries and pharmaceuticals. In contrast, calcium alginate is a cream-colored, gelatinous, water-insoluble substance that can be created by adding aqueous calcium chloride to aqueous sodium alginate.
[0095] When the composition is to be used in a newborn powder, this is preferred in that it does not contain modified starch or polysaccharides such as sodium alginate whereas sodium alginate which is a product extracted from algae is not approved for use in newborn powder for newborns younger than 1 year (newborn formula). For this reason a comparison of several compositions without alginate was prepared and it was found as demonstrated in the examples that these compositions exhibit good stability. Additional comparison experiments were done between compositions with and without alginate and it was found that there is substantially no difference between compositions with and without alginate with regard to stability.
[0096] Important disadvantages using alginate which is a product extracted from algae as described above is that there is a large batch-to-batch variation with reference to viscosity and that the product may contain bacteria meaning that some treatment, for example, heat treatment has to be applied in order to inactivate the bacteria. The heat treatment can be any given combination of temperature and retention time that achieves a reduction of log 6 or more (F0 value equal to or more than 6), ie it can be a batch heat treatment in a pressurized tank with a heating blanket, the use of sterile steam injection/condensation directly into the protective agent solution (either reasonably long duration at 110 to 121°C) or continuously through a UHT treatment unit (as long as short duration at high temperature, this is 30 s at 132°C).
[0097] This treatment leads to alginate depolymerization and heat treatment generally has a negative impact on stability for compositions with sodium alginate as demonstrated in example 9.
[0098] Thus, contrary to conventional teaching in the art to include alginic acid salts such as sodium alginate in compositions to stabilize and protect living bacteria during harsh conditions, it is advantageous for the above reasons to provide compositions that do not comprise alginic acid salts such as sodium alginate. Consequently the compositions of the present invention preferably do not comprise an alginic acid salt such as sodium alginate.
[0099] In a preferred embodiment, the invention relates to a dry composition comprising from 109 to 1013 cfu/g of the composition of lactic acid bacteria cells, wherein the composition is characterized by the fact that it also comprises the following amounts of protective agents (all amounts of protective agents below are given in relation to 1 g of lactic acid bacteria cells in the composition): (i): from 6 to 9 g of trehalose,
[00100] from 0.1 to 1 g of inulin and
[00101] from 0.5 to 3 g of hydrolyzed casein, and
[00102] by the fact that it does not comprise a salt of alginic acid.
[00103] Specifically, the composition does not comprise hydrogen alginate, potassium alginate or sodium alginate.
[00104] In a particularly preferred embodiment the dry composition according to the invention comprises 75 to 80% (w/w) of trehalose, 3 to 10% (w/w) of inulin and 15 to 20% (w/w) of hydrolyzed casein and does not comprise an alginic acid salt such as sodium alginate.
The entire disclosure described in this specification and claims with reference to the compositions and methods is of course included with reference to the preferred embodiment and particularly preferred embodiment described above. Specifically, the invention relates to a powder for a newborn, a food product or a dietary supplement comprising a composition according to the preferred embodiment and particularly preferred embodiment described above.
[00106] The invention further relates to a method for preparing a dry composition according to the invention wherein the method for preparing the dry composition comprises the following steps: (a): fermenting the LAB cell and harvesting the cells for obtaining a LAB cell concentrate comprising the LAB cells and water - wherein the concentrate comprises from 108 to 1014 cfu/g of lactic acid bacteria cell concentrate (LAB) dry matter; (b): mixing a suitable amount of protective agent(s) with the LAB cell concentrate to form a slurry - wherein the slurry comprises an amount of protective agent(s) from 6 to 9 g trehalose, 0.1 to 1 g inulin and 0.5 to 3 g hydrolyzed casein and does not comprise a salt of alginic acid - wherein the amount of protective agent(s) is given in ratio of 1 g of lactic acid bacteria cells in the slurry and both the amount of protective agent(s) and lactic acid bacteria cells is measured as dry matter in a slurry; (c): freezing the slurry to form solid frozen particles/pellets; (d): load a tray with 2 kg/m2 to 50 kg/m2 of the frozen particles/pellets to obtain the material here relevant in the tray; (e): primarily dry the material in the tray under a vacuum pressure of 0.7 to 2 millibar (mbar), at a temperature where the temperature of the material is not so high that more than 75% of the LAB cells are inactivated and for a period of time until at least 90% of the water in the slurry from step (b) has been removed; and (f): secondarily drying the material from step (e) under a vacuum pressure of 0.01 to 0.6 millibar (mbar), at a temperature where the temperature of the material is not so high that more than 75 % of the LAB cells are inactivated and for a period of time sufficient to reduce the aqueous activity (aw) to less than 0.30 and thereby obtain the dry composition comprising: (i): from 109 to 1013 cfu/g da composition of lactic acid bacteria cells, wherein the composition is characterized by the fact that it also comprises the following amounts of protective agents (all amounts of protective agents below are given in relation to 1 g of acid bacteria cells lactic in the composition):
[00107] from 6 to 9 g of trehalose,
[00108] from 0.1 to 1 g of inulin and
[00109] from 0.5 to 3 g of hydrolyzed casein, and
[00110] by the fact that it does not comprise a salt of alginic acid.
[00111] Compared to a method where the composition comprises sodium alginate, the mixing step b) is easier as the slurry has a lower viscosity and is also easier to pellet in step c) meaning the particles they are often homogeneous and of an appropriate size as explained in more detail below with respect to step c) even without a milling step. Addition of other compounds to the composition:
[00112] The dry composition as described herein may comprise other compounds of interest.
[00113] This for example may be vitamins (eg tocopherol) or other compounds that one may be interested in having them present in the final composition. Examples of such compounds can be moisture scavengers such as for example potato starch.
[00114] Although the drying method described above is preferred, alternative methods exist as described above. Depending on which drying method being used, it may be necessary to add a viscosity modifier. If, for example, vacuum belt drying is intended, it may be necessary to increase the viscosity. Conversely, if spray drying is intended, it may be necessary to lower the viscosity.
[00115] Suitable examples of viscosity modifiers are for example water (to lower viscosity), pectin, pregelatinized starch, gums (eg acacia, xanthan, guar gum, locust bean gum), glycerols (eg. glycerin); glycols (for example polyethylene glycols, propylene glycols); plant-derived waxes (eg, carnauba, rice, candelilla), non-plant waxes (beeswax); lecithin; vegetable fibers; lipids; and silicas (eg, silicon dioxide). Use of a composition according to the invention:
[00116] Generally the specific preferred industrial use of a cell containing the composition as described herein would normally depend on the specific characteristics of the cell in question.
[00117] The composition can be provided to a human, an animal or a fish for health promotion purposes. This is generally more relevant if the cell has probiotic properties and is particularly relevant when the cell is a probiotic LAB cell.
[00118] A preferred formulation of the invention is in the form of a powder for newborns, whereby the composition is mixed with powdered milk. As known in the art - milk powder can also comprise other supplements.
[00119] Another use refers to the use of the composition as described herein in cereals, such as musli, or other dry foodstuffs.
[00120] Consequently, in other aspects, the invention relates to a food product, such as a cereal, musli bars, filled chocolate bars or chocolate bars, which incorporates the composition according to the invention. It can also be used in powders (eg so-called sports powders) intended to be mixed into beverages, such as sports drinks or energy drinks.
[00121] In another aspect, the invention relates to a dietary supplement comprising a dry composition as described herein.
[00122] Below are discussed examples of other relevant protective agents suitable here.
The protective agents used here are generally those commonly used as for example cryo-additives in the field, for example saccharides such as trehalose, lactose, maltose, sucrose, raffinose or glucose; myo-inositol; or other so-called cryoprotectants, such as polyethylene glycol, dimethyl sulfoxide, glycerol, or dextran. Preferred protective agents are sucrose, and/or maltodextrin.
[00124] Other additives, for example antioxidants such as ascorbate may also be present. For the purposes of this invention, ascorbate may be termed a protective agent.
[00125] As discussed here - an advantage of the new method described herein for preparing a dry powder composition is that using the method as described here one can efficiently dry such LAB compositions having a relatively high amount of carbohydrates (such as eg saccharides) as protective agents. Ferment the LAB cell to obtain a LAB cell concentrate - Step (a)
[00126] As discussed above - step (a) of the method of the first aspect says: "(a): ferment the LAB cell and harvest the cells to obtain a LAB cell concentrate comprising the LAB cells and water - in that the concentrate comprises from 108 to 1014 cfu/g of dry matter of lactic acid bacteria cell concentrate (LAB)"
[00127] It is routine work for the versed person to ferment a LAB cell of interest in order to eg produce it/cultivate it on a large scale.
[00128] As known in the art - harvesting fermented cells generally involves a centrifugation step to remove relevant parts of the fermentation medium and thereby obtain a LAB cell concentrate.
[00129] As known in the art - for the production of LAB cells relevant here at this stage one can have a LAB cell concentrate with about 10% cell dry matter - this is a so-called 10% concentrate. The rest of the concentrate afterwards is usually mostly water - this will be around 90% water. The LAB cell concentrate can of course also contain sometimes less water - for example around 50% water. Typically - the LAB cell concentrate in step (b) comprises at least 10% (such as at least 20% or at least 50%) water. In some embodiments, the concentrate may comprise even less than 10% dry matter, such as in the range of 5 to 10%, for example about 5%,
[00130] In the present context it is essentially this water from the LAB cell concentrate which is removed by the drying method as described herein to obtain the dry LAB powder composition described herein.
[00131] After cell harvesting - it may be preferred to include an additional washing step in order to remove the greatest number of components/compounds from the fermentation media as such - i.e. obtain a "purer" LAB cell concentrate which essentially just comprises the LAB cells as such.
[00132] Step (a) says: "wherein the concentrate comprises from 108 to 1014 cfu/g of dry matter of lactic acid bacteria cell concentrate (LAB)." The term "dry matter" within the term "cfu/g dry matter" is to be understood as the person skilled in the art would understand in the present context - ie that the LAB concentrate comprises the given amount of LAB cells as relative to the weight of the dry matter weight of the LAB concentrate (ie is, not including the weight of the liquid as present in the LAB concentrate).
[00133] If desired, a step of freezing the LAB concentrate for example in the form of solid frozen particles/pellets can be added and the LAB concentrate can be kept as a frozen concentrate for a period of time before being thawed and the process continued with step b). Alternatively, the process can be started from step (b) e.g. on the basis of a commercially available LAB cell concentrate. Mix the protective agent(s) with the LAB cell concentrate - Step (b):
[00134] As discussed above - step (b) of the method of the first aspect says: "(b): mix an appropriate amount of protective agent(s) with the LAB cell concentrate to form a slurry - wherein the slurry comprises an amount of protective agent(s) from 2 g to 40 g - wherein the amount of protective agent(s) is given in relation to 1 g of lactic acid bacteria cells in the slurry and both the amount of protective agent(s) and lactic acid bacteria cells are measured as dry matter in a slurry”.
[00135] The term "slurry" should be understood as the person skilled in the art would understand it in the present context - this is as a relatively thick suspension of solids in a liquid.
[00136] It is routine work for the skilled person to mix adequate amount of protective agent(s) with the LAB cell concentrate to obtain the desired concentration/amount of protective agent(s) in the slurry. Freeze the slurry to form solid frozen particles/pellets - Step (c)
[00137] As discussed above - step (c) of the method of the first aspect says: "(c): freeze the slurry to form solid frozen particles/pellets"
[00138] It is routine work for the knowledgeable person to manufacture this slurry freeze to form the solid frozen particles/pellets step as such. As known in the art - this can be done by using for example liquid nitrogen, where the slurry is frozen by using liquid nitrogen to obtain the solid frozen particles/pellets.
[00139] As shown in a working example here - the present inventors tested different particle sizes of the frozen particles/pellets and it was found that very large particles did not provide satisfactory drying results. Consequently, in a preferred embodiment at least 95% (more preferably at least 97%) of the frozen particles/pellets in step (c) are particles/pellets which are able to pass through a mesh/sieve with maximum aperture/hole size 10 mm.
[00140] More preferably, at least 95% (more preferably at least 97%) of the particles/pellets frozen in step (c) are particles/pellets that are capable of passing through a mesh with maximum aperture/hole size of 7 .5mm, more preferably with maximum aperture/hole size of 5mm and most preferably with maximum aperture/hole size of 3mm.
[00141] It is routine work for the versed person to sift relevant particles (here the frozen particles/pellets) through a mesh. It is routine work for the versed person to test whether a specific frozen particle/pellet sample is a sample, where at least 95% (more preferably at least 97%) of the frozen particles/pellets are particles/pellets that are able to pass through a mesh with given maximum size of opening/holes.
[00142] As known in the art - one can simply place the specific frozen particle/pellet sample of interest onto a suitable mesh and then stir/stir the mesh in a suitable mode until no other significant amount of frozen particles/pellets are passing through the mesh - if more than 95% of the frozen particles/pellets have passed through the mesh then the specific frozen particle/pellet sample of interest is a sample, where at least 95% of the frozen particles/pellets are particles/pellets which are able to pass through the mesh with the given maximum size of opening/holes. Load a tray with frozen particles/pellets - Step (d):
[00143] As discussed above - step (d) of the method of the first aspect says: "(d): load a tray with 2 kg/m2 to 50 kg/m2 of the frozen particles/pellets to obtain the material here relevant in the tray ”
[00144] In a preferred embodiment - the tray in step (d) of the first aspect is loaded with 5 kg/m2 to 30 kg/m2 of the frozen particles/pellets (such as for example, from 7 kg/m2 to 15 kg/ m2 of frozen particles/pellets).
[00145] In the present context any suitable trays can be used here. As known - there are several suitable trays relevant here available to the person skilled in the art, some of these trays are also commercially available.
[00146] As understood by the person versed in the present context - the tray is normally loaded with frozen particles/pellets in a manner where relatively uniform/similar distributions of the frozen particles/pellets in the tray are obtained - i.e. preferably do not have all the frozen particles/pellets located for example on only one edge of the tray.
[00147] For example, industrially relevant large-scale production of relatively large amounts of a dry powder composition relevant here (eg from 100 kg to 10000 kg of the dry powder composition) - usually it may be preferred to simultaneously use more than that one (such as eg more than 10 or more than 100) tray eg in loading (d) here - ie where in step (d) more than one (such as eg loading) is loaded more than 10 or more than 100) tray with 2 kg/m2 to 50 kg/m2 of frozen particles/pellets. Primary drying - step (e):
[00148] As discussed above - step (e) of the method of the first aspect says: "(e): primarily dry the material in the tray under a vacuum pressure of 0.7 to 2 millibar (mbar), at a temperature in that the temperature of the material does not get so high that more than 75% of the LAB cells are inactivated and for a period of time until at least 90% of the water in the slurry from step (b) has been removed"
[00149] In the present context any suitable vacuum dryer apparatus can be used here.
[00150] As known - there are several suitable vacuum dryer apparatus available to the skilled person, in which some of these vacuum dryer apparatus are also commercially available.
[00151] In a preferred embodiment - the vacuum dryer apparatus is an apparatus, wherein the heating in the apparatus is so-called radiant heating. As known to the skilled person radiation heating is understood by the skilled person to be different from so-called contact heating. As known to the person skilled in the art - heating by radiation can be obtained for example by having a heating plate situated close to (but not in direct contact with) the tray comprising the material to be dried. In other words, in relation to the primary drying step (e) - there is a space (ie vacuum) between the heating plate and the tray - ie the heating of the tray is then based on radiant heating.
[00152] In a preferred embodiment - the tray is situated between two heating plates, wherein both heating plates provide radiant heating to the tray.
[00153] For example, industrially relevant large-scale production of relatively large amounts of a dry powder composition relevant here (eg from 100 kg to 10000 kg of the dry powder composition) - usually it may be preferred to simultaneously use more than that one (such as eg more than 10 or more than 100) tray eg in drying step (e) here - this is where in step (e) more than one is present (such as per example, more than 10 or more than 100) tray comprising the material to be dried.
[00154] In summary, this primary drying (e) can be seen as a step, in which what might be called "free" water (ie, contrary to what might be called "bound" water) is removed. In the present context - it can be said that this "free" water removed in this step (e) represents the majority of the water present in the LAB cell concentrate of step (a). It can also be said that this "free" water is easier to remove than "bound" water so that it can be said to be essentially mostly removed in the subsequent secondary drying step (f) of the drying method of the invention (see below for another discussion of this water problem " turned on”).
[00155] In this primary drying step the vacuum pressure is 0.7 to 2 millibar (mbar) - this can be seen as an essential element of this primary drying step step (e). As discussed in the working examples here - the present inventors have identified that if in this primary step (e) a vacuum pressure that is different from the range of 0.7 to 2 millibar (mbar) is used, a satisfactory drying is not obtained here. a LAB composition as described here (ie with a relatively high amount of protective agents).
[00156] In summary, the present inventors have identified that if the vacuum pressure is below 0.7 mbar then it can be said that the water in the LAB concentrate is so cold that all frozen particles/pellets are still completely frozen during a significant part of the time period of this primary drying step (e) - ie it can be said that this step (e) thereafter would be virtually 100% so called freeze drying step - ie where all the water is removed by sublimation.
[00157] The present inventors have identified that to remove virtually all of the water, in this primary drying step, by sublimation in the present context does not provide a satisfactory result.
[00158] In summary, the present inventors have identified that if the vacuum pressure is higher than 2 mbar then it can be said that the water in the LAB concentrate is so relatively "hot" that a significant part of the frozen particles/pellets thaws (ie no longer frozen) for a significant part of the time period of this primary drying step (e) - and the present inventors have identified that this is not good so that in the present context a satisfactory final drying result is obtained .
[00159] As known to the knowledgeable person - at pressure of 2 mbar the temperature of water (ie ice) is -12°C, at 1 mbar the temperature of water (ie ice) is -20°C and at pressure of 0.7 mbar the temperature of water (ie ice) is -24°C.
[00160] In particular at the beginning of this primary drying step (e) - it is normal for the material to be dried to have a significant amount of water, as the LAB concentrate from step (a) often has around for example , 90% water and around 10% LAB cells as such.
[00161] Consequently, it can be said that in particular at the beginning of this primary drying step (e) it will generally be the water temperature that will be the type of temperature control of the material to be dried.
[00162] It can be said that the present inventors have identified that the vacuum pressure range of 0.7 to 2 mbar is exactly the perfect/ideal range for drying the LAB compositions relevant here (ie with a relatively high amount of protective agents).
[00163] Without being limited to theory - one reason why this vacuum pressure range is especially good may be because at this pressure the water temperature is adequate to obtain a relatively limited thawing of the frozen particles/pellets of the step (c) but there is no very high thawing of frozen particles/pellets. Without being limited to theory - it could be said that if a limited amount of the frozen particles/pellets is thawed (this is obtained in liquid form) - then it may be that at least some parts of the water are removed by evaporation.
[00164] Consequently, a preferred embodiment here refers to that a limited amount (for example, from at least 0.5% to at most 5%, more preferably from at least 1% to at most 4%) of the material in the step (e) is thawed liquid material (ie, unfrozen material). Furthermore, it is preferred that this be so for a significant part (eg for at least 3 hours or for at least 6 hours) of the time period of the primary drying step (e).
[00165] The person skilled in the art is visually able to determine whether a limited amount of the material in step (e) is the thawed liquid material (i.e., unfrozen material) simply by noting that there is liquid water present, for example, on the surfaces of the frozen particles/pellets.
[00166] As discussed above - in step (e) it reads: "at a temperature where the temperature of the material does not get so high that more than 75% of the LAB cells are inactivated"
[00167] It is routine work for the skilled person to continuously measure the temperature of the material as such during the time period of step (e). Typically - you simply have one or more thermometers present in the material as such during the time period of step (e).
[00168] As discussed above - it can be said that in particular at the beginning of this primary drying step (e) it will generally be the temperature of the water which will in some way control the temperature of the material to be dried.
[00169] As discussed above - at the 0.7 to 2 millibar (mbar) pressure used in step (e), the temperature of the water (ie ice) is approximately -24°C to -12°C. At this relatively cool temperature (ie approximately -24°C to -12°C) there is generally no significant inactivation of LAB cells here.
[00170] However, at the end of the time period of step (e) - for example 97% of the so-called "free" water in this step (e) may have been removed - ie exists in this time period of step (e) ) significantly less water present than at the beginning of the time period of step (e).
[00171] Consequently, it can be said that in particular at the end of the time period step (e) it is very important that the possibly applied heating of the tray/material is well controlled - this is so that the temperature of the material does not become very tall.
[00172] In the present context the person versed will generally know what might be called the thermal stability of a LAB of interest here relevant. In other words, for a LAB of interest here, it would be routine work to determine what the maximum temperature of the material should be so as not to get too many of the LAB cells inactivated.
[00173] For example - when the lactic acid bacteria (LAB) cells of point (ii) of the first aspect are Lactobacillus cells - it is preferred that the temperature of the material from steps (e) and (f) of the first aspect is a temperature that does not get higher than 40°C.
[00174] For example - when the lactic acid bacteria (LAB) cells of point (ii) of the first aspect are Bifidobacterium animalis subsp lactis cells - it is believed that the temperature can be a little higher without getting much inactivation cell high - this is the temperature of the material from steps (e) and (f) of the first aspect is a temperature that does not get higher than eg 50°C.
[00175] As evident in the present context - in step (e) and step (f) preferably there should be as little inactivation of the LAB cells as possible.
[00176] Consequently, a preferred embodiment refers to step (e), in which the temperature of the material is not so high that more than 50% of the LAB cells are inactivated, more preferably the temperature of the material is not so high that more than 25% of the LAB cells are inactivated, even more preferably the temperature of the material is not so high that more than 10% of the LAB cells are inactivated and most preferably the temperature of the material is not so high that more than 2% of the LAB cells are inactivated.
[00177] In a preferred embodiment - the primary drying of step (e) of the first aspect is done under a vacuum pressure of 1 to 2 millibar (mbar), such as 1.1 to 1.7 millibar (mbar).
[00178] In a preferred embodiment - in the primary drying step (e) of the first aspect at least 95% of the water is removed from the slurry, more preferably at least 97% of the water is removed from the slurry and most preferably is removed at least 98% of the water in the slurry.
[00179] A preferred embodiment herein refers to wherein the time period of step (e) of the first aspect is a period of 3 hours to 60 hours - more preferably 5 hours to 36 hours, even more preferably 5 hours to 24 hours (such as from 7 to 15 hours).
[00180] Without being limited to theory - it is believed by the fact that for industrially relevant large scale production here it may be difficult to carry out the primary drying step (e) itself in less than 3 hours. Without being limited to theory - using more than 60 hours for the drying step (e) would normally not be ideal for industrially relevant large scale production here. Secondary drying - step (f):
[00181] As discussed above - step (f) of the method of the first aspect says: "(f): secondarily dry the material from step (e) under a vacuum pressure of 0.01 to 0.6 millibar (mbar), at a temperature where the temperature of the material does not get so high that more than 75% of the LAB cells are inactivated and for a period of time sufficient to reduce the aqueous activity (aw) to less than 0.30 and thereby obtain the dry powder composition comprising: (i): from 108 to 1014 cfu/g of the lactic acid bacteria cell (LAB) composition; and (11) an amount of protective agent(s) of 2 g 40 g - in which the amount of protective agent(s) is given in relation to 1 g of lactic acid bacteria cells in the dry composition.”
[00182] As understood by the skilled person - several of the technical problems discussed for the primary drying step (e) above may also be of corresponding relevance with respect to the secondary drying step (f). For example - the vacuum drying apparatus used in step (f) is often the same (or very similar) vacuum drying apparatus as used in step (e).
[00183] In summary, one can observe this secondary drying (f) as a step, in which what can be called "bound" water is removed (this is contrary to what can be called "free" water as usually removed in the step (e) - see discussion above). As known to the person skilled in the art - it can be said that "bound" water is more difficult to remove than "free" water - consequently, more vacuum (less pressure in mbar) is used in step (f) compared to step (and).
[00184] A preferred embodiment herein refers to wherein the secondary drying of step (f) of the first aspect is done under a vacuum pressure of 0.05 to 0.4 millibar (mbar), such as 0.1 at 0.3 millibar (mbar).
[00185] In the present context - a pressure around 0.2 mbar can sometimes be called "total vacuum".
[00186] As discussed above - in step (e) and step (f) preferably there should be as little inactivation of the LAB cells as possible.
[00187] Consequently, a preferred embodiment refers to step (f), in which the temperature of the material is not so high that more than 50% of the LAB cells are inactivated, more preferably the temperature of the material is not so high that more than 25% of the LAB cells are inactivated, even more preferably the temperature of the material is not so high that more than 10% of the LAB cells are inactivated and most preferably the temperature of the material is not so high that more than 2% of the LAB cells are inactivated.
[00188] As discussed in relation to step (e) above - when the lactic acid bacteria (LAB) cells of point (ii) of the first aspect are Lactobacillus cells - it is preferred that the temperature of the material from steps (e) and (f) from the first aspect is a temperature that does not get higher than 40°C.
[00189] For example - when the lactic acid bacteria (LAB) cells of point (ii) of the first aspect are Bifidobacterium animalis subsp lactis cells - it is believed that the temperature can be a little higher without getting too much inactivation high of cells - this is the temperature of the material from steps (e) and (f) of the first aspect is a temperature that does not get higher than, for example, 50°C.
[00190] As discussed above, at the end of the time period of the primary drying step (e) - eg 97% of the so-called "free" water in this step (e) may have been removed - i.e. exists at the end of step (e) significantly less water present than at the beginning of the time period of step (e).
[00191] In line with this - it is evident that during the secondary drying step (f) there is relatively little water present.
[00192] Consequently, if any heating is applied in this secondary drying step (f) - for example, the heating plates used (eg for radiant heating - see above) will generally be set to a heating temperature that is very close to the temperature you would like to have as the temperature of the material (to be dried) as such. For example - if the temperature of the material (to be dried) as such in step (f) should be at most eg 37°C - then for example the heating plates used will not be set at a temperature significantly above this 37 °C.
[00193] A preferred embodiment herein refers to wherein the time period of step (f) of the first aspect is a period of 3 hours to 60 hours - more preferably 5 hours to 36 hours, even more preferably 5 hours to 24 hours (such as from 7 to 15 hours).
[00194] Without being limited to theory - it is believed by the fact that for industrially relevant large-scale production here it may be difficult to carry out the primary drying step (f) properly in less than 3 hours. Without being limited to theory - using more than 60 hours for the drying step (f) would normally not be ideal for industrially relevant large scale production here.
[00195] In a preferred embodiment - the time period of step (f) is a period of time sufficient to reduce the aqueous activity (aw) to less than 0.25, more preferably less than 0.20 and more preferably less than 0.15.
[00196] The skilled person knows how to determine the aqueous activity (aw) of the dry composition as described here. Other optional steps:
[00197] As understood by the person skilled in the art - the method of drying the first aspect as discussed here may comprise other optional steps.
[00198] An obvious relevant additional optional step here would be, for example, properly packaging in step (f) the obtained dry powder composition.
[00199] As known in the art - the packaging for example may be a bottle, box, vial, capsule etc - preferably the packaging is impermeable to keep the aqueous activity of the dry powder composition low.
[00200] Other obvious relevant additional optional steps here include the use of the dry powdered LAB composition obtained in step (f) for a relevant commercial use here.
[00201] Just as an example - commercially relevant uses may for example be as a powder for a newborn, whereby the dry powdered composition of LAB is mixed with powdered milk - or use to manufacture a dairy product. CAPTION FOR THE FIGURES
[00202] Figure 1 shows storage stability for newborn powder compositions of L. casei 431® with aw: 0.3 and at a storage temperature of 35°C.
[00203] Figure 2 shows storage stability for six different L. casei 431® newborn powder compositions with aw: 0.3 and at a storage temperature of 35°C.
[00204] Figure 3 shows storage stability for newborn powder compositions of L. casei 431® with aw: 0.3 and at a storage temperature of 35°C.
[00205] Figure 4 shows storage stability for LGG® newborn powder compositions with aw: 0.3 and at a storage temperature of 35°C.
[00206] Figure 5 shows storage stability for newborn powder compositions of L. casei 431® with aw: 0.3 and at a storage temperature of 40°C.
[00207] Figure 6 shows storage stability for LGG® newborn powder compositions with aw: 0.3 and at a storage temperature of 40°C.
[00208] Figure 7 shows storage stability for LGG® newborn powder compositions with aw: 0.25 and at a storage temperature of 35°C. EXAMPLES Materials and Methods
Lactobacillus rhamnosus LGG® and Lactobacillus paracasei subsp. paracasei L. casei 431® - obtainable from Chr. Hansen A/S, Denmark
[00210] Cargill name Trehalose: Treha 16400
[00211] Enzymatically Hydrolyzed Casein from DMV International
[00212] FMC BioPolymer Na Alginate: Manugel® DMB
[00213] BENEO-ORAFTI Inulin: Orafti®GR
[00214] Maltodextrin: Glucidex IT 12 from Roquette
[00215] Na ascorbate from Northeast Pharmaceutical group Co.,
[00216] Nordic Sugar Sucrose: Granulated Sugar 550
[00217] Remy HC-P starch (pregelatinized rice), baby food grade from Beneo-remy NV
[00218] Newborn powder was EnfaGrow sourced from Mead Johnson LCC, Evansville, IN. EXAMPLE 1: Drying of the LAB composition
[00219] The lactic acid bacteria cell (LAB) was the commercially available Lactobacillus LGG® cell - obtainable from Chr. Hansen A/S, Denmark.
[00220] The vacuum dryer apparatus was an apparatus, in which the heating in the apparatus was so-called radiant heating. The tray was situated between two heating plates, both heating plates providing radiant heat to the tray. Step (a):
[00221] 1 kg of LAB cell concentrate was obtained - it comprises around 10% cell dry matter - this is a so-called 10% concentrate with about 90% water. Step (b):
1 kg of a mixture of protective agents (the mixture comprised 30 g of Sodium Alginate; 50 g of Inulin, 753 g of Trehalose and 167 g of Casein Hydrolysate) was mixed with the LAB cell concentrate.
[00223] Consequently a slurry was obtained which comprised an amount of protective agents around 10 g - in which the amount of protective agents is given in relation to 1 g of lactic acid bacteria cells in the slurry and both the amount of protective agent(s) and lactic acid bacteria cells are measured as dry matter in a slurry. Step (c):
[00224] The slurry was frozen to form solid frozen particles/pellets.
[00225] This was done by the use of liquid nitrogen. Step (d):
[00226] Trays were loaded with 10 kg/m2 of the frozen particles/pellets to obtain the material here relevant on the trays. Step (e):
[00227] The primary drying of the material in the tray was carried out under different vacuum pressures - some were within the vacuum pressure range of 0.7 to 2 millibar (mbar) (eg 1.3 mbar pressure was used) and some were performed outside this range (eg 2.5 mbar pressure was used).
[00228] This step was carried out at a temperature where the material temperature was not higher than 37°C.
[00229] At this maximum temperature significantly less than 50% of the LAB cells were inactivated.
[00230] This step was carried out for a period of time until at least 97% of the slurry water from step (b) was removed - this lasted around 12 hours. Step (f):
[00231] Secondary drying of the material from step (e) was carried out under a vacuum pressure of 0.2 mbar.
[00232] As for step (e) - this step was also carried out at a temperature where the material temperature was not higher than 37°C. This step was carried out over a period of time around 12 hours. Results: Table 1

[00233] From table 1 it is observed that within the products dried at 2.5 mbar the aqueous activity is higher than preferred (< 0.15) whereas the sample dried at 1.3 mbar has an aqueous activity as preferred. Process survival is furthermore higher for the 1.3 mbar sample than for the 2.5 mbar dry samples. Table 2

[00234] From table 2 it is observed that the aqueous activity as well as the process survival are in an unacceptable range when the drying temperature is too high (70°C) whereas there is no difference even on the aqueous activity nor on the survival of the process if the drying temperature is 50 or 60°C. In both cases the values are within an acceptable range.
[00235] For this example - it can be said that the essential parameter here that was varied was in the primary drying step (e) - in which different vacuum pressures were used - some were within the vacuum pressure range of 0, 7 to 2 millibar (mbar) (eg 1.3 mbar pressure was used) and some were performed outside this range (eg 2.5 mbar pressure was used).
[00236] The experimental results essentially demonstrated that when a vacuum pressure outside the range of 0.7 to 2 mbar was used, a satisfactory drying of the LAB composition was not obtained here. The pressure should be selected to be slightly above the formulation transition temperature for the reasons previously explained. The transition temperature of the formulation used in example 1 is about -33°C. At 2.5 mbar the temperature is around -10.5°C which is much higher than the transition temperature. The results in table 1 thus demonstrate that 1.3 mbar is more suitable.
[00237] When the vacuum pressure was within the range of 0.7 to 2 millibar (mbar) (eg 1.3 mbar pressure) then it was possible to do proper drying and efficient drying to obtain the dry formula composition with an aqueous activity (aw) of less than 0.15. Conclusions:
[00238] The results of this Example 1 essentially demonstrate that only by working within the vacuum pressure range of 0.7 to 2 mbar in step (e) that a satisfactory method is obtained here for drying a LAB composition relevant here comprising relatively high amount of protective agents. EXAMPLE 2: Particle size of frozen particles/pellets from step (c)
[00239] An experiment was done essentially as described for example 1 - but in which the vacuum pressure in step (c) was kept constant around a pressure of 1.3 mbar.
[00240] In this experiment the essential variable was the particle size of frozen particles/pellets of step (c).
[00241] Experiments have been done, in which at least 97% of the frozen particles/pellets in step (c) were particles/pellets that were able to pass through a mesh with maximum size opening/holes of different sizes. Results:
[00242] The experimental results essentially demonstrated that when the particle/pellet sizes were above 10 mm then an ideal drying result was not obtained.
[00243] But when the particle/pellet sizes were below 5 mm then very good and efficient drying was obtained. Conclusions:
[00244] The results of this Example 2 essentially demonstrate that it is preferred herein that at least 95% (more preferably at least 97%) of the particles/pellets frozen in step (c) are particles/pellets which are capable of passing through a mesh with 10mm maximum aperture/hole size (preferably with 5mm maximum aperture/hole size). EXAMPLE 3: Drying of other LAB compositions
Example 1 was essentially repeated but using other LAB cells and other protective agents. Drying at pressures of 0.9/0.2 mbar and heating temperatures of 32°C and 37°C was evaluated equally (for formulations with and without alginate) and for both temperatures the aqueous activity of the dry products was < 0 .15 after 24 hours of drying.
[00246] The formulations without alginate and the formulations with sucrose were run as #7 and resulted in dry products with aqueous activity < 0.15 and process survival of ~ 50%. The sucrose formulation resulted in dry products with an aqueous activity <0.3 but by increasing the drying time the aqueous activity could be much lower.
[00247] For step (b) a slurry was obtained in all experiments which comprised an amount of protective agents around 6 g to 15 g - in which the amount of protective agents is given in relation to 1 g of cells of lactic acid bacteria in the slurry and both the amount of protective agent(s) and lactic acid bacteria cells are measured as dry matter in a slurry.
[00248] For all experiments - at least 50% of the protective agents used were saccharides. Conclusions:
[00249] The results of this Example 3 essentially demonstrated the same as in example 1 - this is because only working within the vacuum pressure range of 0.7 to 2 mbar in step (e) that a Satisfactory method here for drying a LAB composition relevant here comprising relatively high amount of protective agents. The exact vacuum pressure range suitable for the individual composition is selected by determining the transition temperature of the composition and correlating it with a water vapor pressure table as explained above.
[00250] It can be said that this Example 3 confirmed this conclusion for different LAB compositions which can be characterized as comprising a relatively high amount of saccharides as protective agents. EXAMPLE 4 Preparation of formulations without alginate
[00251] To one part of LGG® concentrate two parts of demineralized water were added and the concentrate was centrifuged back to the original volume (~1.27x1011 active cells/g). The cell concentrate used below had around 10% cell dry matter - this is a so-called 10% concentrate.
Formulation 1: To 100 g of washed concentrate were added 30 g of sucrose + 17.5 g of maltodextrin (Glucidex IT 12) + 13 g of Na-ascorbate. The mixture was stirred until the additives were dissolved. Afterwards the mixture was dried in vacuum.
Formulation 2: To 100 g of washed concentrate were added 70 g of sucrose + 17.5 g of maltodextrin (Glucidex IT 12) + 13 g of Na-ascorbate. The mixture was stirred until the additives were dissolved. Afterwards the mixture was dried in vacuum.
Formulation 3: To 100 g of washed concentrate were added 37.4 g of sucrose + 60 g of Trehalose + 2.6 g of Na-ascorbate. The mixture was stirred until the additives were dissolved. Afterwards the mixture was dried in vacuum.
Reference 1 (reference formulation): To 100 g of washed concentrate were added 6 g of sucrose + 3.5 g of maltodextrin (Glucidex IT 12) + 2.6 g of Na-ascorbate. The mixture was stirred until the additives were dissolved. Afterwards the mixture was dried in vacuum.
[00256] If the mixtures are dried in a vacuum belt dryer it may be necessary to add a small amount of gelling agent eg pectin to obtain a proper viscosity. EXAMPLE 5
[00257] Testing of alginate-free LGG® formulations in open and powdered newborn bags at 30°C
[00258] The stability of the products was tested in open pouches stored at 30°C and 30% RH and when mixed in powder for newborns with an aqueous activity of 0.3. See stability data in Table 3 and Table 4. As a reference an LGG® containing 20% of the amount of additives in Formulation 1 as outlined in example 4 above is used. The powder was moistened to obtain an aqueous activity of 0.27 to 0.30. Table 3 Storage in open pouches stored at 30°C / 30% RH.

[00259] Samples are measured by Flow Cytometry (active cells/g). Conclusion:
[00260] From table 3 it is observed that the highest loss of % active cells is found in Reference 1. Increasing the amount of protective agents by a factor of 5 stability is significantly increased (comparison of Formulation 1 and Reference 1). Also formulations 2 and 3 demonstrate increased stability. Table 4

[00261] The bags were fluxed with N2 and sealed before storage. Conclusion:
[00262] Newborn powder made with formulations 1 to 3 has better stability than Reference 1. EXAMPLE 6 Testing formulations of L. paracasei subsp. paracasei L. casei 431® without alginate powder for newborn at 35°C
[00263] To a part concentrate of L. paracasei subsp. paracasei L. casei 431® (LC 431) two parts of demineralized water were added and the concentrate was centrifuged back to the original volume (~1.27x1011 active cells/g). The cell concentrate used below had around 10% cell dry matter - this is a so-called 10% concentrate.
Formulation 1LC 431: To 100 g of washed concentrate were added 30 g of sucrose + 17.5 g of maltodextrin (Glucidex IT 12) + 13 g of Na-ascorbate. The mixture was stirred until the additives were dissolved. Subsequently, the mixture was pelleted in liquid nitrogen before it was vacuum-dried.
[00265] The mixture was added to the newborn powder and the stability of the product tested in a newborn powder with an aqueous activity of 0.3 stored at 30% RH at 35°C. See stability data in table 3. As a reference, newborn powder containing LC 431 in Reference 1 as outlined in example 4 above is used. Table 5

[00266] The bags were fluxed with N2 and sealed before storage. Conclusion:
[00267] From table 5 and Figure 1 it is observed that also for L. casei 431® and at a storage temperature of 35°C the highest loss of % active cells is found in Reference 1. Increasing the amount of protective agents by a factor of 5 stability is significantly increased also for L. casei 431®. EXAMPLE 7 Testing of six formulations of L. paracasei subsp. paracasei L. casei 431® different without alginate powder for newborn at 35°C
[00268] To one part of L. casei 431® concentrate two parts of demineralized water were added and the concentrate centrifuged back to the original volume (~1.27x1011 active cells/g). The cell concentrate used below had around 10% cell dry matter - this is a so-called 10% concentrate. The mixture was stirred until the additives were dissolved. Subsequently, the mixture was pelleted in liquid nitrogen before it was vacuum-dried.
[00269] Composition 1: To 1000 g of washed concentrate were added 753 g of trehalose + 191 g of maltodextrin + 26 g of Na ascorbate + 30 g of Remy HC-P.
Composition 2: To 1000 g of washed concentrate were added 753 g of trehalose + 50 g of inulin + 167 g of hydrolyzed casein.
Composition 3: To 1000 g of washed concentrate were added 366 g of trehalose + 213 g of maltodextrin + 26 g of Na-ascorbate.
[00272] Composition 6: To 1000 g of washed concentrate were added 615 g of trehalose + 358 g of maltodextrin + 26 g of Na-ascorbate.
[00273] Composition 7: To 1000 g of washed concentrate were added 459 g of trehalose + 267 g of maltodextrin + 26 g of Na-ascorbate.
Composition 8: To 1000 g of washed concentrate were added 213 g of trehalose + 124 g of maltodextrin + 26 g of Na-ascorbate.
[00275] The various compositions were added to the newborn powder and the stability of the products tested in the newborn powder with an aqueous activity of 0.3 stored at 30% RH at 35°C in sealed bags fluxed with N2. See stability data in table 4 and figure 2. As a reference, newborn powder containing L. casei 431® in Reference 1 described in example 4 above is used. Table 6
Conclusion:
[00276] From table 6 and Figure 2 it is observed that for L. casei 431® and at a storage temperature of 35°C the highest loss of % active cells is found in Reference 1. All tested compositions and in particular composition 2 demonstrates substantially improved stability. EXAMPLE 8 Comparison of compositions with and without alginate for L. paracasei subsp. paracasei (LC 431) and for LGG®
[00277] 10% concentrates of LGG and L. paracasei subsp. paracasei (LC 431) were prepared as described above and added to composition 2 or alginate composition 2 (see below). Two different LGG® concentrates and two different L. casei 431® concentrates were used in order to test reproducibility. In addition, two different formulations were prepared, the difference between the two formulations being that composition 2 does not contain alginate whereas composition 2 alginate contains 30 g of Na alginate per kg of washed concentrate. The mixture was stirred until the additives were dissolved and frozen in liquid nitrogen. Afterwards the mixture was dried in vacuum.
[00278] Composition 2 alginate: To 1000 g of washed concentrate were added 753 g of trehalose + 50 g of inulin + 167 g of hydrolyzed casein + 30 g of Na alginate. Table 7a Storage stability data (log (CFU/g) after storage at 35°C in powder for newborn with aw: 0.3 in sealed bags
Table 7b Storage stability data (log (CFU/g) after storage at 40°C in powder for newborn with aw: 0.3 in sealed pouches.
Conclusion:
[00279] From tables 7a and 7b and Figures 3 to 6 it is shown that there is substantially no difference between compositions with or without Na alginate with regard to stability. EXAMPLE 9 Heat treatment of LGG® compositions with and without alginate
10% LGG® concentrates were prepared and added to composition 2 or alginate composition 2 as described in example 8 above. As heat treatment is relevant for production scale products two of the compositions were subjected to heat treatment in order to compare the stability of the compositions with or without alginate. As a reference, newborn powder containing LGG® in Reference 1 described in example 4 above is used. Table 8 Storage stability data (log (CFU/g) after storage at 35°C in powder for newborn with aw: 0.25 in sealed bags
Conclusion:
[00281] From table 8 and Figure 7 it is observed that for the composition with sodium alginate the heat treatment has a negative impact on stability.

权利要求:
Claims (12)
[0001]
1. Stable dry composition of lactic acid bacteria cells, characterized in that it comprises: (i) from 109 to 1013 cfu of lactic acid bacteria cells per gram of composition, and (ii) protective agents in effective amounts to stabilizing the lactic acid bacteria in the composition comprising: (iii) from 2 to 9 g sucrose per gram of the lactic acid bacteria cells in the composition; (iv) from 1 to 3 g of maltodextrin per gram of the lactic acid bacteria cells in the composition; and (v) i) from 0.75 to 2 g of Na-ascorbate per gram of lactic acid bacteria cells in the composition.
[0002]
2. Dry composition according to claim 1, characterized in that the lactic acid bacteria is at least one selected from the group consisting of: Lactococcus species, Streptococcus species, Enterococcus species, Lactobacillus species, Leuconostoc, Bifidobacterium species, Propioni species and Pediococcus.
[0003]
3. Dry composition according to claim 1 or 2, characterized in that the lactic acid bacteria is at least one selected from the group consisting of: Lactobacillus rhamnosus, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Leuconostoc lactis, Leuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactobacillus casei subsp. casei, Streptococcus thermophilus, Enterococcus faecum, Bifidobacterium animalis, Bifidobacterium lactis, Bifidobacterium longum, Lactobacillus lactis, Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus.
[0004]
4. Dry composition according to any one of claims 1 to 3, characterized in that the lactic acid bacterium is at least one lactic acid bacterium selected from the group consisting of Bifidobacterium animalis subsp lactis deposited as DSM 15954, Bifidobacterium animalis subsp lactis deposited as ATCC 27536, Bifidobacterium animalis subsp lactis deposited as DSM 10140, Lactobacillus acidophilus deposited as DSM 13241, Lactobacillus rhamnosus deposited as ATCC 53103, Lactobacillus rhamnosus deposited as ATCC 55826, Lactobacillus reutericase deposited as Lactospillus 558. paracasei deposited as ATCC 55544, Lactobacillus paracasei deposited as LMG-17806, Streptococcus thermophilus deposited as DSM 15957, Lactobacillus fermentum deposited as NM02/31074, and Lactobacillus paracasei subsp. paracasei deposited as CCTCC M204012.
[0005]
5. Dry composition according to any one of claims 1 to 4, characterized in that the weight of the dry composition is from 50 g to 10,000 kg.
[0006]
6. Dry composition, according to any one of claims 1 to 5, characterized by the fact that the lactic acid bacteria in the composition are stable when stored at 30°C and in 30% relative humidity.
[0007]
7. Dry composition according to any one of claims 1 to 6, characterized in that the lactic acid bacteria in the composition exhibit improved stability when stored at 30°C and at 30% relative humidity compared to a composition comparable lacking effective amounts of protective agents.
[0008]
8. Dry composition according to any one of claims 1 to 7, characterized in that the aqueous activity (aw) of the dry composition is less than 0.30.
[0009]
9. Newborn powder, characterized in that it comprises a composition as defined in any one of claims 1 to 8.
[0010]
10. Newborn formula, follow-on formula, growth milk or special formula or nutritional product for newborns and children to improve their intestinal microflora while simultaneously providing nutrition to the newborn or child, characterized by the fact which comprises the newborn powder as defined in claim 9.
[0011]
11. Food product, characterized in that it comprises a composition as defined in any one of claims 1 to 8.
[0012]
12. Dietary supplement, characterized in that it comprises a composition as defined in any one of claims 1 to 8.

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CN107668211B|2017-11-13|2021-08-10|海普诺凯营养品有限公司|Infant formula goat milk powder for enhancing immune function and preparation method thereof|

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IT201800003128A1|2018-02-28|2019-08-28|Propharma Soc A Responsabilita Limitata Semplificata|PROBIOTIC COMPOSITION|

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AU2019366968A1|2018-10-26|2021-05-27|Danisco Us Inc|Stable microbial composition and drying process|

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CN113025540B|2021-05-28|2021-10-01|中国食品发酵工业研究院有限公司|Lactobacillus agent for fermenting soy protein, preparation method, fermentation method and fermentation product with muscle increasing function|

法律状态:
2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: A61K 35/747 (2015.01), A23L 29/00 (2016.01), A23L |

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-07-09| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-10-08| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2020-09-08| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|

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2021-01-19| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2021-04-20| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-06-29| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 30/06/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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EP11172132.0|2011-06-30|

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EP11172132|2011-06-30|

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EP11172697.2|2011-07-05|

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EP11172697|2011-07-05|

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EP11191955|2011-12-05|

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EP11191955.1|2011-12-05|

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PCT/EP2012/062787|WO2013001089A1|2011-06-30|2012-06-30|Novel compositions|

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