Method for producing kefir grain analogs and method for producing fermented milk using kefir grain analogs

Abstract

To provide a method for producing a kefir grain analogue having a bacterial flora equivalent to that of a natural kefir grain using a kefir grain isolated stock and usable for producing fermented milk.SOLUTION: A kefir grain isolated stock belonging to one or more selected from a group consisting of Lactobacillus kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, and Lentilactobacillus kefiri, and if necessary Lentilactobacillus parakefiri, Lactococcus lactis, Leuconostoc mesenteroides, and yeast is mixed, and cultured in a liquid medium or a solid medium to generate a proto-kefir grain. If necessary, another kefir grain isolated stock necessary for symbiosis is added and planted to succeed, thereby generating a kefir grain analogue. A method for producing a kefir grain analogue from a kefir grain isolated stock is also provided. In addition, a method for producing fermented milk using the kefir grain analogue is provided.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing a kefir grain analog and a method for producing fermented milk using the kefir grain analog. Specifically, it relates to a method for artificially creating a kefir grain analog that functions equivalently to natural kefir grains used in the production of kefir. More specifically, it provides a method for purely separating the microorganisms constituting kefir grains, and relates to a method for creating a kefir grain analog having the same function as natural kefir grains by using the strains separated by this method in combination.

Background Art

[0002] Kefir grains are lumps of elastic microorganisms ranging in size from a few millimeters to a few centimeters, with a milky white cauliflower-like appearance. Kefir grains are symbiotic with special lactic acid bacteria and microorganisms such as yeast, and grow like a single type of microorganism in milk. By fermenting milk or other mammalian milk using this as a starter culture, fermented milk called kefir can be produced.

[0003] The origin of kefir grains is said to be from the mountainous people in the northern Caucasus. Subsequently, kefir spread mainly in Russia and Eastern European countries, but now it is consumed worldwide. Accordingly, kefir grains have also spread around the world and are now easily available through the Internet and the like.

[0004] The main microbial species that make up kefir grains have been isolated and identified. The main microorganisms that make up kefir grains are known as Lactobacillus kefiranofaciens, Lentilactobacillus kefiri, Lentilactobacillus parakefiri, Lactococcus lactis, lactic acid bacteria of the genus Leuconostoc, other lactic acid bacteria, various yeasts, and acetic acid bacteria as lactic acid bacteria (see Non-Patent Document 1). L. kefiranofaciens is further classified into two subspecies: L. kefiranofaciens subsp. kefiranofaciens and L. kefiranofaciens subsp. kefirgranum.

[0005] In this specification, "L. kefiranofaciens" means L. kefiranofaciens subsp. kefiranofaciens and / or L. kefiranofaciens subsp. kefirgranum. Also, in this specification, "L. kefiranofaciens subsp. kefirgranum" means all L. kefiranofaciens other than L. kefiranofaciens subsp. kefiranofaciens that form characteristic viscous colonies among L. kefiranofaciens. The characteristics of these bacteria are described in detail in Bergey’s Manual (Non-Patent Document 2) and the like.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

[0007] However, there are different views among microbiologists regarding the essential flora that is invariably present in kefir grains. Also, since the bacterial species required for grain formation have not been determined, there is currently no report of artificially producing kefir grains using isolated microorganisms.

[0008] Therefore, to produce kefir, natural kefir grains must be obtained. However, the flora of natural kefir grains varies greatly depending on the source, and the quality of the kefir produced using them may be poor. The main cause is the variation in the types of lactic acid bacteria and yeast flora that make up the grains, and there may also be cases where the kefir is contaminated with microorganisms that deteriorate the flavor.

[0009] Therefore, if a method can be established to separate lactic acid bacteria and yeast from natural kefir grains and reconstruct kefir grains using the obtained isolates, problems such as variations in the flavor of kefir and quality deterioration due to contamination can be solved.

[0010] An object of the present invention is to provide a method for constructing a kefir grain analog having a bacterial flora equivalent to that of natural kefir grains and usable for the production of fermented milk by elucidating the mechanism of kefir grain formation using strains isolated from natural kefir grains.

Means for Solving the Problems

[0011] To solve the above problems, the present inventors first performed a bacterial flora analysis of seven types of natural kefir grains derived from Denmark, Belgium, Russia, the United Kingdom, Germany, Japan, and Turkey, identified seven major microorganisms commonly present in all grains, and preserved them as kefir grain isolates.

[0012] Microorganisms commonly present in all kefir grains were Lactobacillus kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri of lactic acid bacteria, and Kazachstania exigua of yeast. In addition, bacterial species detected in most kefir grains and strongly presumed to be commonly present were Leuconostoc mesenteroides subsp. mesenteroides, L. parakefiri, and Lactococcus lactis. The present inventors considered that only the bacterial species commonly present in these kefir grains and the bacterial species detected in most kefir grains must play an essential role in kefir grain formation. Although there are reports claiming the importance of acetic acid bacteria in the role of kefir grain formation, the present inventors considered that acetic acid bacteria were not involved in kefir grain formation because they could not be detected in all kefir grains.

[0013] The inventors investigated the behavior of mixing and culturing L. kefiranofaciens subsp. kefiranofaciens and L. kefir, which are the main flora of kefir grains, among these seven bacterial species. When L. kefiranofaciens subsp. kefiranofaciens and L. kefiri were cultured in reduced whey, it was found that no aggregates were formed when cultured alone, but when mixed and cultured, bacterial masses that could be recovered with a wire mesh were formed. Subsequently, when L. kefiranofaciens subsp. kefiranofaciens, L. kefiri, L. kefiranofaciens subsp. kefirgranum, etc. were mixed and grown on the surface of a solid medium, a special colony with adhesiveness and plasticity was formed. When this colony was cultured in reduced skim milk, it was first found that bacterial masses (hereinafter sometimes referred to as "proto-kefir grains") very similar to kefir grains were produced in the milk. The proto-kefir grains could be filtered and recovered with a wire mesh in the same manner as natural kefir grains. By repeating operations such as subculturing using the proto-kefir grains as a primordium, if they grow in the same manner as natural kefir grains, they can be developed into kefir grains equivalent to natural ones.

[0014] Thus, it has never been known until now that proto-kefir grains can be produced using only L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, etc. as constituent microorganisms. Also, it was found that when L. kefiranofaciens subsp. kefiranofaciens is not included as a constituent microorganism, even if L. kefiranofaciens subsp. kefirgranum is included, proto-kefir grains do not form.

[0015] As a result of intensive research using these isolates, the inventors have found that protokefir grains containing L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, Lactococcus lactis, etc. can be propagated to have a bacterial flora similar to kefir grains, and a kefir grain analogue that can maintain its shape without the grains collapsing or disappearing can be constructed.

[0016] It has been found that the kefir grain analogue thus prepared can stably ferment milk and can be used as a starter for fermented milk in the same manner as natural kefir grains. Based on the above findings, the inventors have completed the present invention.

[0017] That is, the present invention relates to the following method. (1) A method for producing a kefir grain analogue from a kefir grain isolate, comprising mixing kefir grain isolates belonging to one or more selected from the group consisting of Lactobacillus kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, Lentilactobacillus kefiri, and, if necessary, Lentilactobacillus parakefiri, Lactococcus lactis, Leuconostoc mesenteroides, and yeast, culturing them in a liquid medium or a solid medium to form a kefir grain-like mass (protokefir grain), and, if necessary, adding other kefir grain isolates necessary for symbiosis and propagating them to form a kefir grain analogue. (2) Step 1 of culturing a bacterial solution containing Lactobacillus kefiranofaciens and, if necessary, kefir grain isolates of other bacterial species on the surface of a solid medium to form colonies, Collect the colony and inoculate it into a liquid medium. Optionally, further inoculate the liquid medium with a kefir grain isolate and culture it at least once to form proto-kefir grains in the liquid medium (Step 2). Successively transfer the proto-kefir grains multiple times using a liquid medium. Optionally, further add a kefir grain isolate to form a kefir grain analogue (Step 3). A method for producing a kefir grain analogue from a kefir grain isolate, characterized by comprising the above steps. (3) The method according to (2) above, wherein the Lactobacillus kefiranofaciens is Lactobacillus kefiranofaciens subsp. kefiranofaciens alone or a mixture of Lactobacillus kefiranofaciens subsp. kefiranofaciens and Lactobacillus kefiranofaciens subsp. kefirgranum. (4) The method according to (2) or (3) above, wherein the "kefir grain isolate of another bacterial species" in Step 1 belongs to one or more selected from the group consisting of Lactobacillus kefiranofaciens subsp. kefirgranum, Lentilactobacillus kefiri, Lentilactobacillus parakefiri, Leuconostoc mesenteroides, Lactococcus lactis, and yeast. Mixing kefir grain isolates belonging to one or more selected from the group consisting of (5) Lactobacillus kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, Lentilactobacillus kefiri, and, if necessary, Lentilactobacillus parakefiri, Lactococcus lactis, Leuconostoc mesenteroides, and yeast, and culturing in a liquid medium or a solid medium to form proto-kefir grains, and, if necessary, adding other kefir grain isolates necessary for symbiosis and propagating to form a kefir grain analogue, and producing fermented milk using the kefir grain analogue. A method for producing fermented milk, characterized by the above. (6) Step 1 of culturing a bacterial solution containing Lactobacillus kefiranofaciens and, if necessary, a kefir grain isolate of another bacterial species on the surface of a solid medium to form colonies; Step 2 of collecting the colonies and inoculating them into a liquid medium, and, if necessary, further inoculating the liquid medium with a kefir grain isolate and culturing at least once to form proto-kefir grains in the liquid medium; Step 3 of subculturing the proto-kefir grains multiple times using a liquid medium and, if necessary, further adding a kefir grain isolate to form a kefir grain analogue; Step of producing fermented milk using the kefir grain analogue; A method for producing fermented milk, characterized by including the above steps. (7) Mixing kefir grain isolates belonging to one or more selected from the group consisting of Lactobacillus kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, Lentilactobacillus kefiri, and, if necessary, Lentilactobacillus parakefiri, Lactococcus lactis, Leuconostoc mesenteroides, and yeast, and culturing them in a liquid medium or a solid medium to form probiotic kefir grains, and, if necessary, adding other kefir grain isolates necessary for symbiosis and propagating them to form kefir grain analogs, and propagating the kefir grain analogs to form artificial kefir grains indistinguishable from natural kefir grains. A method for producing artificial kefir grains, characterized by the above steps. (8) Step 1 of culturing a bacterial solution containing Lactobacillus kefiranofaciens and, if necessary, kefir grain isolates of another bacterial species on the surface of a solid medium to form colonies; Step 2 of collecting the colonies and inoculating them into a liquid medium, and, if necessary, further inoculating the liquid medium with kefir grain isolates and culturing at least once to form probiotic kefir grains in the liquid medium; Step 3 of propagating the probiotic kefir grains multiple times using a liquid medium and, if necessary, further adding kefir grain isolates to form kefir grain analogs; Step of propagating the kefir grain analogs to form artificial kefir grains indistinguishable from natural kefir grains; A method for producing artificial kefir grains, characterized by including the above steps.

Advantages of the Invention

[0018] According to the present invention, by elucidating the mechanism of the formation of kefir grains, it is possible to provide a method for producing kefir grain analogs having a microbial flora equivalent to that of natural kefir grains and usable for the production of fermented milk.

[0019] In addition, the kefir grain analogs obtained by the present invention have the following advantages. (1) Although they do not grow in milk as single microorganisms, they can stably ferment milk as kefir grain analogs and can be used as starters for fermented milk. (2) Even without propagating kefir grains, if there are preserved isolates, kefir grain analogs can be reconstructed and produced at any time. (3) The flavor and physical properties of fermented milk can be modified by selecting the strains constituting the kefir grain analogs.

Brief Description of the Drawings

[0020]

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Mode for Carrying Out the Invention

[0021] Hereinafter, this embodiment will be described in detail. In the following, “%” means weight % (W / W) unless otherwise specified.

[0022] 〔Method for Producing Kefir Grain Analogs〕 The method for producing kefir grain analogs from kefir grain isolates according to this embodiment is to mix specific kefir grain isolates and culture them in a liquid medium or a solid medium to form a kefir grain-like bacterial mass (protokefir grains), and if necessary, add other kefir grain isolates necessary for symbiosis and subculture to form kefir grain analogs.

[0023] “Kefir grain isolate” refers to lactic acid bacteria or yeast that are stored as a single strain by separating and culturing from natural kefir grains. The kefir grain isolates are not limited to the strains deposited in the strain preservation institution.

[0024] In this embodiment, as the kefir grain isolates to be initially mixed and cultured, “specific” kefir grain isolates, which have been clarified by the present inventors to be essential for the formation of protokefir grains, are used.

[0025] When the specific kefir grain isolate is used, it belongs to at least three species: Lactobacillus kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, and Lentilactobacillus kefiri. Preferably, in addition to these three species, microorganisms belonging to one or more selected from the group consisting of Lentilactobacillus parakefiri, Lactococcus lactis, Leuconostoc mesenteroides, and yeast can be used. As for yeast, if it is a kefir grain isolate, the species is not limited, but preferably, yeast belonging to the genus Kazachstania, particularly Kazachstania exigua, can be used.

[0026] Specifically, the following combinations of kefir grain isolates of different species can be exemplified. · L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, and L. parakefiri. · L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, and Lactococcus lactis. · L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, and Leuconostoc mesenteroides. · L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, and Lactococcus lactis. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, Lactococcus lactis, and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, Lactococcus lactis, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, Leuconostoc mesenteroides, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, Lactococcus lactis, and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, Lactococcus lactis, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, Leuconostoc mesenteroides, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, Lactococcus lactis, Leuconostoc mesenteroides, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, Lactococcus lactis, Leuconostoc mesenteroides, and yeast.

[0027] In the present embodiment, the mixed culture of the above kefir grain isolates is performed using a liquid medium or a solid medium. Examples of the "liquid medium" include (reduced) whey, (reduced) skim milk, animal milk such as cow's milk, and acid whey. Examples of the "solid medium" include the M agar medium, W agar medium, RMW medium, RSMP medium, etc., which will be described later.

[0028] The mixed culture of the kefir grain isolates using the above liquid medium or solid medium can be started by mixing the bacterial solutions of the above specific bacterial species and inoculating the medium therewith. The bacterial solution may contain a plurality of strains of the same bacterial species. The concentration of each bacterial species (strain) contained in the bacterial solution is at least 10 6It is preferably set at CFU / ml or more.

[0029] The inoculum amount of the bacterial solution to the above liquid medium or solid medium is not particularly limited, but usually, it can be about 0.1 to 1 ml with respect to 200 mL of the liquid medium, and about 30 μl at three locations with respect to the solid medium having an inner diameter of 8 cm. Further, as the culture conditions, it can be about 1 to 4 days at 20 to 30°C.

[0030] In the case of the liquid medium, by performing mixed culture in this way, protokefir grains can be formed. The protokefir grains are not coagulated milk components, but are mainly composed of a symbiotic body of lactic acid bacteria, and have elasticity and a viscosity and hardness such that they can be filtered through a wire mesh.

[0031] On the other hand, in the case of the solid medium, by performing mixed culture as described above, first, colonies that become the nuclei for forming protokefir grains are formed. Since these colonies are viscous and plastic, they are collected by peeling them from the surface of the solid medium with a spatula or the like for each colony. Next, the obtained colonies are inoculated into a liquid medium and, if necessary, cultured together with other kefir grain isolates to form protokefir grains in the liquid medium. As the liquid medium, those described above can be used.

[0032] In the present embodiment, next, kefir grain analogs are produced by subculturing protokefir grains in a liquid medium. The subculture interval is preferably about 1 to 2 days, until coagulation in the case of acid coagulation, and less than 4 days in the case of non-acid coagulation. As the subculture method, protokefir grains can be recovered from the culture using a wire mesh or the like and inoculated into a new liquid medium. As the liquid medium, those described above can be used. The culture conditions may be the same as above.

[0033] If necessary, when propagating, add a bacterial solution of other kefir grain isolates necessary for symbiosis. The bacterial solution to be added here can contain one or more bacterial solutions selected from the group consisting of L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, Leuconostoc mesenteroide, Lactococcus lactis, and yeast. The bacterial solution may contain multiple strains of the same bacterial species. The concentration of each bacterial species (strain) contained in the bacterial solution is preferably at least 10 6 CFU / ml or more. This bacterial solution may contain the same bacterial species (strain) as the bacterial solution used in the aforementioned first mixed culture.

[0034] By repeating the propagation in the above-described manner, a kefir grain analog can be obtained. Whether it is a "kefir grain analog" can be determined by whether the grain does not collapse or disappear and can continue to maintain its shape. For example, if the wet weight of the bacterial mass after repeating the propagation 10 times at 22°C has increased by 10% or more compared to the wet weight of the initial protokefir grain, it can be said that the shape of the grain is maintained, and thus the bacterial mass can be determined to be a "kefir grain analog".

[0035] FIG. 1 is a diagram for explaining a method of creating a kefir grain analog from a kefir grain isolate according to the present embodiment. The flow of this method will be described by dividing it into the following three steps.

[0036] That is, the method of the present embodiment is characterized by including Step 1 for forming a colony that becomes the nucleus of protokefir grain formation, Step 2 for forming protokefir grain, and Step 3 for forming a kefir grain analog.

[0037] "Step 1" is a step of culturing a bacterial solution (bacterial solution 1) containing Lactobacillus kefiranofaciens and, if necessary, a kefir grain isolate of another bacterial species on the surface of a solid medium to form colonies.

[0038] First, "bacterial solution 1" containing at least L. kefiranofaciens is dropped at several locations on the surface of the solid medium. For example, in the case of a petri dish with an inner diameter of 8 cm, about 30 μl is dropped at about 3 locations.

[0039] "Bacterial solution 1" only needs to contain at least L. kefiranofaciens subsp. kefiranofaciens, and preferably contains a mixture of L. kefiranofaciens subsp. kefiranofaciens and L. kefiranofaciens subsp. kefirgranum.

[0040] "Bacterial solution 1" may further contain one or more bacterial solutions selected from the group consisting of L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, Lactococcus lactis, Leuconostoc mesenteroides, and yeast as "kefir grain isolates of another bacterial species" necessary for the formation of proto-kefir grains.

[0041] Specific examples of "bacterial solution 1" can be combinations of kefir grain isolates of the following bacterial species. · Only L. kefiranofaciens subsp. kefiranofaciens. · L. kefiranofaciens subsp. kefiranofaciens and L. kefiranofaciens subsp. kefirgranum. · L. kefiranofaciens subsp. kefiranofaciens and L. kefiri. ·L. kefiranofaciens subsp. kefiranofaciens and L. parakefiri. ·L. kefiranofaciens subsp. kefiranofaciens and Lactococcus lactis. ·L. kefiranofaciens subsp. kefiranofaciens and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens and yeast. ·L. kefiranofaciens subsp. kefiranofaciens and L. kefiranofaciens subsp. kefirgranum and L. kefiri. ·L. kefiranofaciens subsp. kefiranofaciens and L. kefiranofaciens subsp. kefirgranum and L. parakefiri. ·L. kefiranofaciens subsp. kefiranofaciens and L. kefiranofaciens subsp. kefirgranum and Lactococcus lactis. ·L. kefiranofaciens subsp. kefiranofaciens and L. kefiranofaciens subsp. kefirgranum and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens and L. kefiranofaciens subsp. kefirgranum and yeast. ·L. kefiranofaciens subsp. kefiranofaciens and L. kefiri and L. parakefiri. ·L. kefiranofaciens subsp. kefiranofaciens and L. kefiri and Lactococcus lactis. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiri, and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiri, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. parakefiri, and Lactococcus lactis. ·L. kefiranofaciens subsp. kefiranofaciens, L. parakefiri, and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens, L. parakefiri, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, Lactococcus lactis, and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens, Lactococcus lactis, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, Leuconostoc mesenteroides, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, and L. parakefiri. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, and Lactococcus lactis. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. parakefiri, and Lactococcus lactis. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. parakefiri, and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. parakefiri, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, Lactococcus lactis, and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, Lactococcus lactis, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, Leuconostoc mesenteroides, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiri, L. parakefiri, and Lactococcus lactis. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiri, L. parakefiri, and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiri, L. parakefiri, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiri, Lactococcus lactis, and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiri, Lactococcus lactis, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiri, Leuconostoc mesenteroides, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. parakefiri, Lactococcus lactis, and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens, L. parakefiri, Lactococcus lactis, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. parakefiri, Leuconostoc mesenteroides, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, Lactococcus lactis, Leuconostoc mesenteroides, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, and Lactococcus lactis. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, Lactococcus lactis, and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, Lactococcus lactis, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, Leuconostoc mesenteroides, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. parakefiri, Lactococcus lactis, and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. parakefiri, Lactococcus lactis, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. parakefiri, Leuconostoc mesenteroides, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, Lactococcus lactis, Leuconostoc mesenteroides, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiri, L. parakefiri, Lactococcus lactis, and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiri, L. parakefiri, Lactococcus lactis, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiri, L. parakefiri, Leuconostoc mesenteroides, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiri, Lactococcus lactis, Leuconostoc mesenteroides, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. parakefiri, Lactococcus lactis, Leuconostoc mesenteroides, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, Lactococcus lactis, and Leuconostoc mesenteroides. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, Lactococcus lactis, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, Leuconostoc mesenteroides, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, Lactococcus lactis, Leuconostoc mesenteroides, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. parakefiri, Lactococcus lactis, Leuconostoc mesenteroides, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiri, L. parakefiri, Lactococcus lactis, Leuconostoc mesenteroides, and yeast. ·L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, Lactococcus lactis, Leuconostoc mesenteroides, and yeast.

[0042] The bacterial solution may contain multiple strains of the same bacterial species. The concentration of each bacterial species (strain) contained in the bacterial solution is preferably at least 10 6 CFU / ml or more.

[0043] The "solid medium" that can be used is as described above. Subsequently, anaerobic culture is carried out at 20 - 30°C, preferably 22°C, for about 5 - 10 days.

[0044] "Step 2" is a step of collecting the colonies obtained in Step 1, inoculating them into a liquid medium, and if necessary, further inoculating the liquid medium with a kefir grain isolate (bacterial solution 2), culturing at least once, and forming proto-kefir grains in the liquid medium.

[0045] First, since the colonies are viscous and plastic, they are collected by peeling them off the surface of the solid medium with a spatula or the like for each colony. This is inoculated into about 200 ml of liquid medium. The "liquid medium" that can be used is as described above. Specifically, 10% (W / V) reduced skim milk or the like sterilized in boiling warm water for about 15 minutes can be used.

[0046] If necessary, "Bacterial liquid 2" can also be inoculated into the liquid medium. "Bacterial liquid 2" can contain a bacterial liquid of one or more kinds among L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, Leuconostoc mesenteroide, Lactococcus lactis and yeast. The bacterial liquid may contain a plurality of strains of the same bacterial species. The concentration of each bacterial species (strain) contained in the bacterial liquid is preferably at least 10 6 CFU / ml or more. Also, "Bacterial liquid 2" may contain the same bacterial species (strain) as "Bacterial liquid 1".

[0047] Subsequently, in "Step 2", the liquid medium inoculated with the colonies is cultured at 20 to 30 °C, preferably 22 °C for about 1 to 4 days. The criterion for the end of the culture is that in the case of skim milk, if it has acid coagulated, it ends at that time, and if it has not coagulated, the culture is stopped after about 4 days. When using whey or the like, it is judged by visually confirming the growth status. The first protokefir grains can be recovered by filtering the culture with a wire mesh or the like.

[0048] "Step 3" is a step of subculturing the protokefir grains obtained in Step 2 a plurality of times using a liquid medium, and adding a kefir grain isolate (bacterial liquid 3) as necessary to form a kefir grain analog.

[0049] In Step 3, the probiotic kefir grains can be grown and stabilized by subculturing them in a liquid medium. As a practical matter, once the probiotic kefir grains can be formed, then by adding the other necessary bacterial species (bacterial solution 3) and subculturing, they can stably grow to obtain a kefir grain analog similar to natural kefir grains.

[0050] The subculture interval is preferably about 1 to 2 days until acid coagulation occurs if acid coagulation occurs, or less than 4 days if acid coagulation does not occur. As a method of subculture, the probiotic kefir grains can be recovered from the culture using a wire mesh or the like and inoculated into a new liquid medium. The liquid medium and culture conditions that can be used are the same as in Step 2.

[0051] If necessary, microorganisms to be further symbiotically associated can be added as "bacterial solution 3" during subculture. "Bacterial solution 3" can contain one or more bacterial solutions of L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, Leuconostoc mesenteroide, Lactococcus lactis, and yeast. The bacterial solution may contain multiple strains of the same bacterial species. The concentration of each bacterial species (strain) contained in the bacterial solution is preferably at least 10 6 CFU / ml or more. Also, "bacterial solution 3" may contain the same bacterial species (strain) as "bacterial solution 1" or "bacterial solution 2".

[0052] By repeating subculture about 10 times by the above method, a kefir grain analog can be obtained. The criteria for determining whether it is a "kefir grain analog" are as described above.

[0053] The kefir grain analog thus constructed is somewhat different morphologically from natural kefir grains, but has a similar flora, can grow by propagation, and can be recovered by filtration using a wire mesh, so it can be used as a starter in the production of fermented milk.

[0054] 〔Method for producing fermented milk〕 That is, according to this embodiment, a method for producing fermented milk using a kefir grain analog is provided. The method for producing fermented milk can be carried out in the same manner as the general method for producing kefir, except that a kefir grain analog is used instead of natural kefir grains. For example, about 3% of the kefir grain analog is inoculated into milk or other mammalian milk or their skim milk, and after fermenting at about 22°C for about 18 hours, the kefir grain analog is filtered off to produce fermented milk.

[0055] 〔Natural kefir grains as the separation source〕 The "natural kefir grains" used as the separation source of the kefir grain isolate in this embodiment can be purchased commercially available ones. The "natural kefir grains" are kefir grains for fermented milk (also called daily kefir grains), and kefir grains of Caucasian origin are preferred. On the other hand, kefir grains mainly composed of acetic acid bacteria, such as water kefir grains or sugar kefir grains, cannot be used as the separation source in this embodiment.

[0056] Normal "natural kefir grains" increase in weight by several percent to several tens of percent in a single propagation in milk. If the weight does not seem to increase, it is necessary to propagate many times until the weight increases stably. If there is no increase in weight even after propagating 10 times or more, it cannot be used as the separation source.

[0057] 〔Propagation method〕 The propagation of natural kefir grains (as well as kefir grain analogs obtained according to this embodiment and protokefir grains which are their precursors) as the separation source can be carried out by conventional methods. An example of the propagation method is shown below.

[0058] All operations need to be carried out hygienically using instruments that have been sterilized, such as dry heat sterilization. First, inoculate fresh kefir grains into sterilized 10% (W / V) reduced skim milk (sterilized in boiling warm water at about 90 °C for 10 - 30 minutes and cooled to around 22 °C before use). It is advisable to use about 20 - 50 times the amount of reduced skim milk relative to the wet weight of the kefir grains. When cultured for 1 to 2 days, it will coagulate due to acid, so stir this and filter it through a stainless steel wire mesh (about 16 - 40 meshes), then the kefir grains will remain on the wire mesh and can be recovered. The filtered liquid part (the liquid that passed through the wire mesh) is fermented milk (kefir). Also, this filtered fermented milk can be used as a bulk starter to ferment a larger amount of milk. The kefir grains remaining on the wire mesh are propagated to the next batch of milk and maintained.

[0059] The kefir grains can be propagated daily by the above method, or can be maintained permanently by propagating at least once a week. For normal kefir grains, the weight increases by a few percent each time they are propagated. When storing kefir grains for a long time, an appropriate amount of sterilized 80% glycerin can be added and stored frozen at about -80 °C. The kefir grain analogs of this embodiment or protokefir grains which are their precursors can be handled in the same way as the natural kefir grains described above.

[0060] [Separation of lactic acid bacteria and yeast from natural kefir grains] The method for separating constituent microorganisms from natural kefir grains will be described below. First, fresh kefir grains are washed with sterilized physiological saline or sterilized skim milk, and then crushed with a sterilized ultra-disperser or a stomacher to form a uniform dispersion. Although it is difficult to achieve complete emulsification, it is not a problem for implementation as long as it becomes approximately uniform. Kefir grains generally contain lactic acid bacteria at a level of 10 9 CFU / g and yeast at a level of 10 4 to 10 6 CFU / g. Therefore, the uniform dispersion is diluted with sterilized physiological saline to an appropriate concentration and spread on the following media.

[0061] · Selective medium for lactic acid bacteria For the isolation of lactic acid bacteria of the genus Lactobacillus from kefir grains, Rogosa CW medium or the like that has already been reported can be used (see Kojima, SW. et al. Biosci. Biotech. Biochem. 1993. 57, 119-120). In this specification, in order to clearly describe the preparation method of the medium, an example of separation using Rogosa Milk Whey (RMW) medium, which is a slightly modified Rogosa CW medium, will be mainly shown.

[0062] In addition, L. kefiranofaciens has a slow growth rate, and anaerobic culture at a minimum of 22°C - 30°C for 5 days or more, preferably around 10 days, is desirable. L. kefiranofaciens subsp. kefiranofaciens has particularly slow growth, so anaerobic culture for 7 days or more is desirable for stable separation. By extending the culture time, the colony morphology can be characterized and the discrimination of bacterial species can be facilitated. Other lactic acid bacteria of the genus Leuconostoc also grow in the RMW medium. The composition and preparation method of the medium are as follows.

[0063] · RMW medium 5 g of Bacto Tryptone, 5 g of BBL Trypticase peptone, 5 g of Bacto Yeast Extract, 20 g of glucose, 6 g of potassium dihydrogen phosphate, 2 g of diammonium citrate, 1 ml of Tween 80, 15 g of sodium acetate trihydrate, 15 g of agar, and 5 ml of salt solution are added to 1 liter of milk whey, sterilized at 118°C for 15 minutes, and poured onto plates to form plate media. When used as broth, dispense into test tubes without adding agar and sterilize at 118°C for 15 minutes.

[0064] The preparation method of milk whey is as follows. Dissolve 300 g of skim milk powder in 3 liters of deionized water and warm it with boiling warm water. Add 7.8 ml of lactic acid [90% lactic acid solution (for food additives)] to this (the pH will be about 5.5), and leave it in boiling warm water for 15 minutes. Then, since casein in the milk aggregates, remove it with a wire mesh (16 mesh and then 40 mesh), and almost transparent milk whey can be obtained. The salt solution is prepared by dissolving 11.5 g of MgSO4·7H2O, 2.4 g of MnSO4·2H2O, and 0.68 g of FeSO4·7H2O in 100 ml of water.

[0065] For the isolation of lactic acid bacteria, a dilution of the sample was surface smeared on the plate medium with a Conrage rod and anaerobically cultured at 30°C for 5 to 14 days.

[0066] ·RSMP medium RSMP (Rogosa Skim Milk Powder) medium was developed as a selective medium that can also detect Lactobacillus and Leuconostoc. This medium has the same components as the RMW medium (Bacto Tryptone 5 g, BBL Trypticase peptone 5 g, Bacto Yeast Extract 5 g, glucose 20 g, potassium dihydrogen phosphate 6 g, diammonium citrate 2 g, Tween80 1 ml, sodium acetate trihydrate 15 g, agar 15 g, the aforementioned salt solution 5 ml). Dissolve these components in 700 ml of deionized water, sterilize at 118 °C for 15 minutes, then cool to near 50 °C and add 1.32 ml of glacial acetic acid. Next, dissolve 50 g of skim milk powder in 300 ml of deionized water, sterilize at 118 °C for 15 minutes, cool to near 50 °C, and mix with the above-mentioned 700 ml of sterilized medium, then pour it onto plates and let it solidify. The pH of the medium is about 5.5. The culturing method for lactic acid bacteria is the same as that for the RMW medium.

[0067] ·Selective medium for lactic acid bacteria of the genus Lactococcus Lactic acid bacteria of the genus Lactococcus can be isolated using commercially available M17 agar medium (manufactured by Merck). By performing anaerobic culture at 30 °C for about 5 days, relatively large characteristic colonies are formed compared to other colonies that may occur, so if present, they can be easily isolated.

[0068] ·YMX medium YMX medium was used as a selective medium for yeast. The preparation method of YMX medium is as follows. Autoclave commercially available YM agar medium (121 °C, 15 minutes), cool to 50 °C, and add 0.7% (V / V) of 1N HCl. Add 2.5 ml of 2% X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) solution dissolved in dimethylformamide per liter, and pour it onto plates to prepare a plate medium.

[0069] The formation of colonies other than yeast is suppressed by the addition of hydrochloric acid, and lactose-utilizing yeast turns dark blue by the addition of X-Gal. After smearing the specimen on the plate medium, it was aerobically cultured at 22 °C or 25 °C for about 5 days. Natural kefir grains contain both lactose-fermenting yeast and lactose-non-fermenting yeast, but Kazachstania exigua used in this embodiment does not ferment lactose and thus does not turn blue.

[0070] · Skim milk agar medium (M agar medium) As a solid medium for forming colonies that serve as the nuclei for proto-kefir grain formation from isolates, M agar medium can be used. Add 1.5% agar to 10% (W / V) reduced skim milk, sterilize at 118 °C for 15 minutes, and pour it onto plates to solidify. It can be refrigerated and stored at 5 °C until use.

[0071] · Whey agar medium (W agar medium) As a solid medium for forming colonies that serve as the nuclei for proto-kefir grain formation from isolates, W agar medium can be used. Add 90% lactic acid or the like to 10% (W / V) reduced skim milk to lower the pH to 5.5, heat in boiling warm water for 15 minutes, remove the aggregates with a wire mesh (16 mesh then 40 mesh) to obtain milk whey. Add 1.5% agar to this milk whey, sterilize at 118 °C for 15 minutes, and pour it onto plates to solidify. It can be refrigerated and stored at 5 °C until use.

[0072] 〔Storage of strains and preparation of bacterial solutions〕 The bacterial solution used in this embodiment can be prepared as follows. This bacterial solution can be stored long-term at about -80 °C as a preserved strain.

[0073] First, Lactobacillus and Leuconostoc lactic acid bacteria derived from kefir are streaked on RMW agar medium or RSMP agar medium for pure isolation, and then anaerobically cultured at 30°C for about 5 to 7 days to form sufficiently large colonies (about several millimeters to 7 millimeters in diameter). Visually confirm that there is no contamination in these colonies. If it is a petri dish with a standard size of 8 cm in diameter, aseptically add about 5 ml of sterilized skim milk (10% W / V) to the surface of the medium, and disperse the colonies in the skim milk while peeling them off with a spreader stick. Aseptically collect this dispersion with a pipette. Add sterilized 80% (V / V) glycerol to this at a ratio of about 20%, mix well, dispense into vials, etc., and store frozen at -80°C. Such a bacterial solution is called a glycerol stock.

[0074] Note that since L. kefiranofaciens subsp. kefiranofaciens requires time for culture, anaerobically culture for about 5 to 10 days until large slime-like colonies (about 5 millimeters) are formed. When the dispersion is too viscous and difficult to handle when adding sterilized skim milk, further add sterilized skim milk (about 10 ml in total) to reduce the viscosity and prepare a glycerol stock. In the case of Lactococcus lactis, streak-culture on M17 agar medium at 30°C for about 5 days, prepare a colony dispersion in the same manner as above, and make it into a glycerol stock in the same manner as described above. In the case of yeast, aerobically culture on YM agar medium at around 22 - 25°C for about 5 days, collect the resulting colonies in the same manner as above, and also make it into a glycerol stock.

[0075] For any bacterial species, a bacterial solution of approximately 10 8 CFU / ml or so can be obtained by the above method. It is advisable to measure the number of bacteria in the bacterial solution by a culture method if necessary.

Example

[0076] The present invention will be specifically described below with reference to examples and comparative examples.

[0077] (Example 1) Isolation and Identification of Lactic Acid Bacteria from Kefir Grains The method by which the present inventors isolated constituent microorganisms from seven types of natural kefir grains derived from Denmark, Belgium, Russia, the United Kingdom, Germany, Japan, and Turkey will be described below.

[0078] First, a serially diluted homogenate of kefir grains was spread on a plate medium of RMW medium, and anaerobic culture was performed. Desirably, less than 30 colonies were grown on a single standard-sized (inner diameter 8 cm) petri dish. Then, lactic acid bacteria were identified by generally used microbiological techniques, and after streak isolation, they were stored as single strains. In this example, in order to discriminate the main bacterial species by colony morphology, the culture time is required to be at least about 5 to 14 days at 30 °C, and around 8 days is desirable. Usually, a 10 to the 7th power dilution is prepared, and about 0.1 ml is spread on the RMW medium.

[0079] 57 to 96% of the colonies formed on the RMW medium by this method were colonies of L. kefiranofaciens. The rest were mainly L. kefiri and L. parakefiri. L. mesenteroides was also detected depending on the kefir grains. By roughly classifying based on colony morphology, L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, and L. mesenteroides required for this embodiment can be easily obtained. Figure 2 shows the characteristics of representative colony morphologies formed on the RMW medium and the corresponding bacterial species.

[0080] The colony morphology of L. kefiranofaciens is highly diverse. However, translucent colonies with a diameter of several millimeters on RMW medium that are ropy (ropy, viscous and stringy in texture) and mucoid when touched with an inoculation loop are all presumed to be L. kefiranofaciens subsp. kefiranofaciens. Depending on the kefir grains, the abundance of L. kefiranofaciens subsp. kefiranofaciens may be low and it may be difficult to isolate. In such cases, by culturing for more than 10 days using the required number of plate media at a dilution factor such that less than 30 colonies are formed on a petri dish, ropy and mucoid colonies can be discovered. At a dilution factor where the number of colonies formed on a single petri dish exceeds 30, L. kefiranofaciens subsp. kefiranofaciens usually cannot be detected. Most of the other colonies are L. kefiranofaciens subsp. kefirgranum. The colony polymorphism of L. kefiranofaciens subsp. kefirgranum is also remarkable, but relatively large colonies are formed after culturing for about 8 days, so separation is easy.

[0081] The remaining colonies form relatively small colonies, and most of them are L. kefiri. The proportion is about 5% - 24% of the total colonies that appear. However, some of them may be L. parakefiri. L. mesenteroides forms relatively smaller colonies than other colonies on RMW medium and is easy to identify because it is a Gram-positive coccus. Also, L. mesenteroides is abundant in the culture filtrate and can be easily isolated if present. On the other hand, if L. lactis is present, it can be easily isolated on M17 agar medium. Also, the yeast K. exigua forms non-blue, convex to pulvinate colonies on YMX medium and can be easily isolated if present.

[0082] Since isolates cannot be identified solely by colony morphology, they also need to be identified by standard microbiological methods. It is common to observe the bacterial morphology, extract DNA from the colonies, and perform identification based on the 16sDNA sequence. In this example, the colonies were further identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

[0083] In the identification by 16sDNA sequence and MALDI-TOF MS, L. kefiranofaciens subsp. kefiranofaciens and L. kefiranofaciens subsp. kefirgranum cannot be distinguished. However, among the strains identified as L. kefiranofaciens, those that form semi-transparent and ropey colonies may be identified as L. kefiranofaciens subsp. kefiranofaciens. All other L. kefiranofaciens can be presumed to be L. kefiranofaciens subsp. kefirgranum.

[0084] · Floc formation of L. kefiranofaciens subsp. kefirgranum As shown in Fig. 2, the subspecies L. kefiranofaciens subsp. kefirgranum also exhibits strong colony polymorphism. When compared with the JCM strains, JCM8570 often forms smooth-surfaced, umbonate, and slightly viscous colonies. On the other hand, the reference strain JCM8572 forms colonies with a smooth surface, convex (convex-rounded) elevation, slightly loose texture, or rough surface and undulate (mid-convex) elevation. Also, in liquid culture, JCM8570 shows overall turbid, uniform, thin floc-like growth that does not sediment much, while JCM8572 shows strong floc formation and sedimentation (Fig. 3A). The sedimentation of the flocs is thought to indicate strong hydrophobicity and aggregability of the strain. This trait must be affecting the physical structure stability of the kefir grains. When observing the flocs under a microscope, it can be seen that they grow in a filamentous form (Fig. 3B). Therefore, the JCM8572-type bacterial species seems to play a role in maintaining the form in kefir grains.

[0085] When isolated from natural kefir grains, the proportion of strains showing floc formation of the JCM8572 type among L. kefiranofaciens subsp. kefirgranum varied depending on the grains. Since the growth states of the colony traits also differed greatly, in this embodiment, JCM8570 and JCM8572 were used separately. Even when using strains isolated from natural kefir grains, these strains need to be distinguished by the difference in floc formation, and if they exist, both strains should be used.

[0086] Fig. 3 is a photographic image diagram showing the difference in floc formation of Lactobacillus kefiranofaciens subsp. kefirgranum. In Fig. 3A, the left shows the JCM8570 strain and the right shows the JCM8572 strain. In both cases, the medium used was the RMW medium, and the cultures were incubated at 22 °C for 5 days. Fig. 3B is a diagram of the flocs of the JCM8572 strain observed without staining.

[0087] ·Elucidation of the basic flora of kefir grains To reconstruct kefir grains from isolated strains, the flora analysis of seven different kefir grains was carried out. The origins were Denmark, Japan, Russia, the UK, Germany, Belgium, and Turkey. The results are summarized in Table 1-1 and Table 1-2. The main flora commonly present in all kefir grains and the bacterial species strongly suggested to be commonly present were as follows.

[0088] In the method for detecting flora using the culture method, even if it is not detected in the homogenate of kefir grains, if it is detected in the culture filtrate, the bacteria present in the culture solution are originally derived from the grains, so it means that they were present in the grains. This is simply because in the flora analysis of kefir grains, it is difficult to detect due to the overwhelming number of other lactic acid bacteria present at the same time.

[0089] Only the bacterial species that are necessarily present in any kefir grains must be involved in grain formation. Therefore, it was predicted that at most, these seven bacterial species would be sufficient to reconstruct kefir grains.

[0090]

Table 1-1

[0091]

Table 1-2

[0092] (Isolated strains used in the examples) The isolated strains used in the following examples are shown in Table 2 and Table 3. Table 2 shows the strains isolated from six of the above seven kefir grains by bacterial species. Using the strains in Table 2, glycerol stocks were prepared by the preparation method already shown. The names of the source kefir grains are the storage names in the laboratory of the applicant.

[0093]

Table 2

[0094] Table 3 shows the types, strain names, concentrations of bacterial suspensions, and sources of acquisition used in the examples. The JCM strains were obtained from the Microbial Material Development Laboratory of the RIKEN BioResource Center (RIKEN BRC), National Institute of Advanced Industrial Science and Technology. In the following examples, basically, the results with kefir grain analogs prepared using isolated strains were consistent with the results obtained using the reference strains of the strain preservation institutions. Therefore, it was considered that the results of the examples were not limited to individual strains and could be generalized to the same bacterial species.

[0095]

Table 3

[0096] (Example 2) Mixed culture of L. kefiranofaciens subsp. kefiranofaciens and L. kefiri in reduced whey To confirm the interaction between L. kefiranofaciens subsp. kefiranofaciens and L. kefiri, mixed cultures were performed in reconstituted whey using L. kefiranofaciens subsp. kefiranofaciens GA11 and L. kefiri GA8. Both GA11 and GA8 are strains isolated by the inventors from kefir grains derived from Belgium (see Table 2).

[0097] First, whey powder was dissolved in water at 10% (W / V) and sterilized at 63°C for 30 minutes to obtain reconstituted whey. Next, fresh cultures of the above strains were inoculated into 300 ml of reconstituted whey, and static culture was performed at 22°C for 3 days. Thereafter, shaking culture was further performed at 100 rpm for 1 day, and the culture was filtered through a 40-mesh wire net. As a control, GA11 and GA8 were each inoculated into reconstituted whey alone for comparison.

[0098] Figure 4 is a photographic image diagram of the aggregates formed when L. kefiranofaciens subsp. kefiranofaciens GA11 and L. kefiri GA8 were mixed and cultured in reconstituted whey. The results of GA11 monoculture, GA11 and GA8 mixed culture, and GA8 monoculture are shown in order from the left in Figure 4. The inner diameter of the wire net shown in Figure 4 is approximately 7.5 cm.

[0099] As a result, aggregates similar to kefir grains were recovered only from the mixed culture (Figure 4). Therefore, it was found that proto-kefir grains can be formed in a liquid medium simply by mixing and culturing L. kefiranofaciens subsp. kefiranofaciens and L. kefiri.

[0100] (Example 3) Symbiotic relationship among the main flora of kefir grains L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, and L. kefiri, which are the main flora of kefir grains, grow extremely slowly in reconstituted skim milk when alone. These bacteria can grow well in milk only when they coexist as kefir grains. On the other hand, Lactococcus lactis grows well and causes acid coagulation even alone in reconstituted skim milk. To understand these basic relationships, the growth properties in reconstituted skim milk were investigated.

[0101] The following strains were used. All of these strains were isolated by the inventors from kefir grains derived from Belgium (see Table 2). L. kefiranofaciens subsp. kefiranofaciens GA11 L. kefiranofaciens subsp. kefirgranum GG004 L. kefiri GA8 Lactococcus lactis GF019

[0102] 18 ml of 10% (W / V) reconstituted skim milk was dispensed into each sterilized test tube and sterilized by boiling for 15 minutes. 200 μL of fresh bacterial solutions of the above strains were inoculated into each of them. The inoculation combinations are as shown in Table 4.

[0103]

Table 4

[0104] Cultivation was carried out at 22 °C for 7 days. The pH of the culture solution was measured 1, 2, 3, and 7 days after the start of cultivation. After the cultivation was completed, the culture solution was inoculated into RSMP medium (for measuring the number of Lactobacillus bacteria) and M17 medium (for measuring the number of Lactococcus bacteria) and cultured to examine the number of bacteria.

[0105] Figure 5 shows the change in pH of reconstituted skim milk with each formulation shown in Table 4. In Figure 5, the vertical axis represents pH and the horizontal axis represents the number of days of culture. Also, white markers indicate the pure cultures of A1 to A4, and black markers indicate the mixed cultures of A5 to A9.

[0106] As can be seen from Figure 5, L. kefiranofaciens subsp. kefiranofaciens GA11 (A1), L. kefiranofaciens subsp. kefirgranum GG004 (A2), and L. kefiri GA8 (A3) grow extremely slowly in skim milk alone, and the decrease in pH is slow. Nevertheless, the pH of GA11 has decreased to some extent after 7 days. On the other hand, Lactococcus lactis GF019 (A4) also shows a decrease in pH alone. In the formulations (A5, A6, A7, A8, and A9) in which these are mixed, the pH has decreased more than that of Lactococcus lactis alone (A4), and it was presumed that growth was promoted by the mixed culture. When the cell count was measured, this decrease in pH was considered to be mainly due to the promotion of the growth of L. kefiranofaciens subsp. kefiranofaciens and L. kefiranofaciens subsp. kefirgranum.

[0107] (Example 4) Growth property of L. kefiranofaciens subsp. kefiranofaciens in skim milk L. kefiranofaciens subsp. kefiranofaciens has poor growth in skim milk. However, it was confirmed what effect it has on growth when the pH of skim milk is lowered. The pH was adjusted with lactic acid and glacial acetic acid, but since the results were almost the same, only the results adjusted with glacial acetic acid are shown in this example (Table 5).

[0108] First, acetic acid was sterilized by filtration and added to sterilized reduced skim milk to adjust the pH to 6.19, 5.82, 5.47, 5.01, and 4.54. Fresh L. kefiranofaciens subsp. kefiranofaciens TWA-1 was inoculated into this, and after culturing at 30°C for 5 days, the number of bacteria in the culture solution was measured using RMW medium (average of duplicates). Note that TWA-1 is a strain isolated by the inventors from kefir grains derived from Denmark (see Table 2).

[0109] The results are shown in Table 5. Table 5 shows the effect of pH on the growth of L. kefiranofaciens subsp. kefiranofaciens TWA-1 in skim milk (after culturing at 30°C for 5 days). From Table 5, it was found that the growth of the L. kefiranofaciens subsp. kefiranofaciens TWA-1 strain was promoted simply by lowering the pH of the skim milk to around 5.5. Although L. kefiranofaciens subsp. kefiranofaciens is considered difficult to culture and separate, the fact that growth is promoted simply by lowering the pH of skim milk is an important finding in considering the formation of kefir grains. In particular, it was a result that complemented Example 3 above, in which Lactococcus lactis, the main acid-producing bacterium of kefir grains, promotes the growth of L. kefiranofaciens subsp. kefiranofaciens.

[0110] [Table 5]

[0111] (Example 5) Formation conditions of proto-kefir grains If proto-kefir grains are formed, the only issues are whether to transfer them as they are to make them into kefir grain analogs, or, if the bacterial species are insufficient, how to add the insufficient bacterial species and transfer them. Then, the main outline of the results using mainly type species regarding the conditions under which proto-kefir grains are formed is summarized below.

[0112] Explaining in the flow shown in FIG. 1, when "Bacterial solution 1" contains only L. kefiranofaciens subsp. kefiranofaciens, if "Bacterial solution 2" is L. kefiranofaciens subsp. kefirgranum alone, or L. kefiri alone, or L. parakefiri alone, or a mixture of L. kefiranofaciens subsp. kefirgranum and L. kefiri, or a mixture of L. kefiranofaciens subsp. kefirgranum, L. kefiri and L. parakefiri, in most cases, protokefir grains were formed.

[0113] Also, when "Bacterial solution 1" contains L. kefiranofaciens subsp. kefiranofaciens and L, kefiranofaciens subsp. kefirgranum, or L. kefiranofaciens subsp. kefiranofaciens and L. kefiri, or L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum and L. kefiri, in most cases, protokefir grains were formed.

[0114] On the other hand, when "Bacterial solution 1" is only L. kefiranofaciens subsp. kefirgranum, or L. kefiranofaciens subsp. kefirgranum and L. kefiri or L. kefiranofaciens subsp. kefirgranum and L. parakefiri, even if "Bacterial solution 2" contains L. kefiranofaciens subsp. kefiranofaciens, almost no protokefir grains were formed. Yeast, whether present or not as a whole, did not have much effect on the formation of protokefir grains.

[0115] Therefore, in the present embodiment, it was essential that L. kefiranofaciens subsp. kefiranofaciens was included in "Bacterial liquid 1". Several examples are shown below.

[0116] (Example 5-1) MIX003 First, as a preliminary test, using the method shown in FIG. 1, it was tested whether protokefir grains were formed when L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, and Lactococcus lactis were present in "Bacterial liquid 1" (MIX003). Table 6 shows the composition of "Bacterial liquid 1" MIX003 of this example. Note that RG201, RG202, and RG7VS01 are strains isolated by the applicant from kefir grains derived from Russia, YRC3780 is a strain isolated from kefir grains derived from Denmark (see Tables 2 and 3). "Bacterial liquid 2" was not used in this example.

[0117] [Table 6]

[0118] FIG. 6 shows a photographic image diagram of a special colony (about 13 mm in diameter) that occurred when a mixed bacterial liquid (MIX003) of L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, and Lactococcus lactis was cultured in an RSMP medium in "Step 1" (see FIG. 1). In FIG. 6, from left to right, the 6th day and the 10th day of culture are shown.

[0119] In this example (MIX003), "Bacterial liquid 1" containing L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, and Lactococcus lactis was inoculated into RMW medium and anaerobically cultured at 30 °C for 13 days ("Step 1"). Then, semi-transparent characteristic colonies appeared on the RSMP medium (Figure 6). The appearance was significantly different on the 6th and 10th days of culture. The colonies that were milky white on the 6th day became shiny semi-transparent colonies on the 10th day. These were collected and subcultured in 10% (W / V) reduced skim milk ("Step 2"). "Bacterial liquid 2" and "Bacterial liquid 3" were not used.

[0120] Figure 7 shows the shapes of the protokefir grains and kefir grain analogs at the time of subculture. In Figure 7, from left to right, the first subcultured protokefir grain and the kefir grain analog at the 7th subculture (size is approximately 0.95 cm) are shown. The wire mesh shown on the right in Figure 7 is a stainless steel wire mesh with an inner diameter of 7.5 cm and 40 meshes used for the subculture operation.

[0121] The special colonies generated in MIX003 grew in skim milk in the same way as natural kefir grains, were elastic, and their morphology was extremely similar to that of natural kefir grains. The number of grains increased by subculture and did not collapse. Therefore, it can be said that kefir grain analogs were constructed in this example.

[0122] Figure 8 is a graph showing the growth of the kefir grain analogs of MIX003. The vertical axis represents the grain weight (g), and the horizontal axis represents the number of subcultures. MIX003 took about 2 days to solidify skim milk, and the weight also increased slightly (Figure 8).

[0123] Also, it should be noted that protokefir grains were formed even without yeast. Since the kefir grain analogues created by propagating this protokefir grain contained Lactococcus lactis, a lactic acid bacterium with strong acid-producing ability, the skim milk fermented with this had a good kefir-like flavor and was fermented milk.

[0124] (Example 5-2) MIX022, MIX023, MIX024 In the test of Example 5-1 (MIX003), it was shown that it was possible to create protokefir grains with a limited number of lactic acid bacteria. Therefore, in this example, mainly using the reference strains of the strain preservation institution, it was tested whether Lactobacillus kefiranofaciens subsp. kefiranofaciens, Lactobacillus kefiranofaciens subsp. kefirgranum, Lactobacillus kefiri, and Lactococcus lactis were included as "Bacterial solution 1". The formulations of "Bacterial solution 1" (MIX022, MIX023, MIX024) in this example are as shown in Table 7. Among Table 7, Lactococcus lactis YRC3780 is a strain isolated by the applicant from kefir grains derived from Denmark, while the other strains are all strains derived from kefir grains obtained from the RIKEN BioResource Research Center (see Table 3).

[0125]

Table 7

[0126] For the bacterial suspensions of L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, and L. kefiri, 10 ml each of the culture solutions obtained by culturing in a liquid medium corresponding to the RMW medium for 3 days were used. For Lactococcus lactis, 10 ml of the culture solution obtained by culturing in M17 broth for 3 days was used. These bacterial suspensions were mixed in the combinations shown in Table 7 to obtain "Bacterial suspension 1". 0.3 ml of this "Bacterial suspension 1" was dropped onto 6 M agar media and anaerobically cultured at 22°C for 6 days ("Step 1").

[0127] Figure 9 is a photographic image diagram of colonies formed by culturing MIX022 on an M agar medium. As shown in Figure 9, when L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, and L. kefiri were mixed and cultured on an M agar medium, special colonies that could be rolled into a sheet-like shape were formed in most cases.

[0128] This colony was inoculated into 200 ml of 10% (W / V) reduced skim milk and subcultured at 22°C ("Step 2"). Figure 10 shows the appearance of the first proto-kefir grains formed in the first subculture. From left to right in Figure 10, MIX022, MIX023, and MIX024 are shown. All of them are similar to natural kefir grains, indicating that the method of this embodiment is also applicable to the reference strains. That is, it means that anyone can produce proto-kefir grains using the reference strains of the strain preservation institution.

[0129] Furthermore, the obtained proto-kefir grains were repeatedly subcultured at 22°C ("Step 3"). Since there may still be missing bacterial species, although there were increases and decreases in the grain weight, they did not disappear rapidly (Figure 11). Therefore, it can be said that kefir grain analogs were constructed even in this example using the reference strains.

[0130] Figure 11 is a graph showing the weight change (MIX022, MIX023, MIX024) due to the propagation of kefir grain analogs using a reference strain. The vertical axis represents the grain weight (g), and the horizontal axis represents the number of propagation times.

[0131] As shown in Figure 11, the weight of the grains did not increase stably, suggesting that these formulations still do not have the essential flora. However, since all of these kefir grain analogs contain Lactococcus lactis, the fermented liquid fermented with these kefir grain analogs had a good flavor and had no problem as fermented milk. In addition, since all the strains used in MIX023 and MIX024 are reference strains, it was considered that the method of this embodiment could be implemented only with the reference strains.

[0132] (Example 5-3) MIX333, MIX334, MIX335 The formation of probiotic kefir grains was confirmed with MIX333, MIX334, and MIX335 when only JCM6985, which is the reference strain of L. kefiranofaciens subsp. kefiranofaciens, was included as "Bacterial solution 1" (Table 8). As "Bacterial solution 2", only JCM5818, which is the reference strain of L. kefiri, was used in MIX333, JCM5818 of L. kefiri and JCM8572, which is the reference strain of L. kefiranofaciens subsp. kefirgranum, were used in MIX334, and JCM5818 of L. kefiri, JCM8572 and JCM8570 (non-reference strain) of L. kefiranofaciens subsp. kefirgranum were added in MIX335. Table 8 shows the strains and compounding amounts of "Bacterial solution 1" and "Bacterial solution 2" of MIX333, MIX334, and MIX335.

[0133]

Table 8

[0134] First, 30 μL of "Bacterial liquid 1" was dropped at three locations on each of 6 W agar media, and anaerobically cultured at 22°C for 7 days ("Step 1"). The resulting colonies were collected, inoculated into 200 ml of 10% skim milk medium, and the total amount shown in Table 8 of "Bacterial liquid 2" was added, followed by subculture at 22°C ("Step 2").

[0135] Figure 12 shows the appearance and weight of the protokefir grains of MIX333, MIX334, and MIX335 that first occurred under these conditions. (The weight of the protokefir grains is shown in parentheses.)

[0136] It was found that protokefir grains are easily formed if at least L. kefiranofaciens subsp. kefiranofaciens is included as "Bacterial liquid 1" and L. kefiri is included as "Bacterial liquid 2". As shown in this example, protokefir grains were formed with L. kefiranofaciens subsp. kefiranofaciens alone in the reference strain, but protokefir grains were not formed with L. kefiranofaciens subsp. kefiranofaciens alone in many isolates. Although not shown here, from other test results as well, when "Bacterial liquid 1" is L. kefiranofaciens subsp. kefiranofaciens alone, the presence of L. kefiri as "Bacterial liquid 2" was an essential condition for protokefir grain formation.

[0137] In addition, when L. kefiranofaciens subsp. kefirgranum was included as "Bacterial solution 2" (MIX334, MIX335), it was found that more elastic protokefir grains were formed. Also, in the case where both JCM8572 type and JCM8570 type of L. kefiranofaciens subsp. kefirgranum were included (MIX335), the shape of the protokefir grains was more similar to that of natural kefir grains. Since the MIX in this example did not contain Lactococcus lactis, the fermenting power was weak and milk coagulation did not occur. However, by subculturing every 2 - 4 days, the grain weight gradually increased.

[0138] (Example 5 - 4) MIX319, MIX320, MIX323 Furthermore, in order to confirm the conditions for protokefir grain formation, the formation of protokefir grains was observed when L. kefiranofaciens subsp. kefiranofaciens and L. kefiri or L. parakefiri were used as "Bacterial solution 1" (Table 9). Table 9 shows the mixing ratios of "Bacterial solution 1" for MIX319, MIX320, and MIX323.

[0139] [Table 9] Symbol M: M agar medium was used as the solid medium for "Step 1". Symbol W: W agar medium was used as the solid medium for "Step 1".

[0140] Seven pieces each of M agar medium and W agar medium were used as solid media. 30 μL each of the "Bacterial solution 1" in Table 9 was dropped at three locations on the solid medium and anaerobically cultured at 22°C for 6 days ("Step 1"). Then, the colonies formed were collected and inoculated into 10% (W / V) reduced skim milk. "Bacterial solution 2" was not added. After culturing at 22°C for 5 days, the protokefir grains were collected with a 40 - mesh wire mesh ("Step 2") (Figure 13).

[0141] Figure 13 shows the appearance and weight of the protokefir grains of MIX319, MIX320, and MIX323. (The weight of the protokefir grains is shown in parentheses.) The symbol M indicates that the M agar medium was used as the solid medium in "Step 1", and the symbol W indicates that the W agar medium was used. * indicates that the measurement was taken at the second subculture. Figure 14 shows the microscopic images (Gram staining) of the protokefir grains of MIX319, MIX320, and MIX323.

[0142] Thus, when L. kefiranofaciens subsp. kefiranofaciens and L. kefiri or L. parakefiri were used as "Bacterial solution 1", colonies were formed on the M agar medium and the W agar medium, and then inoculated into skim milk, protokefir grains with extremely similar elasticity to kefir grains were formed without exception (Figure 13). When these protokefir grains were sampled, Gram-stained, and observed under a microscope, it was found that all the protokefir grains had a high bacterial density similar to that of natural kefir grains, and that long rod-shaped bacteria considered to be L. kefiranofaciens, which are characteristic of kefir grains, were the main component (Figure 14).

[0143] These protokefir grains did not contain L. kefiranofaciens subsp. kefirgranum, which is considered essential for kefir grains, and did not contain Lactococcus lactis in their flora, so their fermenting power was weak and they did not produce enough lactic acid to cause acid coagulation of milk. In this example, "Bacterial solution 2" was not added when inoculating the colonies into the liquid medium, but the L. lactis YRC3780 strain was added to all the grains as "Bacterial solution 3" at the first subculture. However, the grain weight decreased rapidly during several subsequent subcultures.

[0144] (Example 5-5) MIX39LC, MIX39LC+ From the previous examples, if "Bacterial liquid 1" contains all the bacterial species essential for forming kefir grains, it is expected that by transplanting the protokefir grains, artificial kefir grains equivalent to natural kefir grains can be obtained directly. Therefore, tests were conducted using the Giacomo grain isolate produced in Belgium (Table 10).

[0145] Table 10 shows the bacterial liquid formulations of MIX39LC and MIX39LC+. MIX39LC+ was obtained by transplanting the protokefir grains formed using MIX39LC as "Bacterial liquid 1" ten times, then dividing the grains into two, and adding Leuconostoc mesenteroides and Kazachstania exigua as "Bacterial liquid 3" for transplantation.

[0146]

Table 10

[0147] Note that the "JCM8570 type" refers to strains that do not form flocs like the aforementioned L. kefiranofaciens subsp. kefirgranum JCM8570, and the "JCM8572 type" refers to strains that show strong floc formation like the aforementioned L. kefiranofaciens subsp. kefirgranum JCM8572.

[0148] M agar medium was used as the solid medium. 30 μL of "Bacterial liquid 1" of MIX39LC was dropped at three locations on the solid medium and anaerobically cultured at 22°C ("Step 1"). Then, the resulting colonies were collected and inoculated into 10% (W / V) reduced skim milk and cultured at 22°C. "Bacterial liquid 2" was not added ("Step 2"). Thereafter, transplantation was repeated at 22°C ("Step 3"). At the tenth transplantation, half of the kefir grain analog was collected, and transplantation was started after adding "Bacterial liquid 3" of MIX39LC+.

[0149] Figure 15 shows the appearance of kefir grain analogs. For comparison, the appearance of natural Giacomo grains, which are the source of separation, is also shown. In Figure 15, the upper left shows the natural Belgian Giacomo grains, which are the source of separation, the upper right shows MIX39LC after 24 transfers, and the lower part shows MIX39LC+ after 7 transfers. These kefir grain analogs were different in that they were slightly softer than the natural Giacomo grains.

[0150] Figure 16 shows the weight changes of kefir grain analogs by subculturing MIX39LC and MIX39LC+. The vertical axis represents the grain weight (g), and the horizontal axis represents the number of subcultures. From Figure 16, it was found that both kefir grain analogs generally showed an increasing trend.

[0151] (Examples 5-6) MIX41C, MIX42C, MIX43C, MIX44C, MIX45C, MIX46C Considering the possibility that the diversity of bacterial species is important for forming complete kefir grains from the isolated strains, the following tests were conducted.

[0152] A "bacterial solution 1" was prepared by mixing 0.5 ml of each of the multiple bacterial species shown in Table 11. It was dropped onto the center of each of 10 M agar media and anaerobically cultured at 22°C for 13 days ("Step 1"). The resulting colonies were collected with a spatula and inoculated into 200 ml of RSM broth and cultured until coagulation ("Step 2"). Subculturing was repeated at 22°C ("Step 3"), and the weight changes of the kefir grain analogs were examined. "Bacterial solution 2" and "bacterial solution 3" were not used.

[0153] The composition of the "bacterial solution 1" used is shown in Table 11. MIX41C mainly used the reference strain, MIX42C mainly used the Russia grain isolate from Russia, MIX43C mainly used the YCHR2 grain isolate from Japan, MIX44C mainly used the Maria grain isolate from the UK, MIX45C mainly used the Giacomo grain isolate from Belgium, and MIX46C mainly used the ALT grain isolate from Turkey.

[0154]

Table 11

[0155] Figure 17 shows a state where a part of the special colonies formed after dropping the "Bacterial liquid 1" of MIX41C, 42C, 43C, 44C, 45C, 46C onto M agar medium and culturing at 22°C for 13 days was peeled off with a spatula. In each case, the colony diameter was about 1.5 cm. All the colonies formed on the M agar medium grew very well and were specific colonies that could be peeled off in a sheet form (Figure 17).

[0156] Figure 18 shows the appearance of the proto-kefir grains initially formed with MIX41C, 42C, 43C, 44C, 45C, 46C. The shapes of the initially formed proto-kefir grains were all elastic and resembled kefir grains, but were slightly softer (Figure 18).

[0157] Figure 19 shows the weight change due to subculture of the kefir grain analogs of MIX41C, 42C, 43C, 44C, 45C, 46C. The vertical axis indicates the grain weight (g), and the horizontal axis indicates the number of subcultures. The kefir grain analogs of MIX41C and MIX46C completely disintegrated upon subculture. However, MIX42C, MIX43C, MIX44C, and MIX45C existed stably for a certain period of subculture (Figure 19). From these results, it was considered that increasing the diversity of the "Bacterial liquid 1" does not necessarily lead to a stable increase in kefir grain analogs, and the balance of bacteria at the proto-kefir grain stage has a great influence.

[0158] So far, regarding the formation of kefir grains, it has been reported that L. kefiranofaciens subsp. kefiranofaciens produces a large amount of EPS. Also, it has been reported that L. kefiri has strong adhesiveness. However, for the first time in this specification, it has been shown that kefir grain analogs can be formed only with at least L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofacicens subsp. kefirgranum, L. kefiri, L. parakefiri, Lactococcus lactis, and Leuconostoc mesenteroides.

[0159] It is still unknown whether such kefir grain analogs will develop to the extent that they are indistinguishable from natural kefir grains. However, the examples so far have shown that kefir grain analogs grow in the same way as natural kefir grains, ferment milk, and can be used as a starter for fermented milk. Also, a specific method for artificially constructing kefir grain analogs that were not previously known could be provided. Also, for the first time, it could be shown that although yeast is recognized as essential for symbiosis in kefir grains, it is not essential for grain formation.

[0160] It is known that there are significant morphological differences among natural kefir grains, but the kefir grain analogs of the present invention were more viscous, softer, or had smaller grains than natural kefir grains. Therefore, it was judged that they are still at the stage of kefir grain "analogs".

[0161] Also, when forming proto-kefir grains using "bacterial solution 1" and "bacterial solution 2", it was considered that the order in which the bacterial species are added also has a great influence on whether the proto-kefir grains will become kefir grain analogs that grow stably.

[0162] Protocatechuic acid grains without Lactococcus lactis did not coagulate milk. Therefore, it was found that L. lactis is an essential element when using kefir grain analogs as starters. Therefore, it seemed desirable to add it to "Bacterial solution 1" from the beginning.

[0163] Also, as can be seen in the example of MIX41C, even if "Bacterial solution 1" was simply prepared by adding all the bacterial species, the resulting protocatechuic acid grains did not grow smoothly to become kefir grain analogs. This may be due to high strain dependence, but it may also be affected by differences in the formulation of "Bacterial solution 1" and "Bacterial solution 2", or the type of solid medium used to culture "Bacterial solution 1".

[0164] The following became clear from the above examples. (1) It is possible to reconstruct kefir grain analogs from kefir grain isolates if there are at most seven bacterial species: L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, L. kefiri, L. parakefiri, L. mesenteroides, L. lactis, and yeast. (2) In particular, it was considered that L. kefiranofaciens subsp. kefiranofaciens, L. kefiranofaciens subsp. kefirgranum, and L. kefiri play a central role in protocatechuic acid grain formation. (3) Some strains of L. kefiranofaciens subsp. kefirgranum form strong flocs, and it is easier to form kefir grain analogs when using both strains with weak floc-forming ability and strains with strong floc-forming ability. (4) For using kefir grain analogs as starters for fermented milk, it is essential to contain L. lactis. (5) Yeast is not basically required for the formation of protocatechuic acid grains itself.

[0165] The kefir grain analog of the present invention still has morphological differences from natural kefir grains. Although the present invention leaves unknown parts regarding the formation of kefir grains, it is clearly disclosed methodologically, and has clarified the basic technology necessary for the artificial formation of kefir grains.

Industrial Applicability

[0166] By using the kefir grain analog of the present invention, it becomes possible to produce fermented milk containing L. kefiranofaciens and L. kefiri in the same way as when using natural kefir grains, and it has great applicability in the dairy field. Further, according to the present invention, a technique for generating a kefir grain analog has been established, and a basic technique for reconstructing a kefir grain analog indistinguishable from natural kefir grains using this technique can be provided.

Claims

**Claim 1**: A method for producing a kefir grain analog, characterized by mixing kefir grain isolates belonging to Lactobacillus kefiranofaciens subsp. kefiranofaciens, Lactobacillus kefiranofaciens subsp. kefirgranum, and Lentilactobacillus kefiri, culturing them in a liquid medium or a solid medium to form a kefir grain-like bacterial mass (proto-kefir grain), and propagating it to form a kefir grain analog. The kefir grain analog has a bacterial flora similar to that of kefir grains, and when repeated subculturing 10 times, the wet weight of the bacterial mass increases by 10% or more. A method for producing a kefir grain analog from kefir grain isolates. **Claim 2**: The method according to claim 1, wherein the kefir grain isolates belonging to Lactobacillus kefiranofaciens subsp. kefiranofaciens, Lactobacillus kefiranofaciens subsp. kefirgranum, Lentilactobacillus kefiri, and one or more selected from the group consisting of Lentilactobacillus parakefiri, Lactococcus lactis, Leuconostoc mesenteroides, and yeast are mixed and cultured in the liquid medium or the solid medium to form the kefir grain-like bacterial mass (proto-kefir grain). **Claim 3**: Step 1 of culturing a bacterial liquid containing kefir grain isolates belonging to Lactobacillus kefiranofaciens subsp. kefiranofaciens, Lactobacillus kefiranofaciens subsp. kefirgranum, and Lentilactobacillus kefiri on the surface of a solid medium to form colonies. Step 2 of collecting the colony, inoculating it into a liquid medium, culturing it at least once, and forming protokefir grains in the liquid medium; Step 3 of subculturing the protokefir grains multiple times using a liquid medium to form kefir grain analogs; The method according to claim 1, comprising:

4. The method according to claim 3, wherein the "bacterial solution" in Step 1 contains Lactobacillus kefiranofaciens subsp. kefiranofaciens and a kefir grain isolate belonging to one or more selected from the group consisting of Lactobacillus kefiranofaciens subsp. kefi granum, Lentilactobacillus kefiri, Lentilactobacillus parakefiri, Leuconostoc mesenteroides, Lactococcus lactis, and yeast.

5. The kefir grain analog (protokefir grain) is subcultured by adding a kefir grain isolate belonging to one or more selected from the group consisting of Lactobacillus kefiranofaciens subsp. kefiranofaciens, Lactobacillus kefiranofaciens subsp. kefi granum, Lentilactobacillus kefiri, Lentilactobacillus parakefiri, Lactococcus lactis, Leuconostoc mesenteroides, and yeast as another kefir grain isolate necessary for symbiosis to form the kefir grain analog. The method according to any one of claims 1 to 4.

6. A method for producing fermented milk, characterized by producing a kefir grain analog by the method according to any one of claims 1 to 5 and using the kefir grain analog to produce fermented milk.

7. A method for producing artificial kefir grains, characterized by producing a kefir grain analog by the method according to any one of claims 1 to 5 and subculturing the kefir grain analog to form artificial kefir grains indistinguishable from natural kefir grains.

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