METHOD FOR PRODUCING FERMENTED LIQUID COMPRISING SHORT-CHAIN FATTY ACID
20220248718 · 2022-08-11
Assignee
Inventors
Cpc classification
A23L33/105
HUMAN NECESSITIES
A23L11/50
HUMAN NECESSITIES
A23V2002/00
HUMAN NECESSITIES
A23L33/135
HUMAN NECESSITIES
International classification
A23L11/50
HUMAN NECESSITIES
A23L33/105
HUMAN NECESSITIES
Abstract
A method for producing a fermented liquid having an acidity of pH 3 to 4, including colloidal particles having a particle size not exceeding 50 nm and short-chain fatty acids. The method includes providing fermentation apparatus including a plurality set of temperature-controlled fermentation containers at each stage of the multi-stage fermentation process; using a soft water as a starter, a seed bacterial liquid comprising seven species of fermentation bacteria (International Accession No. NITE BP-02945 to NITE BP-02951) including spore-forming Clostridium bacteria, controlled under a low temperature condition, and three kinds of fermented media derived from natural materials, produced by individually fermenting respective first medium of dried soybeans, second medium of mixed medium of dried plants consisting Taiso, Kukoshi, and Ukon, and third material of honey material; and producing the fermented liquid by multi-stage fermentation process.
Claims
1. A method for producing a fermented liquid having an acidity of pH 3 to 4, comprising colloidal particles having a particle size not exceeding 50 nm and short-chain fatty acids, the short-chain fatty acids comprising butyric acid, propionic acid and lactic acid, the method comprising the steps of: preparing: a soft water having pH 7.0 to 7.6; three kinds of fermented media consisting of a first fermented medium having pH 4.2 to 4.8 produced from dried soybeans, a second fermented medium having pH 4.5 to 5.1 produced from dried plants comprising Lycium Fruit (Kukoshi), Jujube (Taiso) and Turmeric (Ukon), and a third fermented medium having pH 4.1 to 4.7 produced from a honey material; and a seed bacterial liquid having pH 5.0 to 5.6 comprising seven species of fermentation bacteria (International Accession Numbers NITE BP-02945, NITE BP-02946, NITE BP-02947, NITE BP-02948, NITE BP-02949, NITE BP-02950, and NITE BP-02951), the fermentation bacteria producing organic acids comprising short-chain fatty acids; performing a Process A comprising the steps of: putting the soft water, the first fermented medium, and the seed bacterial liquid in a volumetric ratio of 1100 to 1300 L of the soft water, 280 L of the first fermented medium, and 270 to 450 L of the seed bacterial liquid, into a first fermentation tank and mixing them to produce a turbid liquid having pH 6.1 to 6.7; fermenting the turbid liquid at a fermentation temperature of 37° C. to 40° C. for 50 to 100 days to form three layers consisting of a supernatant sponge layer containing fermentation gas, a first deposition layer at a bottom, and an intermediate layer of a translucent fermented liquid having pH 5.0 to 5.6 between the sponge layer and the first deposition layer; and extracting the translucent fermented liquid from the three layers to produce a first fermented liquid having pH 5.0 to 5.6; performing a Process B comprising the steps of: transferring the first fermented liquid having pH 5.0 to 5.6 into a second fermentation tank, fermenting the first fermented liquid at a fermentation temperature of 37° C. to 40° C. for 3 to 5 days in a fermentation environment without using any fermented medium to form three layers consisting of a supernatant first surface layer containing fermentation gas bubbles, a second deposition layer at a bottom in which a residue of the first fermented medium is deposited, and an intermediate layer of a translucent fermented liquid having pH 4.7 to 5.3 between the first surface layer and the second deposition layer; and extracting the translucent fermented liquid from the three layers to produce a second fermented liquid having pH 4.7 to 5.3; performing a Process C comprising the step of: transferring the second fermented liquid having pH 4.7 to 5.3 into a third fermentation tank; enclosing the second fermented medium in a bag-shaped filter, suspending the bag-shaped filter inside of the third fermentation tank, and fermenting the second fermented liquid at a fermentation temperature of 37° C. to 40° C. for 8 or 9 days, with circulating the second fermented liquid, to form two layers consisting of a supernatant second surface layer containing fermentation gas bubbles, and a bottom layer of a turbid fermented liquid having pH 4.2 to 4.8; and extracting the turbid fermented liquid from the two layers to produce a third fermented liquid having pH 4.2 to 4.8; performing a Process D comprising the steps of: transferring the third fermented liquid having pH 4.2 to 4.8 into a fourth fermentation tank, and mixing the third fermented liquid and the third fermented medium in a volumetric ratio of 20 to 1 to produce a turbid liquid; fermenting the turbid liquid at a fermentation temperature of 37° C. to 40° C. for 30 to 60 days to form three layers consisting of a supernatant third surface layer, a third deposition layer at a bottom, and an intermediate layer of a translucent fermented liquid having pH 3.4 to 4.0 between the third surface layer and the third deposition layer; and extracting the translucent fermented liquid from the three layers to produce a fourth fermented liquid having pH 3.4 to 4.0; performing a Process E comprising the steps of: transferring the fourth fermented liquid having pH 3.4 to 4.0 into a fifth fermentation tank; fermenting the fourth fermented liquid at a fermentation temperature of 37° C. to 40° C. for 30 to 60 days in a fermentation environment without using any fermented medium to produce a translucent fermented liquid having pH 3.3 to 3.9 and a fourth deposition layer; and extracting the translucent fermented liquid to produce a fifth fermented liquid having pH 3.3 to 3.9; and performing a Process F comprising the steps of: transferring the fifth fermented liquid having pH 3.3 to 3.9 into a reserve tank of a cooling unit, and rapidly cooling the fifth fermented liquid at the fermentation temperature of 37° C. to 40° C. to 5° C. or lower by the cooling unit; returning the rapidly cooled fifth fermented liquid to an ordinary temperature condition to produce an ordinary temperature fermented liquid; fermenting the ordinary temperature fermented liquid at an ordinary temperature for 180 to 240 days to produce a transparent fermented liquid having pH 3.0 to 3.6; and extracting the transparent fermented liquid to produce a sixth fermented liquid having pH 3.0 to 3.6.
2. The method according to claim 1, wherein the Process A is a Process A′ comprising the steps of: preparing two first fermentation tanks; putting the soft water, the first fermented medium, and the seed bacterial liquid in a volumetric ratio of 1100 to 1300 L of the soft water, 280 L of the first fermented medium, and 270 to 450 L of the seed bacterial liquid, into each of the two first fermentation tanks equally, and mixing them to produce a turbid liquid having pH 6.1 to 6.7; fermenting the turbid liquid at a fermentation temperature of 37° C. to 40° C. for 50 to 100 days to form three layers consisting of a supernatant sponge layer containing fermentation gas, a first deposition layer at a bottom, and an intermediate layer of a translucent fermented liquid having pH 5.0 to 5.6 between the sponge layer and the first deposition layer; and extracting the translucent fermented liquid from the three layers to produce the first fermented liquid having pH 5.0 to 5.6; and wherein the Process B is a Process B′ comprising the steps of: transferring each of the first fermented liquids having pH 5.0 to 5.6 into one second fermentation tank, and mixing them to produce a first fermented liquid having a uniform acidity; fermenting the first fermented liquid at a fermentation temperature of 37° C. to 40° C. for 3 to 5 days in a fermentation environment without using any fermented medium to form three layers consisting of a supernatant first surface layer containing fermentation gas bubbles, a second deposition layer at a bottom in which a residue of the first fermented medium is deposited, and an intermediate layer of a translucent fermented liquid having pH 4.7 to 5.3 between the first surface layer and the second deposition layer; and extracting the translucent fermented liquid from the three layers to produce the second fermented liquid having pH 4.7 to 5.3.
3. The method according to claim 1, wherein the fermented liquid having an acidity of pH 3 to 4 comprises 0.5 to 0.6 g of the butyric acid per 100 mL of the fermented liquid.
4. The method according to claim 1, wherein, when the first fermented liquid formed in the three layers in the first fermentation tank is assumed to be 1200 L, a process for producing the first fermented medium having pH 4.2 to 4.8 comprises the steps of: soaking and reconstituting the dried soybeans corresponding to 6.5 kg in at least 20 L of the soft water in each of four or more fermentation bottles provided with a lid having valve functionality, and putting 250 g of a sugar chain and 140 cc of the seed bacterial liquid into each of the fermentation bottles; fermenting the contents of the fermentation bottles at a fermentation temperature of 37° C. to 40° C. for three days (68 to 74 hours) to produce a first preliminary fermented liquid and fermented soybeans, corresponding to 35 L in each fermentation bottle; removing the fermented soybeans from each of the fermentation bottles, grinding the fermented soybeans into a paste by a grinding means, and mixing the ground soybeans and the first preliminary fermented liquid to produce the first preliminary fermented liquid and a soybean paste, corresponding to 35 L; transferring the first preliminary fermented liquid and the soybean paste, corresponding to 35 L produced in each fermentation bottle, into a heating kettle, and gradually heating them to 55 to 60° C. to produce the first preliminary fermented liquid and the soybean paste, corresponding to 180 L in total; and transferring the first preliminary fermented liquid and the soybean paste, corresponding to 180 L, from the heating kettle into a first medium tank, with avoiding exposure to external air, wherein the soft water for cooling corresponding to 100 L has been preliminarily put into the first medium tank, and stirring them to produce the first fermented medium corresponding to 280 L.
5. The method according to claim 1, wherein, when the first fermented liquid formed in the three layers in the first fermentation tank is assumed to be 1200 L, the turbid liquid having pH 6.1 to 6.7 produced in the first fermentation tank is a second preliminary fermented liquid having pH 6.1 to 6.7 corresponding to 1800 L, produced by putting the first fermented medium having pH 4.5 to 5.1 corresponding to 280 L, the seed bacterial liquid having pH 5.0 to 5.6 corresponding to 270 to 450 L, and the soft water having pH 7.0 to 7.6 corresponding to 1100 to 1300 L into the first fermentation tank and stirring and mixing them.
6. The method according to claim 1, wherein, when the third fermented liquid corresponding to 1050 to 1100 L is assumed to be produced, a process for producing the second fermented medium having pH 4.5 to 5.1 comprises the steps of: mixing 200 to 210 g of the Jujube, 110 to 120 g of the Lycium Fruit, and 25 to 30 g of the Turmeric per 35 L of the second fermented liquid in total to produce a mixed medium; putting the mixed medium and 35 L of the second fermented liquid into a second medium tank, and fermenting the mixed medium and the second fermented liquid for 2 or 3 days to produce a preliminary fermented mixed medium and a third preliminary fermented liquid having pH 4.5 to 5.1; and removing the preliminary fermented mixed medium from the second medium tank, grinding the removed preliminary fermented mixed medium by a grinding means, returning the ground preliminary fermented mixed medium to the second medium tank, and blending the preliminary fermented mixed medium and the third preliminary fermented liquid to produce the second fermented medium having pH 4.5 to 5.1 corresponding to 35 L.
7. The method according to claim 1, wherein, when the fourth fermented liquid corresponding to 1000 to 1050 L is assumed to be produced, a process for producing the third fermented medium comprises the steps of: putting the honey material corresponding to 3 to 5% of the third fermented liquid into a third medium tank, transferring the third fermented liquid corresponding to four times as much as the honey material into the third medium tank with avoiding exposure to external air, and stirring them to produce a preliminary fermented medium; and fermenting the preliminary fermented medium at a fermentation temperature of 37° C. to 40° C. for 2 or 3 days to produce the third fermented medium having pH 4.1 to 4.7.
Description
BRIEF DESCRIPTION OF DRAWINGS
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DESCRIPTION OF EMBODIMENTS
[0163] An embodiment of the present invention is described as follows. The embodiment of the present invention provides a method for producing a fermented liquid having an acidity of pH 3 to 4 comprising colloidal particles having a particle size not exceeding 50 nm and short-chain fatty acids, by multi-stage fermentation process. The short-chain fatty acids may comprise butyric acid, propionic acid, and lactic acid. The fermented liquid produced by the method of the present invention may contain the colloidal particles at a content of 6.5 to 7.5%, and 0.5 to 0.6 g of the butyric acid per 100 mL of the fermented liquid. The fermented liquid produced by the method of the present invention may be used, by itself, a functional food or a raw material liquid for functional food.
Details of a First Fermentation Line f1
[0164] More particularly, the multi-stage fermentation starts at a first process p.sub.11 configuring a first fermentation line f1, as shown in
[0165] When a charge amount for producing a first fermented liquid n1 in the first fermentation line f1, that is an amount of a second preliminary fermented liquid pn2 is assumed to be 1800 L, a charge amount as a starter and a final amount to be produced for each process can be estimated as follows.
[0166] As shown in
[0167] In the first pre-treatment process, dried soybeans corresponding to 6.5 kg are soaked and reconstituted in 20 L of a soft water w of pH 7.3± in each of four pottery fermentation bottles 10 shown in
[0168] In the second pre-treatment process, the contents of the fermentation bottles 10 are fermented for 3 days (68 to 74 hours) to produce a first preliminary fermented liquid pn1 and fermented soybeans fs, corresponding to 35 L per each fermentation bottle 10.
[0169] As shown in
[0170] As shown in
[0171] A second process p.sub.12 of the first fermentation line f2 further comprises a fourth preliminary process pp.sub.14 to sixth preliminary process pp.sub.16.
[0172] In the fourth preliminary process pp.sub.14 of the second process p.sub.12, the first preliminary fermented liquid pn1 and the soybean paste gfs produced in the heating kettle 30 in the third preliminary process pp.sub.13 of the first process p.sub.11, corresponding to 180 L, is transferred, with avoiding exposure to external air, into a first medium tank 20 preliminarily containing soft water w corresponding to 100 L for cooling, and stirred to produce a first fermented medium m1 of pH 4.5± corresponding to 280 L.
[0173] As shown in
[0174] In the fifth preliminary process pp.sub.15 and the sixth preliminary process pp.sub.16 of the second process p.sub.12 shown in
[0175] In each fermentation tank 100, the sponge layer sp swelled by the long-term fermentation and the paste-like first deposition layer dep1 occupy a volume corresponding to 300 L in total, and thus the first fermentation liquid n1 of pH 5.3±, which is the translucent first intermediate layer liquid, may correspond to 600 L. The supernatant sponge layer sp and the paste-like first deposition layer dep1 may become base materials for the seed bacterial liquid b.
[0176] The first fermentation line f1 may further comprises a third process p.sub.13. In the third process p.sub.13, the first fermented liquid n1 of pH 5.3±, which is the first intermediate layer liquid corresponding to 600 L produced in each first fermentation tank 100, is transferred into one second fermentation tank 200 as shown in
[0177] The third process p.sub.13 uses the first fermented liquid n1 of pH 5.3±corresponding 1200 L transferred into the one second fermentation tank 200 as a starter, and the first fermented liquid n1 is fermented for 3 to 5 days by fermentation bacteria b1 of the seed bacterial liquid b contained in the first fermented liquid n1 to produce a second fermented liquid n2 of PH 5.0±.
Technical Problem for Producing the Second Fermented Liquid n2
[0178] The third process p.sub.13 has a feature in that the first fermented liquid n1 is fermented only by the fermentation bacteria b1 of the seed bacterial liquid b, in an environment without using medium or fermented medium. Here, a technical problem for the third process p.sub.13 of the first fermentation line f1 is considered. The two lots of first fermented liquid n1 separately produced in the two fermentation tanks 100 may have different fermentation environments each other, due to symbiosis of the fermentation bacteria b1 of the seed bacterial liquid b. The symbiosis and antagonism of the fermentation bacteria b1 of the seed bacterial liquid b contained in the first fermentation liquid n1 may be further stimulated by transferring the two lots of first fermented liquid n1 into one second fermentation tank 200 and mixing them to be unified. This may be estimated from the fact that the first fermented liquid of pH 5.3± is acidified to the second fermented liquid n2 of pH 5.0±.
[0179] Therefore, as indicated by two routes shown in
[0180] In the case when the process directly proceeds from the second process p.sub.12 of the first fermentation line f1 to a second fermentation line f2, one fermentation tank (not shown) having a size similar as that of the second fermentation tank 200 may be used. The inventors found that, from their steady practical work for many years, when the process does not go through the third process p.sub.13 of the first fermentation line f1, the symbiosis and antagonism of the fermentation bacteria b1 remains low level, that is, secretion and fermentation of acidic materials by the fermentation bacteria b1 cannot sufficiently proceed, and thus, it is not so easy to stably produce the first fermented liquid n1 of pH 5.3±corresponding 1200 L in the one first fermentation tank. Therefore, in order to preliminarily ferment the first fermented liquid n1 of PH 5.3±until it becomes the second fermented liquid n2 of pH 5.0±, it may be necessary to further modification and more fermentation days. Based on such knowledge, the inventors have addressed such a problem and conceived an idea that the third process p.sub.13 is integrated in the first fermentation line f1 to solve the problem.
[0181] In fact, in the third process p.sub.13 of the first fermentation line f1, the first fermented liquid n1 of pH 5.3± is fermented for 3 to 5 days only by the fermentation bacteria b1 with maintaining a fermentation condition at 37° C. to 40° C. to form a supernatant first surface layer sf1 of fermentation gas bubbles. The first surface layer sf1 prevents oxygen from dissolving into the liquid to further facilitate secretion and fermentation by the fermentation bacteria b1. A paste-like second deposition layer dep2 is formed at a bottom layer, in which residue of the first fermented medium m1 is deposited. Between the first surface layer sf1 and the second deposition layer dep2, a translucent second intermediate layer liquid corresponding 1140 to 1240 L is formed. The translucent second intermediate layer liquid is the second fermented liquid n2 of pH 5.0±. Whether the third process p.sub.13 is integrated in the first fermentation line f1 or not is an issue of choice.
Technical Feature of the First Fermentation Line f1
[0182] A technical feature of the first fermentation line f1 is that, in contrast to conventional soybeans fermentation, dried soybeans ds are preliminarily fermented in fermentation bottles 100 to produce a first preliminary fermented liquid pn1 and a soybean paste gfs, and a first fermented medium m1 are produced from the first preliminary fermented liquid pn1, the soybean paste gfs, a soft water w, and a part of the seed bacterial liquid b. The preliminarily fermented first fermented medium m1 of pH 4.5± and a soft water w are used to produce a first fermented liquid n1 of pH 5.3±.
[0183] Usually, fermentation products using soybeans are produced in a condition in which soybeans ds are soaked in a liquid such as a material to be fermented or water. On the other hand, the inventors have achieved the present invention by focusing on producing a fermented liquid via a process for preliminary fermenting the first fermented medium m1 using dried soybeans, to produce the first fermented liquid n1 of pH 5.3±.
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[0185] The photograph A shows the first fermented liquid A produced by fermenting the sample A for 6 days, wherein the sample A is prepared by preliminarily fermenting 60 g of soybeans, grinding the preliminarily fermented soybeans to produce a soybean paste, heating the soybean paste and a soft water w to 55° C., then cooling them to 37° C., adding 0.8 L of seed bacterial liquid b, and stirring them, with assuming 4 L of first fermented liquid n1 is to be produced. As apparent from the photograph A, three layers are clearly formed in the first fermented liquid A, including a supernatant sponge layer sp, a deposition layer dep, and, between these two layers, an highly transparent intermediate liquid n1 corresponding to 4 L.
[0186] The photograph B shows the fermented liquid B for compering with the photograph A, the fermented liquid B being produced by fermenting the sample B for 6 days, wherein the sample B is prepared by soaking 60 g, i.e, same amount as that of sample A, of soybeans in water for 18 hours as it is, grinding the soybeans to produce a soybean paste without being preliminarily fermented, heating the soybean paste and a soft water w are heated to 55° C., then cooling them to 37° C., adding 0.8 L of seed bacterial liquid b, and stirring them. As apparent from the photograph B, although intermediate liquid having lower transparency and a deposition layer are formed, almost no supernatant sponge layer sp is formed, the sponge layer being necessary to fermentation environment for fermentation bacteria b1, as a result of producing the fermented liquid B under a condition using soybean paste without preliminary fermentation, different from that of the sample A.
[0187] The result of the comparative test shown in
[0188] It is apparent from the result of the test shown in photographs A and B in
Details of a Second Fermentation Line f2
[0189] A fermentation line f2 is configured by:
[0190] a first process p.sub.21 for preliminarily producing a second fermented medium m2 of pH 4.8± in a second medium tank 40, as shown in
[0191] a second process p.sub.22 for producing a third fermented liquid n3 of pH 4.5± in a third fermentation tank 300 and a third fermentation system s3, as shown in
[0192] The first process p.sub.21 of the second fermentation line f2 includes, as a preliminary step, preparing a second medium tank 40, provided with a perforated inner lid 41 having bores through which a fermentation gas passes, and to be sealed with a sheet 42, as shown in enlarged view of
[0193] The first process p.sub.21 further includes,
[0194] as shown in
[0195] fermenting them for two or three days with maintaining a fermentation condition at 37° C. to 40° C. to produce a preliminary fermented mixed medium pfm2 and a third preliminary fermented liquid pn3, as shown in
according to processes not shown,
[0196] grinding the preliminary fermented mixed medium pfm2 by a grinding means to the extent that seeds of Jujube are not collapsed, and returning the ground preliminary fermented mixed medium pfm2 to the second medium tank 40, and
[0197] blending the ground preliminary fermented mixed medium pfm2 with the third preliminary fermented liquid pn3 to produce a second fermented medium m2 of pH 4.8±.
[0198] The preliminarily prepared mixed medium pm2 is a mixed medium in which sterilized dried plants consisting of Jujube, Lycium Fruit, and Turmeric are mixed in a weight ratio of 200 to 210 g of Jujube, 110 to 120 g of Lycium Fruit, and 25 to 30 g of Turmeric for the second fermented liquid n2 corresponding to 35 L in total. Jujube (Taiso), Lycium Fruit (Kukoshi), and Turmeric (Ukon) are medicinal natural products and listed in crude drugs in the Japanese Pharmacopoeia. Taiso is a fruit of jujube, Kukoshi is a fruit of Lycium, and Ukon is a rhizome of a plant of Zingiberaceae. Based on inventor's steady practice for many years, these plants were selected from many crude drugs.
Technical Feature of the Second Fermentation Line f2
[0199] Based on their try-and-error practice for many years, the inventors have achieved the first process p.sub.21 of the second fermentation line f2, in which the mixed medium pm2 is prepared by mixing 200 to 210 g of Jujube, 110 to 120 g of Lycium Fruit, and 25 to 30 g of Turmeric (weight ratio of 7:4:1) per 35 L of the second fermented liquid n2 in total, the mixed medium pm2 is preliminarily fermented in the second medium tank 40 to produce the preliminary fermented mixed medium pfm2 and the third preliminary fermented liquid pn3, as shown in
[0200] When the third process p.sub.13 is integrated in the first fermentation line f1, a process partly overlapped with the process p.sub.13 for fermenting the second fermented liquid n2 is preferably adopted as the first process p.sub.21 of the second fermentation line f2.
[0201] First technical reason for adopting a concurrent parallel arrangement in which the first process p.sub.21 and the process p.sub.13 for fermenting the second fermented liquid n2 are partly overlapped, instead of linearly arranging the first process p.sub.21 and the process p.sub.13, is for changing a fermentation environment of the fermentation bacteria b1 during the fermentation process. By changing the fermentation environment, secretion and fermentation of acidic material by the fermentation bacteria b1 are further facilitated, and the second fermented medium m2 can be produced more rapidly.
[0202] Second technical reason is for adjusting a start time of the process p.sub.22 for fermenting the third fermented liquid n3 to an end time of the process p.sub.13 for fermenting the second fermented liquid n2. It makes possible to start the first process p.sub.21 of the second fermentation line f2 for producing the second fermented medium m2, without waiting the end of the process p.sub.13 for fermenting the second fermented liquid n2, which is the third process of the first fermentation line f1. As a result, the second process p.sub.22 of the second fermentation line f2 for producing the third fermented liquid n3 is started no later than the end of the third process p.sub.13 for fermenting the second fermented liquid n2 of the first fermentation line f1.
[0203] A technical meaning of the second fermentation line f2 using the second fermented medium m2 prepared by preliminarily fermenting the dried plants pm2 of the selected three crude drugs in the first process p.sub.21 is demonstrated from a result of a comparative test for samples A and B of fermentation models of the third fermentation liquid n3 shown
[0204] The second fermentation line f2 comprises a process p.sub.21 for producing a second fermented medium m2. The process p.sub.21 comprises a preliminary process pp.sub.21. In the preliminary process pp.sub.21, a mixed medium pm2 is preliminarily fermented with a part of the second fermented liquid n2 in a second fermentation tank 40, to produce a third preliminary fermented liquid pn3 and a preliminary fermented mixed medium pfm2, wherein the mixed medium pm2 is prepared by mixing 200 to 210 g of Jujube, 110 to 120 g of Lycium Fruit, and 25 to 30 g of Turmeric (weight ratio of 7:4:1) per 35 L of the second fermented liquid n2 in total. The process p.sub.21 further comprises a preliminary process pp.sub.22. In the preliminary process pp.sub.22, the preliminary fermented mixed medium pfm2 are ground by a grinding means to the extent that seeds of Jujube are not collapsed, returned to the second medium tank 40, and blended with the third preliminary fermented liquid pn3 to produce the second fermented medium m2 of pH 4.8±.
[0205] The second fermentation line f2 further comprises a process p.sub.22 for producing a third fermented liquid n3 of pH 4.5±. The process p.sub.22 shown in
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[0207] The sample A is a fermented liquid A prepared by preliminarily fermenting 1 L of second fermented liquid n2 and a mixed medium pm2 consisting 5.71 g of Jujube, 3.14 g of Lycium Fruit, and 0.85 g of Turmeric (weight ratio of 7:4:1) corresponding to 1 L of the second fermented liquid n2, enclosing the preliminarily fermented second fermented medium m2 in a filter for crude drags, as a substitute for the bag-shaped medium filter, suspending the filter inside of a test container A, putting the second fermented liquid n2 and a third preliminary fermented liquid pn3 corresponding to 1 L as a starter into the test container A, stirring them, and fermenting them for 5 days with maintaining fermentation condition at 38° C. to 40° C. to form the fermented liquid A consisting of two layers including a supernatant fermentation gas layer and a layer of turbid third fermented liquid n3 (photograph A in
[0208] The sample B is a fermented liquid B prepared by, without preliminary fermentation, enclosing 1 L of second fermented liquid n2 and a mixed medium pm2 consisting 5.71 g of Jujube, 3.14 g of Lycium Fruit, and 0.85 g of Turmeric (weight ratio of 7:4:1) corresponding to 1 L of the second fermented liquid n2 in a filter for crude drags, as a substitute for the bag-shaped medium filter, suspending the filter inside of a test container B, putting the second fermented liquid n2 and a third preliminary fermented liquid pn3 corresponding to 1 L as a starter into the test container B, stirring them, and fermenting them for 5 days with maintaining fermentation condition at 38° C. to 40° C. to form the fermented liquid B consisting of a third fermented liquid n3 in which almost no supernatant fermentation gas layer is formed (photograph B in
[0209] The result of the comparative test demonstrates that the difference between the fermented liquids A and B may result from a difference in degree of fermentation due to secretion and metabolism of acidic materials by the fermentation bacteria b1. More specifically, the fermentation of the fermented liquid A due to secretion and metabolism by the fermentation bacteria b1 further progresses than that of the fermentation of liquid B.
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[0211] The graph of
[0212] In the first preliminary process pp.sub.21 of the second process p.sub.22 of the second fermentation line f2, as shown in
[0213] the second fermented medium m2 of pH 4.8± produced in the first process p.sub.21 is equally divided and enclosed in a plurality of steam-sterilized bag-shaped medium filters 320 suspended inside of the third fermentation tank 300,
[0214] the third preliminary fermented liquid pn3 produced in the first process p.sub.21 is transferred from the second medium tank 40 into the third fermentation tank 300,
[0215] the second fermented liquid n2 of pH 5.9± is transferred from the second fermentation tank 200 into the third fermentation tank 300 via the extraction unit 210 of the second fermentation tank 200, coupled to the third fermentation tank 300.
[0216] In the second preliminary process pp.sub.22 of the second process p.sub.22 of the second fermentation line f2,
[0217] a circulating pump unit 330 shown in
[0218] through the second fermented medium m2 of pH 4.8± enclosed in the bag-shaped medium filter 320 suspended inside of the third fermentation tank 300, such that the second fermented medium m2 is not mixed into other materials,
[0219] the third preliminary fermented liquid pn3 of pH 4.8± and the second fermented liquid n2 of pH 5.0± as a second starter in the third fermentation tank 300, and
[0220] ferment them for 8 to 9 days with maintaining fermentation condition at 37° C. to 40° C. to produce a third fermented liquid n3 of pH 4.5± consisting of a turbid liquid with supernatant second surface layer sf2.
[0221] In the second process p.sub.22 of the second fermentation line f2, the second fermented medium m2 is enclosed in fine mesh bag-shaped medium filter 320, and thus, any residue of the second fermented medium m2 is not deposited at the bottom of the third fermentation tank 300. It is confirmed that, during the second fermented liquid n2 changes to the third fermented liquid n3, the acidity of the third fermented liquid n3 proceeds to pH 5 or lower.
Details of a Third Fermentation Line f3
[0222] A third fermentation line f3 shown in
[0223] An overview of the third fermentation line f3 is shown in
[0224] The third fermentation line f3 includes a process [c] in which a part of the third fermented liquid n3 of the third fermentation tank 300 and an adequate amount of a honey material pm3 is put into a third medium tank 50, stirred, and fermented for 2 or 3 days with maintaining fermentation condition at 37° C. to 40° C. to produce a third fermented medium m3.
[0225] The third fermentation line f3 further includes processes [d] to [g] in which the third fermentation liquid n3 transferred from the third fermentation tank 300 and the third fermented medium m3 are put into a fourth fermentation tank 400, stirred, and fermented for 30 to 60 days with maintaining fermentation condition at 37° C. to 40° C. to produce a fourth fermented liquid n4.
[0226] More specifically, the first process p.sub.31 comprises a first preliminary process pp.sub.31 in which a honey material pm3 corresponds to 3 to 5% of 1050 to 1100 L of the third fermented liquid n3 produced as shown in
[0227] The first process p.sub.31 further comprises a second preliminary process pp.sub.32 in which the preliminary fermented medium pfm3 produced in the first preliminary process pp.sub.31 is fermented for 2 or 3 days with maintaining fermentation condition at 37° C. to 40° C. to produce a third fermented medium m3 of pH 4.4± from the honey material pm3.
[0228] In the second process p.sub.32 of the third fermentation line f3, as shown in
[0229] the third fermented medium m3 of pH 4.4± is put into a fourth fermentation tank 400,
[0230] the third fermented liquid n3 of pH 4.5± is transferred into the fourth fermentation tank 400 by a extraction unit 310 of the third fermentation tank 300, coupled to the fourth fermentation tank 400,
[0231] the third fermented medium m3 of pH 4.4± and the third fermented liquid n3 of pH 4.5± are mixed and stirred as a fourth starter, and fermented for 30 to 60 days with maintaining fermentation condition at 37° C. to 40° C. to form a supernatant third surface layer sf3 and a third deposition layer dep 3 containing a deposition at a bottom layer, and
[0232] produce a translucent third intermediate layer liquid corresponding to 1000 to 1050 L between the third surface layer sf3 and the third deposition layer dep 3. The translucent third intermediate layer is a fourth fermented liquid n4 of pH 3.7±.
Technical Feature of the Third Fermentation Line f3
[0233] The inventors conceived the third fermentation line f3 based on their steady try-and-error practice for many years, and have repeated try-and error to enhance growth and ability of secretion and metabolism of the fermentation bacteria b1 by providing certain amount of sugar to the fermentation bacteria b1, and achieve continuous fermentation due to further secretion and metabolism of acidic materials.
[0234] With repeating the try-and-error, the inventors found that a suitable sugar amount for maintaining the ability of the secretion and metabolism of the fermented bacteria b1 may correspond to a honey material pm3 corresponding to 3 to 5% of the third fermented liquid n3, based on a degree of water activity (if it is too low, activity of bacteria in water is inhibited and the bacteria cannot grow). The honey material pm3 was mixed and stirred with the third fermented liquid n3 four times as much as the amount of the honey material pm3 to produce a preliminary fermented medium pfm3. The preliminary fermented medium pfm3 was fermented for 2 or 3 days with maintaining fermentation condition at 37° C. to 40° C. to produce a third fermented medium m3.
[0235] Further, the third fermented medium m3 was mixed and stirred with a third fermented liquid n3 twenty times as much as the amount of the third fermented medium m3, and fermented for 30 to 60 days with maintaining fermentation condition at 37° C. to 40° C. to facilitate the fermentation due to the secretion and metabolism of acidic material by fermentation bacteria b1. In this manner, the inventors succeeded to produce a fourth fermented liquid n4 of pH 3.7±. This is the third fermentation line f3. The technical feature of the third fermentation line f3 is shown in
[0236]
[0237] The sample A is a fermented liquid A, which is a fourth fermented liquid n4 prepared by mixing a third fermented medium m3 produced by preliminarily fermenting a honey material pm3 with a third fermented liquid n3 twenty times as much as the volume of the third fermented medium m3 such that total amount becomes 1 L, stirring them, and fermenting them for 4 days with maintaining fermentation condition at 38° C. to 40° C. The sample B is a fermented liquid B, which is a fourth fermented liquid n4 prepared by mixing a honey material pm3 (third medium pm3) as it is with a third fermented liquid n3 twenty times as much as the volume of the third medium pm3 such that total amount becomes 1 L, stirring them, and fermenting them for 4 days.
[0238] The sample A is the fermented liquid A prepared by putting the third fermented medium m3 produced by preliminarily fermenting the honey material pm3 into one test container, stirring them, and fermenting them for 4 days to form three layers including a supernatant third surface layer sf3 of fermentation gas layer, a third deposition layer dep 3 containing deposition deposited at bottom layer, and a translucent intermediate layer liquid between the third surface layer sf3 and the third deposition layer dep 3. The translucent third intermediate layer liquid corresponds to the fourth fermented liquid n4.
[0239] The sample B is the fermented liquid B prepared by putting the honey material pm3 as it is into other test container, stirring them, and fermenting them for 4 days. From photograph B, it is confirmed that a supernatant third surface layer sf3 is formed, but much thinner than that of the sample A. It reveals that the fermentation of the sample B due to secretion and metabolism of acidic materials by the fermentation bacteria b1 is less active than that of the sample A. This is confirmed from
[0240]
[0241]
Details of a Fourth Fermentation Line f4
[0242] In the fourth fermentation line f4 shown in
[0243] The process p.sub.41 of the fourth fermentation line f4 is a process for producing the fifth fermented liquid n5 via following processes.
[0244] In the first fermentation line f1, the first fermented liquid n1 is produced in two first fermentation tanks 100 by using the first fermented medium m1 and transferred into one second fermentation tank 200 in the first process p.sub.11 and the second process p.sub.12, and the second fermented liquid n2 is produce in a fermentation environment without using any fermented medium in the third process p.sub.13. In the second fermentation line f2, the third fermented liquid n3 is produced by using the second fermented liquid n2 produced in the third process p.sub.13 of the first fermentation line f1 and the second fermented medium m2 produced in the second medium tank 40. In the third fermented line f3, the fourth fermented liquid n4 is produced by using the third fermented liquid n3 produced in the second fermentation line f2 and the third fermented medium m3 produced in the third medium tank 50. In the fourth fermentation line f4, the fifth fermented liquid n5 is produced in a fermentation environment without using any fermented medium. A fifth starter for the process p.sub.41 of the fourth fermentation line f4 is only the fourth fermented liquid n4, and any new medium or fermented medium is not used.
Technical Feature of the Fourth Fermentation Line f4
[0245] It seems that there is no significant change between an acidity of the fourth fermented liquid n4 of pH 3.7± and an acidity of the fifth fermented liquid n5 of pH 3.6±. However, as apparent from
[0246] A technical intention for the fourth fermentation line f4 is referred. The inventors once considered in the course of their steady try-and-error practice for many years, that their intended method for producing a fermented liquid has been completed by the third fermentation line f3. At that time, the inventor did not suppose a technical necessity of the fourth fermentation line f4 following the third fermentation line f3. However, activity of secretion and metabolism of acidic materials by the fermentation bacteria b1 did not settle even at the end of the third fermentation line f3. Therefore, the inventors had repeatedly tried to stably produce the fourth fermented liquid n4 at the third fermentation line f3 by making various modifications such as heat sterilization of the fermentation bacteria b1. As a result, the inventors obtained a technical knowledge that the fermentation bacteria b1 including spore-forming fermentation bacteria can form spores and survive even under severe conditions such as high temperature, dryness, and poor nutritional status, and thus, it is difficult to treat such bacteria by heat sterilization etc.
[0247] Based on such technical knowledge, the inventors focused on a technical problem whether it is necessary to terminate the active secretion and metabolism of acidic materials by the fermentation bacteria b1 at the end of the third fermentation line f3, or continue it even taking longer time. Then the inventors got to find a technical value of secretion and metabolism of acidic materials by the fermentation bacteria b1 at or after the fourth fermentation line f4, to successfully achieve the present invention.
Details of a Fifth Fermentation Line f5
[0248] The fifth fermentation line f5 shown in
[0249] a first process p.sub.51 for once rapidly cooling the fifth fermented liquid n5 produced in the fifth fermentation tank 500 in the fourth fermentation line f4, and returning the rapidly cooled fifth fermented liquid n5 to an ordinary temperature condition, and
[0250] a second process p.sub.52 for fermenting the fifth fermented liquid n5 by the fermented bacteria b1 contained in the fifth fermentation liquid n5, under the ordinary temperature condition, to produce a sixth fermented liquid n6 of pH 3.3±corresponding to 1000 to 1050 L.
[0251] The sixth fermented liquid n6 is produced by fermenting the fifth fermented liquid n5 only by the fermented bacteria b1 contained in the fifth fermented liquid n5. It is similar manner as that of the fifth fermented liquid n5 produced by fermenting the fourth fermented liquid n4 only by the fermentation bacteria b1 contained in the fourth fermented liquid n4 in the fourth fermentation line f4. However, a fermentation environment for producing the sixth fermented liquid n6 is critically different from that for the fifth fermented liquid n5 in the following two respects. There exist technical features of the fifth fermentation line f5.
Technical Feature of the Fifth Fermentation Line f5
[0252] The first technical feature of the fifth fermentation line f5 is shown in
[0253] A technical intention of the fifth fermented line f5 is shown in
[0254] The fifth fermented liquid n5, containing the fermentation bacteria b1 terminating its secretion and metabolism by rapid cooling show in
[0255] The second technical feature of the fifth fermentation line f5 is to create a fermentation environment at an ordinary temperature, specifically 5° C. to 28° C. according to the seasons, at which the fermentation bacteria b1 hardly act, rather than a fermentation condition at 37° C. to 40° C. at which the fermentation bacteria b1 can easily act, and perform long-term fermentation for 180 to 240 days (6 to 8 month) as shown in stage 3 of the
Function and Effect of the Invention
[0256] In principle, a first fermented liquid n1 of pH 5.3 produced according to the present invention is an initial fermented liquid for producing the final product, that is a sixth fermented liquid n6 of pH 3.3±. The first fermented liquid n1 is produced by fermenting an original solution θ for producing a fermented liquid having an acidity of pH 3 to 4 and comprising colloidal particles having a particle size not exceeding 50 nm and short-chain fatty acids, as a starter, for 50 to 100 days with maintaining fermentation condition at 37° C. to 40° C. as shown in stage 1 in
[0257] The reason why the turbid liquid (the second preliminary fermented liquid pn2) used as a starter for the first fermented liquid n1 is referred as an original solution θ is that the soft water w and the seed bacterial liquid b including the spore-forming fermented b1 are only used in producing the first fermented liquid n1. No soft water and seed bacterial liquid b are newly added in producing the second fermented liquid n2 to the six fermented liquid n6. Each of the processes for producing the second fermented liquid n2 to the six fermented liquid n6 establishes suitable fermentation environment to activate secretion and metabolism of acidic materials by the seven species of fermentation bacteria b1 including spore-forming fermentation bacteria contained in the first fermented liquid n1, and facilitate fermentation at each process to increase acidity of each fermented liquid.
[0258] The seed bacterial liquid consisting of seven species of fermentation bacteria b1 including spore-forming fermentation bacteria b1 is an essential component of the method for producing a fermented liquid having an acidity of pH 3 to 4 and comprising colloidal particles having a particle size not exceeding 50 nm and short-chain fatty acids according to the present invention. The seed bacterial liquid b consists of seven species of fermentation bacteria b1 including spore-forming fermentation bacteria b1 shown in
[0259] The inventors had repeated try-and-error in the course of their practice for many ears, and finally found and selected the seven species of fermented bacteria b1 shown in
[0260] The seed bacterial liquid b is produced from the three layers formed in the two first fermentation tanks 100, including the sponge layer sp, the translucent intermediate layer, and the first deposition layer dep. More particularly, the supernatant sponge layer sp and the first deposition layer dep deposited at the bottom are removed from the first fermentation tanks 100 and mixed to form paste, which is the seed bacterial liquid b. Any of the three layers formed in the two first fermentation tanks 100 includes the seven species of the fermentation bacteria b1 including spore-forming bacteria configuring the seed bacterial liquid b.
[0261] The sponge layer sp and the first deposition layer dep excluding the translucent intermediate layer formed in the first fermentation tanks 100 becomes the seed bacterial liquid b, such that, when a charge amount for each of the first fermentation tanks 100 is assumed to be 900 L, ⅓ of the charge amount, that is 300 L of materials become a raw material for the seed bacterial liquid b, and when corresponding amount of paste-like seed bacterial liquid b prepared from the row material is used as a seed bacterial liquid b for the next charge, three layers having similar quality to the former charge is formed in the first fermentation tank 100 to produce the first fermented liquid n1 of pH 5.3±.
[0262] The paste-like seed bacterial liquid b may be cryopreserved. It may be confirmed from the fact that when necessary amount of the cryopreserved seed bacterial liquid b is returned to a fermentation condition at 37° C. to 40° C. and used as a paste-like seed bacterial liquid b for the next charge, three layers having similar quality to the former charge is formed in the first fermentation tank 100, and the first fermented liquid n1 of the translucent intermediate liquid layer thus formed always has an acidity of pH 5.3±
[0263] Although the paste-like seed bacterial liquid b may be preserved for long period in a large refrigerator, it may also be used for next fermentation line f1 as it is at the fermentation temperature, immediately after its 50 to 100 days fermentation. According to the method for producing a fermented liquid comprising short-chain fatty of the present invention, it takes at least 303 days (10 months) to 477 days (16 months) to produce the sixth fermented liquid n6 of the final product. However, continuous production may be performed at an interval of within about 100 days, for the period the production of the first fermented liquid n1 being completed. Basically, the sixth fermented liquid n6 of pH 3.3±, which is the final product according to the present invention, may be continuously produced at an interval of within 100 days.
Fermented Liquid Produce by the Invention
[0264] The fermented liquid produced according to the present invention is a fermented liquid produced by using a soft water w as a starter, a seed bacterial liquid b comprising seven species of fermentation bacteria b1 including spore-forming fermentation bacteria, and three kinds of fermented medium derived from natural materials including dried soybeans, dried plants consisting of three kinds of crude drugs of Jujube, Lycium Fruit, and Turmeric, and a honey material, and fermenting them.
[0265] For the final product of sixth fermented liquid n6 of pH 3.3± produced according to the present invention, following analysis is performed, and activities of acidic materials produced due to secretion and metabolism by the fermentation bacteria b1 during the fermentation of the first fermented liquid n1 to the sixth fermented liquid n6 is considered, and components configuring the fermented liquid produced according to the present invention are estimated.
[0266] (1) Analysis of acidity of pH value of the first fermented liquid n1 to the sixth fermented liquid n6;
[0267] (2) Particle size analysis of the sixth fermented liquid n6
[0268] (3) Analysis of sugar concentration of the fourth fermented liquid n4 to the sixth fermented liquid n6; and
[0269] (4) Analysis of short-chain fatty acids for the second fermented liquid n2 to the sixth fermented liquid n6.
[0270] (1) Change of Acidity of pH Value of the First Fermented Liquid n1 to the Six Fermented Liquid n6:
[0271] The Inventors have daily produced the fermented medium and the fermented liquid according to the present invention, with monitoring the acidity of pH value of the fermented medium and the fermented liquid produced in each process described above.
[0272] As shown in
[0273] It should be noted that the first fermented medium m1 of pH 4.5± produced in the first fermentation tank 20, the seed bacterial liquid b, and large amount of the soft water w are put into the two first fermentation tanks 100 and stirred to produce a second preliminary fermented liquid pn2, which becomes an origin of, that is to say a starting point of the fermented liquid produced according to the present invention. After this point, any additional seed bacterial liquid b and soft water w will not be added again. Therefore, the second preliminary fermented liquid pn2 of pH 6.4± corresponds to an original solution θ for producing a sixth fermented liquid n6 of pH 3.3±, which is the final product produced according to the present invention. The second preliminary fermented liquid pn2 of pH 6.4± is fermented for 50 to 100 days at an appropriate fermentation temperature to produce a first fermented liquid n1 of pH 5.3±.
[0274] It should be also noted that the first fermented liquid n1 is fermented only by fermented bacteria b1 for 3 to 5 days at an appropriate fermentation temperature to produce a second fermented liquid of pH 5.0± in the second fermentation tank 200 at the last section of the stage 1. The last section overlaps with the process for producing a second fermented medium m2 of pH 4.8± by 2 or 3 days fermentation configuring the second fermentation line f2.
[0275] As shown in
[0276] The following two change of the acidity should be noted. The first change is that, in the second fermentation line f2 using the second fermented medium m2 and the third fermentation line f3 using the third fermented medium m3, secretion and metabolism of acidic materials by the fermentation bacteria b1 is activated, and the acidity proceeds from pH 5.0± of the third fermented liquid n3 to pH 3.7± of the fourth fermented liquid n4. The second change is that, in the fourth fermentation line f4, only the fourth fermented liquid n4 is used as a starter and fermented for 30 to 60 days with maintaining fermentation condition at 37° C. to 40° C. to produce the fifth fermented liquid n5, an acidity is not so changed from pH 3.7± of the fourth fermented liquid n4 to pH 3.6± of the fifth fermented liquid n5.
[0277] Even at the end of the third fermentation line f3, an activity of the fermentation bacteria b1 for secretion and metabolism of acidic materials does not settle down. The inventors have utilized this fact, and found a technical value of the fermentation process of the fourth fermentation line f4, in which secretion and metabolism of acidic materials by the fermentation bacteria b1 may be facilitated even in mild fermentation, instead of terminating the active secretion and metabolism of acidic materials by the fermentation bacteria b1 at the end of the third fermentation line f3. Since the fermentation bacteria b1 including spore-forming fermentation bacteria can form spores and survive even under severe conditions such as high temperature, dryness, and poor nutritional status, it is difficult to treat such bacteria by heat sterilization etc., and the inventors have noticed the merit of facilitating the fermentation of the fourth fermented liquid n4 even it takes longer time.
[0278] As shown in
[0279] It seems that the fifth fermentation line f5 is technically similar to a process for maturing the fifth fermented liquid n5 of pH 3.6±. However, in fact, the fifth fermentation line f5 is a process for facilitating secretion and metabolism of acidic materials by the fermentation bacteria b1, while allowing the metabolism activity settle down, and finally terminating secretion and metabolism of the fermentation bacteria b1 to produce a sixth fermented liquid n6 pH 3.3± in which the spore-forming fermentation bacteria b1 is in dormant state.
[0280] The change of acidity of pH value in each fermentation period is as follows. At a start of the practical fermentation in the two first fermentation tanks 100, the acidity is pH 6.4±, which is an acidity of the original solution θ, that is the second preliminary fermented liquid pn2. At the end of the first stage, the acidity proceeds to pH 5.0±. From the end of the first stage, the fermentation proceeds due to the secretion activity of the fermentation bacteria b1 included in the second fermented liquid n2. To the end of the second stage, the acidity proceeds to pH 3.6± with maintaining appropriate fermentation temperature at 37° C. to 40° C. During the process, even if the fermented liquid is contaminated with any non-spore-forming fermentation bacteria included in the seed bacterial liquid b or any aerobic bacteria, growth of such bacteria will be suppressed under the highly acidic environment, and killed bacteria will be deposited as a deposition layer at the bottom of each tank.
[0281] In the third stage of the long-term fermentation in the fermentation environment of ordinary temperature condition according to seasons, the fermentation proceeds with facilitating secretion activity of spore-forming fermentation bacteria included in the fermentation bacteria b1, and finally leads the secretin activity of the fermentation bacteria b1 including the spore-forming fermentation bacteria to almost the end of its activity. The fermentation no more proceeds and enters its stable state at acidity of pH 3.3±.
[0282] (2) Particle Size Analysis of the Sixth Fermented Liquid n6
[0283] The inventors have noted, based on their steady practical work, that the fermented liquid produced according to the present provides extremely high absorption ratio in vivo in mammalians. The inventors requested an analysis for configuration of the fermented liquid to certain professional laboratory and confirmed that the fermented liquid contains about 6.5% to 7.5% of colloidal particles. In particular, the inventors requested a particle size distribution analysis based on a cumulant analysis for following two kinds of fermented liquids produced according to the present invention to Industrial Technology Center of Gifu Prefectural Government and obtained results of the analysis.
[0284] The two kinds of the fermented liquids produced according to the present invention are a fourth fermented liquid n4 of pH 3.7± and a sixth fermented liquid n6 of pH 3.3±. The particle size distribution analysis based on the cumulant analysis are prepared as follows.
[0285] The two kinds of samples provided to the Center are a first sample (Calbio-5) and a second sample (Calbio-7). The first sample (Calbio-5) was prepared by a process in which the fourth fermented liquid n4 of pH 3.7± produced from the fermentation using the fermentation media m1 to m3 was heat-sterilized, then cooled, and settled for one day, and 200 mL of sample from the supernatant of the settled fermented liquid was sterilized, and then divided into two 100 mL bottles. The second sample (Calbio-7) was prepared by a process in which the sixth fermented liquid n6 of pH 3.3± produced from the long-term fermentation of the fifth fermented liquid n5 of pH 3.6± for 180 days (6 months) to 240 days (8 months) in ordinary temperature fermentation environment, the fifth fermented liquid n5 being produced from the fermentation only by the fermentation bacteria b1 without using any fermented medium, was heat-sterilized, then cooled, and settled for one day, and 200 mL of sample from the supernatant of the settled fermented liquid was sterilized, and then divided into two 100 mL bottles.
[0286] In particular, the first sample (Calbio-5) is prepared as follows. The last fermented medium m3 (pH 4.4±) and a third fermented liquid n3 (pH 4.5±) as a starter is fermented for 30 to 60 days with maintaining fermentation condition at 37° C. to 40° C. to form a supernatant third surface layer, a third deposition layer dep3 containing deposition at a bottom layer, and a translucent third intermediate layer liquid between the third surface layer sf3 and the third deposition layer dep 3. The translucent third intermediate layer liquid is the fourth fermented liquid n4 of pH 3.7±, which becomes the first sample. The first sample corresponds to a fermented liquid produced in last fermentation process using fermented medium.
[0287] In particular, the second sample (Calbio-7) is prepared as follows. The fifth fermented liquid n5 of pH 3.6± is used as a starter, without using any medium or fermented medium, and the fifth fermented liquid n5 maintained at appropriate fermentation condition at 37° C. to 40° C. is once rapidly cooled to 4 to 5° C. or lower, that is an environment in which the fermentation bacteria b1 hardly activate the secretion and metabolism, and then the environment is shifted to an ordinary temperature environment in which the fermentation bacteria b1 can moderately activate the secretion and metabolism. The fermentation proceeds with facilitating secretion activity of spore-forming fermentation bacteria included in the fermentation bacteria b1, and finally leads the secretin activity of the fermentation bacteria b1 including the spore-forming fermentation bacteria to almost the end of the activity. The fermentation no more proceeds and provide the final product of the sixth fermented liquid n6 having an acidity of pH 3.3±. The sixth fermented liquid n6 becomes the second sample.
[0288]
[0289] x=0.375 μm (y=0.24%) to 18.5 μm (y=0.17%).
[0290]
[0291] x=1.1 nm (y=5.7%) to 6.6 nm (y=0.1%).
[0292] Difference between
[0293] The inventors once considered that their intended method for producing a fermented liquid has been completed by the third fermentation line f3 and tried to treat the fermentation bacteria b1 in order to terminate metabolism activity for secreting acidic materials. The inventors also noted that spore-forming fermentation bacteria included in the fermentation bacteria b1 can form spores and survive even under severe conditions, and thus it is difficult to treat such bacteria to terminate activity thereof. After that, the inventors, via the course of their try-and-error practice, utilize the metabolism activity of the fermentation bacteria b1 including active spore-forming fermentation bacteria and ferment the fourth fermented liquid n4 containing micron size particles as a starter for 30 to 60 days to produce the fifth fermented liquid n5 of pH 3.6±.
[0294] The inventors fermented the fifth fermented liquid n5 as a last starter for long term of 180 days (6 months) to 240 days (8 months) at a fermentation environment at an ordinary temperature to gradually settle down secretion activity of the fermentation bacteria b1, and consequently, gradually increase an acidity due to natural fermentation by the fermentation bacteria b1 including spore-forming fermentation bacteria, to finally succeed in stabilizing the acidity at pH 3.3±. Furthermore, unexpected result can be achieved in that the sixth fermented liquid n6 containing colloidal particles of nanometer unit in size can be obtained. It goes without saying that when such fermented liquids are used as fermentation foods, significant difference in an absorption ratio in vivo occurs between the fermented liquid containing colloidal particles of micron unit in size and the fermented liquid containing colloidal particles of nanometer unit in size.
[0295] Second analysis of performance of the fourth fermented liquid n4 and the sixth fermented liquid n6 is to measure amount of sugar contained in each fermentation liquid. The inventors produced the third fermented medium m3 from the honey material pm3, mixed the third fermented medium m3 with the third fermented liquid n3 corresponding twenty times as much as the fermented medium m3, stirred them to establish an fermentation environment having a sugar concentration of 3 to 5%, which is the best fermentation environment for secretion activity of the fermentation bacteria b1, fermented them for 30 to 60 days with maintaining fermentation condition at 37° C. to 40° C. to further facilitate secretion and metabolism of acidic materials by the fermentation bacteria b1, and succeeded in producing the fourth fermented liquid n4 of pH 3.7±.
[0296] (3) Analysis of Sugar Concentration of the Fourth Fermented Liquid n4 to the Sixth Fermented Liquid n6
[0297]
[0298] c1=3.39 g/100 mL
[0299] c2=2.88 g/100 mL
[0300] c3=1.19 g/100 mL
[0301] The inventors conceived the third fermentation line f3 based on their steady try-and-error practice for many years, and have repeated try-and error, to enhance growth and ability of secretion and metabolism of the fermentation bacteria b1 by providing certain amount of fermented medium obtained by fermenting honey materials to the fermentation bacteria b1, and achieve continuous fermentation due to further secretion and metabolism of acidic materials.
[0302] The inventors found that a certain sugar amount for maintaining the ability of the secretion and metabolism of the fermented bacteria b1 may correspond to a honey material pm3 corresponding to 3 to 5% of the third fermented liquid n3, based on a degree of water activity. The honey material pm3 are mixed and stirred with the third fermented liquid n3 four times as much as the amount of the honey material pm3 to produce a preliminary fermented medium pfm3. The preliminary fermented medium pfm3 are fermented for 2 or 3 days with maintaining fermentation condition at 37° C. to 40° C. to produce a third fermented medium m3. Further, the third fermented medium m3 are mixed and stirred with a third fermented liquid n3 twenty times as much as the amount of the third fermented medium m3, and fermented for 30 to 60 days with maintaining fermentation condition at 37° C. to 40° C. to facilitate the fermentation due to the secretion and metabolism of acidic material by fermentation bacteria b1 In this manner, the inventors successfully produced the fourth fermented liquid n4 of pH 3.7±. This is the third fermentation line f3.
[0303]
[0304] For the sample A, three layers are formed in a test container, wherein the tree layers include a translucent third intermediate layer liquid between a third surface layer sf3 and a third deposition layer dep 3, and translucent third intermediate layer liquid corresponds to the fourth fermented liquid n4. For the sample B, it is confirmed from photograph B that a surface layer sf3 is formed in a test container, but much thinner than that of the sample A. It reveals that the fermentation of the sample B due to secretion and metabolism of acidic materials by the fermentation bacteria b1 is less active than that of the sample A. This is confirmed from
[0305]
[0306] The inventors requested sugar concentration analysis on the fourth to sixth fermented liquid samples by Somogyi modified method to Japan Food Research Laboratories. The fourth fermented liquid n4 of pH 3.7± was produced as described above. The fifth fermented liquid n5 of pH 3.6± was produced by fermenting the fourth fermented liquid n4 of pH 3.7± as a starter, only by the fermentation bacteria b1 without using any fermented medium. The sixth fermented liquid n6 of pH 3.3± was produced by fermenting the fifth fermented liquid n5 of pH 3.6± as a starter for long term of 180 days (6 months) to 240 days (8 months), only by the fermented bacteria b1 without using any fermented medium. All fermented liquids were heat-sterilized, then cooled, and settled for one day. Each of 200 mL of supernatant of the settled fermented liquids was sterilized, then divided into two 100 mL bottles, and provided to the Japan Food Research Laboratories.
[0307] The result of the analysis is as described above. The fermented liquid n4 of pH 3.7± was prepared by using the honey material pm3 corresponding to 5% of the third fermented liquid n3, and thus 5 g of sugar is added per 100 mL of the third fermented liquid n3. The sugar amount was decreased to finally become 3.93 g per 100 mL in the fourth fermented liquid n4 after the fermentation. It may be a result of depletion of sugar due to the activity of the fermentation bacteria b1. The sugar amount of 2.88 g in the fifth fermented liquid n5 and 1.19 g in the sixth fermented liquid n6 may also be the result of the depletion of the sugar due to the activity of the fermentation bacteria b1.
[0308] From the result, it may be understood that the sugar concentration of the fourth fermented liquid n4 is nearly 4%, and the metabolism activity of the fermentation bacteria b1 of secreting acidic materials hardly settles down in the third fermentation line f3, and also it is difficult to perform various treatment such as heat-sterilization to the fermentation bacteria b1.
[0309] Also, the sugar concentration of the fifth fermented liquid n5 of 2.88 g/100 mL and the sixth fermented liquid n6 of 1.19 g/100 mL indicate that the sugar concentration may be decreased so as to facilitate the active secretion and metabolism by the fermentation bacteria b1 while readily leading the activity to settle down by long-term fermentation without performing severe treatment such as heat-sterilization.
[0310] The process of the forth fermentation line f4 for producing the fifth fermented liquid by 30 to 60 days fermentation and the process of the fifth fermentation lane f 5 for producing the sixth fermented liquid n6 by the long-term 180 days (6 months) to 240 days (8 months) fermentation are both the process for facilitating secretion activity of spore-forming fermentation bacteria contained in the fermentation bacteria b1 to allowing them forming spore, and finally provide an environment which is nearly dormant state for the fermentation bacteria b1.
[0311] (4) Analysis of Short-Chain Fatty Acids for the Second Fermented Liquid n2 to the Sixth Fermented Liquid n6
[0312] Constituents of the present invention are acidic materials produced by the secretion activity of the seven species of fermentation bacteria b1 in the seed bacterial liquid b consisting of the fermentation bacteria (International Accession No. NITE BP-02945 to NITE BP-02951) including spore-forming Clostridium bacteria maintained under low temperature state. As seen from acidic materials shown in
[0313] The second fermented liquid n2 is the final product of the first fermentation line f1. In the first fermentation line f1, the first fermented liquid n1 produced in the second process p.sub.12 is transferred from two first fermentation tanks 100 into one second fermentation tank 200 as shown in schematic diagram of
[0314] Change of each final product of fermentation lines was observed, for acidic materials produced by the secretion activity of the seven species of fermentation bacteria b1 in the seed bacterial liquid b consisting of the fermentation bacteria (International Accession No. NITE BP-02945 to NITE BP-02951) including spore-forming Clostridium bacteria maintained under low temperature state.
[0315] Samples are as follows:
[0316] (1) the second fermented liquid n2 of pH 5.0± produced in the first fermentation line f1 for 55 to 108 days;
[0317] (2) the third fermented liquid 3 of pH 4.5± produced by fermenting the second fermented liquid n2 as a starter for 8 to 9 days;
[0318] (3) the fourth fermented liquid n4 of pH 3.7± produced by fermenting the third fermented liquid n3 as a starter for 30 to 60 days;
[0319] (4) the fifth fermented liquid n5 of pH 3.6± produced by fermenting the fourth fermented liquid n4 as a starter for 30 to 60 days; and
[0320] (5) the sixth fermented liquid n6 of pH 3.3± produced by fermenting the fifth fermented liquid n5 as a starter for 180 to 240 days.
[0321] Each of the second fermented liquid (1) to the fifth fermented liquid n5 (4) is the final product of the fermentation with maintaining fermentation condition at 37° C. to 40° C. The sixth fermented liquid n6 (5) is the final product of the fermentation at an ordinary temperature fermentation environment different from that of (1) to (4).
[0322] All fermented liquids (1) to (5) were heat-sterilized, then cooled, and settled for one day. Each of 200 mL of supernatant of the settled fermented liquids was sterilized, then divided into two 100 mL bottles, and provided to the Japan Food Research Laboratories for analyzing short-chain fatty acids by high performance liquid chromatography (HPLC) The result of measurement of contents of short-chain fatty acids, including lactic acid, propionic acid, and butyric acid, contained in the second fermented liquid n2 to the sixth fermented liquid n6 was obtained.
[0323] The results are shown in
TABLE-US-00001 Kind of short-chain fatty acid Lactic acid Propionic acid butyric acid 2nd fermented liquid n2 ND (0 g) 0.01 g 0.18 g 3rd fermented liquid n3 0.08 g 0.01 g 0.30 g 4th fermented liquid n4 0.20 g 0.02 g 0.54 g 5th fermented liquid n5 0.13 g 0.02 g 0.59 g 6th fermented liquid n6 0.21 g 0.02 g 0.58 g
[0324] For the second fermented liquid n2, no lactic acid is detected, and 0.01 g/100 mL of propionic acid and 0.08 g/100 mL of butyric acid are detected. The fermented liquid produced according to the present invention is produced by fermenting natural materials. Among the short-chain acid, the contents of the butyric acid is 0.30 g/100 mL in the third fermented liquid n3, 0.54 g/100 mL in the fourth fermented liquid n4, and almost reaching 0.60 g/100 mL in the fifth fermented liquid n5 and the sixth fermented liquid n6 produced only by the fermentation bacteria b1 contained in the fourth fermented liquid n4 and the fifth fermented liquid n5 without using any medium or fermented medium. Evaluation of such high butyric acid contents will be made by basic research in future, but the content of 6 g of butyric acid per 1000 mL may be sufficiently expected to have considerable effect on at least generation of regulatory T-cells, related to anti-inflammation effect, improvement of bone strength, anti-obesity effect, anticancer effect, etc.
[0325] Finally, the sixth fermented liquid n6 is filtered through 0.2 μm-mesh filter to extract accumulated fermentation metabolite including short-chain fatty acid. The extract is used as a natural fermentation material for various application including a raw material for food or foodstuff itself. An example of the applicability of the fermented liquid is shown below.
Action and Effect of the Fermented Liquid Produced According to the Present Invention
[0326] The fermented liquid produced according to the present invention is a fermented liquid made from natural material, having an acidity of pH 3 to 4, comprising colloidal particles having a particle size not exceeding 50 nm and 5 g or more butyric acid per 1000 mL of the fermented liquid. Routine ingestion of the fermented liquid may activate calcium metabolism in living body, and significantly enhance bone strength and bone metabolism. It was confirmed by several tests such as “Test for calcium absorption evaluation by everted gut sac method” conducted by Tennen Sozai Tansaku Kenkyusyo, Inc., and a detailed test using osteoporosis-model mice for testing functional foods conducted by Department of Physiology, Gifu University School of Medicine.
[0327] Tennen Sozai Tansaku Kenkyusyo, Inc. (Representative Director: AOYAMA, Yoshiko) presented report of “Test for calcium absorption evaluation by everted gut sac method” on November 2015. The test was conducted on test animals, which were male rats of SD strain, and fed with pellet CRF-1 (Oriental Yeast Co., Ltd.) and distilled water from water bottles in free-feeding during preliminary keeping period. Test sample was the sixth fermented liquid n6 (Calbio-7).
[0328] Three test groups of test animals were set as follows:
[0329] Control group including 5 animals (9 gut sacs; 3 gut sacs for each of reactions for 30, 60, and 90 min.);
[0330] Group added with 0.1% of the sixth fermented liquid n6 (Calbio-7) including 5 animals (9 gut sacs; 3 gut sacs for each of reactions for 30, 60, and 90 min); and
[0331] Group added with 0.5% of the sixth fermented liquid n6 (Calbio-7) including 5 animals (9 gut sacs; 3 gut sacs for each of reactions for 30, 60, and 90 min.)
[0332] The test items and method were as follows:
(1) Preliminary keeping period was set to be 6 days. Animals had been preliminarily kept and conditioned with free-feeding of pellet CRF-1 before an actual keeping period starts.
(2) Then, absorption test with the everted gut sac method was conducted. The test includes:
a) preparing inside solution and outside solution, consisting of preparation of reagents and preparation of the inside and outside solutions; b) preparing everted gut sacs; c) subjecting the everted gut sacs to reaction for 30, 60, and 90 minutes; d) performing calcium measurement; and e) performing statistical analysis, wherein a test result was determined to be significant when the level of significance was 5% or lower. The experimental result of the calcium absorption test with the everted gut sac method is shown in
[0333]
[0334] In summarizing the test result, Calbio-7-added group of each concentrations presents significantly higher value in the calcium increment amount than that of the control group at all reaction period, and transfer from the outside solution to the inside solution is highest at a reaction period of 30 min. On the other hand, Calbio-7-added group of each concentration also present significantly higher values in calcium absorption ratio than that of the control group at all reaction period, and the absorption ratio is highest at a reaction period of 60 min.
[0335] The test using osteoporosis-model mice was performed at the Department of Physiology, Gifu University School of Medicine, and the detailed report of the test was made by Chikara ABE at the Department of Physiology and sealed by Shinya MINATOGUCHI, a director of the Gifu University School of Medicine on February 2017. This test was performed for an effect of administration of test material Calbio-7 on a bone mineral density and a bone metabolism marker using osteoporosis-model mice, considering the preceding test report of the “Test for calcium absorption evaluation by everted gut sac method” on November 2015. A part of the contents of this test are extracted and presented below.
[0336] Problems to be tested is related to an effect of administration of test materials on a bone mineral density and a bone metabolism marker using osteoporosis-model mice. An overview of the test is that the test materials were administered continuously for eight weeks to osteoporosis-model mice in estrogen-deficiency state caused by ovariectomy (OVX) to the ovaries both sides, and effects of the administration on prevention of osteoporosis were evaluated.
[0337] The experimental method is as follows:
[0338] As experimental animals, twelve-weeks-old rats C57BL/6 (female, n=24) were used. Four test groups were used, and each group included six rat, including normal Sham rats and ovariectomized OVX rats as follows: [0339] Sham+water (SWW), n=6; [0340] Sham+Calbio-7 (0.5 mL/day) (SCC), n=6; [0341] OVX+water (OWW), n=6; and [0342] OVX+Calbio-7 (0.5 mL/day) (OCC), n=6.
Administration plan of the test material was as follows
TABLE-US-00002 TABLE 1 Week 11 12 13 14 15 16 17 18 19 20 SWW water water water water water water water water water sampling SCC water C7 C7 C7 C7 C7 C7 C7 C7 OWW water water water water water water water water water OCC water C7 C7 C7 C7 C7 C7 C7 C7 C7: test material Calbio-7 (sixth fermented liquid n6) C7 was adjusted based on data of amount of drinking water, such that 0.5 mL was taken per day.
[0343] Test items relate to bone structure, and include acquiring data of the ratio of Gla-Osteocalcin, i.e., bone formation marker, to Glu-Osteocalcin, i.e., a bone resorption marker in blood, as well as acquiring data from tests for degree of effect on bone weight, bone strength and bone mineral density.
[0344]
[0345]
[0346] The left graph of
[0347] The left view of
[0348] The foregoing is an extracted part of this report, and at the conclusion section of the report, the experimenter indicates evaluation results (1) to (3) as follow.
[0349] (1) In this experiment, the ingestion of Calbio-7 improved the bone strength in OCC group. However, no difference was found between OCC and OWW groups in qualitative evaluation of the dried bone weight and the spongy bone trabeculae, and thus some factors other than bone structure (trabecular density) may be considered to exert an effect on recovery of the bone strength. Regarding states of bone formation and bone resorption, there was a tendency that OCC group had higher value than that of OWW group at eighth week. Some factor may be considered to activate calcium metabolism.
[0350] (2) Variation of female hormone balance due to Ovariectomy (OVX) may extremely strong, and thus calcium amount contained in normal feed may supposed to be insufficient to supplement consumed amount of calcium. The result of the test for calcium absorption effect using the everted gut suck method shows that the increment amount and the absorption rate of calcium are higher than that of control group on every calcium sources.
[0351] (3) Considering (1) and (2) and the fact that the ingestion of Calbio-7 can significantly prevent the bone strength reduction caused by Ovariectomy (OVX), it may be desirable to perform additional studies for improving variations of numerical values such as increased bone resorption caused by OVX, by using, in particular, calcium lactate-added feeds, since the calcium lactate provided pronounce increment in the absorption and increment rate (with reference to “Test for calcium absorption evaluation by everted gut sac method” on November 2015),
[0352] For functions of the fermented liquid made from natural material, having an acidity of pH 3 to 4, comprising colloidal particles having a particle size not exceeding 50 nm and 5 g or more butyric acid per 1000 mL of the fermented liquid according to the present invention to exert effects on living bodies, (1) test for effect of Calbio-7 ingestion on intestinal bacterial flora, and (2) test for anti-cancer effect were conducted on May 2014 by Itech Lab Inc. (a parson in charge of experiment is Masaki MATSUURA, and report writer is Shin NAKAMURA), at Kaizu city, Gifu prefecture, and reported. The test sample for these tests were also Calbio-7.
[0353] In the test (1), C57BL/6 mice (male ♂, 7-weeks old) were divided into a control group and Calbio-7-administered group (n=10), and water for injection was orally administered to the control group and Calbio-7 was orally administered to the Calbio-7-administered group at 0.5 mL/body for 28 days continuously.
[0354] In the test (1), after the administration period had completed, contents of colon were collected and analyzed or frozen and stored. Bacterial DNA was then extracted from the contents of colon using a kit dedicated for extracting bacterial nucleic acid. Concentrations of the resultant DNA were measured, and their purities were checked. When DNAs were extracted, equal amounts of samples from three or four individuals in the same group were pooled, and DNA was extracted from the pools. The bacterial DNA was quantified using primers specific for each of Lactobacillus, Bifidobacterium and Clostridium, and a universal primer to calculate relative copy number of each bacterial DNA, as well as each ratio to that of the control group.
[0355] Results of the test (1) as to effects of the ingestion of Calbio-7 on intestinal bacterial flora are shown in
[0356] Upper graphs of
[0357]
[0358] In the test (2), Lewis Lung carcinoma cell line, that is an epithelial cancer cell line, was used as a cancer tissue of the Calbio-7-administered mice, and comparative analysis is performed for gene expression of Krt6b as a tumor growth factor, compared with control group to which water for injection was administered. As a result, expression of keratinocyte (keratinized cell) gene group, such as keratin, is observed. That is, the Krt6b is a maker for identifying squamous-cell carcinoma in lung cancer, and inhibition of the expression of the Krt6b gene is observed as shown in
Summary of the Test Results
[0359] For functions of the fermented liquid made from natural material, having an acidity of pH 3 to 4, comprising about 6.5 to 7.5% of colloidal particles having a particle size not exceeding 50 nm and 5 g or more butyric acid per 1000 mL of the fermented liquid according to the present invention to exert effects on living bodies, the fermented liquid may be used as functional foods itself or ram material liquid for functional foods. First summary is that, as apparent from the data of “increased amount and absorption rate”, which is a result of the calcium absorption test with the everted gut sac method shown in
[0360] Second summary is that, as apparent from data in
Supplemental Information
[0361] Seven species of fermentation bacteria deposited to National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (domestically deposited on May 16, 2019, and transferred to the international deposit on Apr. 22, 2020) From the results of partial base sequencing (about 1500 bp) of 16S rDNA (16S rRNA gene) for isolated and cultured fermentation bacteria, used in producing fermented product by Higher Mount Co., Ltd., for identifying the bacteria, it was confirmed that the following bacteria are innocuous to human and animals, and able to be reconstituted from their dried or freeze-dried form.
NITE BP-02945
[0362] Type of microorganism: bacteria
Cell form: bacillus
Characteristics: spore-forming negative (non-spore-forming)
Taxonomical position: Aneurinibacillus sp.
Culture condition: standard agar medium
Culture temperature: 30° C.
Culture period: 48 hours
Culture method: aerobic
NITE BP-02946
[0363] Type of microorganism: bacteria
Cell form: bacillus
Characteristics: spore-forming negative (non-spore-forming)
Taxonomical position: Brevibacillus sp.
Culture condition: standard agar medium
Culture temperature: 30° C.
Culture period: 48 hours
Culture method: aerobic
NITE BP-02947
[0364] Type of microorganism: bacteria
Cell form: bacillus
Characteristics: spore-forming negative (non-spore-forming)
Taxonomical position: Pseudoclavibacter sp.
Culture condition: standard agar medium
Culture temperature: 30° C.
Culture period: 72 hours
Culture method: aerobic
NITE BP-02948
[0365] Type of microorganism: bacteria
Cell form: bacillus
Characteristics: spore-forming positive (spore-forming)
Taxonomical position: Paenibacillus sp.
Culture condition: standard agar medium
Culture temperature: 30° C.
Culture period: 72 hours
Culture method: aerobic
NITE BP-02949
[0366] Type of microorganism: bacteria
Cell form: bacillus
Characteristics: spore-forming positive (spore-forming)
Taxonomical position: Clostridium sp.
Culture condition: GAM Broth “Nissui” agar
Culture temperature: 30° C.
Culture period: 48 hours
Culture method: anaerobic
NITE BP-02950
[0367] Type of microorganism: bacteria
Cell form: bacillus
Characteristics: spore-forming positive (spore-forming)
Taxonomical position: Clostridium sp.
Culture condition: GAM Broth “Nissui” agar
Culture temperature: 30° C.
Culture period: 48 hours
Culture method: anaerobic
NITE BP-02951
[0368] Type of microorganism: bacteria
Cell form: bacillus
Characteristics: spore-forming positive (spore-forming)
Taxonomical position: Clostridium sp.
Culture condition: GAM Broth “Nissui” agar
Culture temperature: 30° C.
Culture period: 48 hours
Culture method: anaerobic
REFERENCE SIGNS LIST
[0369] 1 fermented liquid having an acidity of pH 3 to 4 comprising colloidal particles having a particle size not exceeding 50 nm and short-chain fatty acids [0370] n1 to n6 first fermented liquid to sixth fermented liquid [0371] pn1 to pn3 first preliminary fermented liquid to third preliminary fermented liquid [0372] w soft water [0373] m fermented medium [0374] m1 to m3 first fermented medium to third fermented medium [0375] ds dried soybeans [0376] fs fermented soybeans [0377] gfs soybean paste [0378] pm2 mixed medium [0379] pfm2 preliminary fermented mixed medium [0380] pm3 honey material [0381] pfm3 preliminary fermented medium [0382] b seed bacterial liquid [0383] b1 fermentation bacteria (International Accession Numbers NITE BP-02945 to NITE BP-0291) contained in the seed bacterial liquid [0384] sp sponge layer [0385] sf1 to sf4 first surface layer to fourth surface layer [0386] dep1 to dep4 first deposition layer to fourth deposition layer [0387] f fermentation stage [0388] f1 to f5 first fermentation line to fifth fermentation line [0389] p.sub.11 to p.sub.13 first process to third process of f1 [0390] p.sub.21/p.sub.22: first process/second process of f2 [0391] p.sub.31/p.sub.32 first process/second process of f3 [0392] p.sub.41 process of f4 [0393] p.sub.51 process of f5 [0394] 10 fermentation bottle (pottery) [0395] 20 first medium tank in f1 [0396] 30 heating kettle in f1 [0397] 40 second medium tank in f2 [0398] 50 third medium tank in f3 [0399] 100 (two) first fermentation tanks in f1 having a volume of 1000 liter [0400] 101 shut-off valve for first fermentation tank, utilizing Pascal's principle and hydrostatic equilibrium [0401] 110 extraction unit having a shut-off valve for first fermentation tank [0402] 200 second fermentation tank in f1 having a volume of 2000 liter [0403] 201 shut-off valve for second fermentation tank, utilizing Pascal's principle and hydrostatic equilibrium [0404] 210 extraction unit having a shut-off valve for second fermentation tank [0405] 300 third fermentation tank in f2 having a volume of 2000 liter [0406] 301 shut-off valve for a third fermentation tank, utilizing Pascal's principle and hydrostatic equilibrium [0407] 310 extraction unit having a shut-off valve for a third fermentation tank [0408] 320 bag-shaped medium filter in f2 [0409] 330 circulating pump unit in f2 [0410] 400 fourth fermentation tank in f3 having a volume of 2000 liter [0411] 401 shut-off valve for fourth fermentation tank, utilizing Pascal's principle and hydrostatic equilibrium [0412] 410 extraction unit having a shut-off valve for fourth fermentation tank [0413] 500 fifth fermentation tank in f4 having a volume of 2000 liter [0414] 501 shut-off valve for fifth fermentation tank, utilizing Pascal's principle and hydrostatic equilibrium [0415] 510 extraction unit having a shut-off valve for fifth fermentation tank [0416] 600 sixth fermentation tank in f5 having a volume of 2000 liter [0417] 610 cooling unit in f5 [0418] 620 reserve tank in f5