Method for improving gastric acid and bile acid resistance of lactobacillus paracasei

Abstract

The present invention provides a novel method for improving gastric acid and bile acid resistance of Lactobacillus paracasei, which is characterized in that lactic acid bacteria and Lactobacillus paracasei are mixed and cultured, and the Lactobacillus paracasei and Lactobacillus paracasei are mixed and cultured so as to obtain Lactobacillus paracasei with gastric acid and bile acid resistance. The lactic acid bacteria have a pH value of 6.0-6.5 when the culture time is 4 hours, a pH value of 5.0-6.0 when the culture time is 8 hours, a pH value of 4.5-5.5 when the culture time is 12 hours, and a pH value of 4.0-4.5 when the culture time is 24 hours when a pre-culture bacteria solution of 0.01 mass% lactic acid bacteria is inoculated in a 10 mass% skimmed milk culture medium and the culture medium is left to stand in a thermostatic bath at 37 DEG C for culture. Or inoculating a culture medium containing 0.1-20% by mass of skimmed milk with a pre-cultured bacterial solution of Lactobacillus paracasei, and culturing in such a manner that the pH value is 5.5-6.25 when the culture time is 8 hours, the pH value is 4.25-5.5 when the culture time is 16 hours, and the pH value is 4.0-4.75 when the culture time is 24 hours.

Description

Technical Field

[0001] The present invention relates to a method for improving the gastric acid and bile acid tolerance of Lactobacillus paracasei. Background Art

[0002] In recent years, as probiotics, microorganisms that improve the balance of intestinal flora and have beneficial effects on host health have attracted attention. As such microorganisms, lactic acid bacteria and bifidobacteria have been used, and various physiological effects such as improvement of constipation and diarrhea, infection defense and allergy suppression effects based on improved immune function, and prevention of arteriosclerosis have been reported by taking these species.

[0003] Regarding the definition of probiotics, in recent years, the viewpoint of not only including live bacteria but also dead bacteria has been proposed. However, as the mechanism of action of probiotics, by multiplying useful microorganisms in the intestinal flora and producing various metabolites to exclude competing harmful microorganisms, inhibiting proliferation, it is possible to more effectively exert its function, so it is believed that it is more advantageous to exist in the intestine in a live bacteria state. In this way, it is expected that the ingested useful microorganisms arrive in the intestine in a state of keeping alive, but before arriving in the intestine, there are various factors such as temperature, pH, oxygen that inhibit the growth of microorganisms, especially digestive juices such as gastric juice and bile, in addition to gastric acid and bile acid, also contain various digestive enzymes. If exposed to these digestive juices, they will be subject to death or even if they survive, they will lose the ability to form colonies (proliferation ability) and other serious injuries.

[0004] Therefore, the technology of protecting useful microorganisms from the influence of digestive juice and improving the survivability in the digestive tract has been studied. For example, a method (patent document 1) for enhancing the gastric acid bile acid resistance of microorganisms themselves by destroying the hrcA gene of microorganisms is disclosed, and a method (patent document 2) etc. for improving survivability by enclosing lactic acid bacteria in enteric-coated capsules to avoid direct contact with gastric juice etc. However, the former requires cumbersome genetic engineering technology, and the latter produces restrictions on dosage form, etc., and it is difficult to say that these technologies have high versatility. On the other hand, as a simpler method, it is reported that the bile acid resistance of Lactococcus lactis is improved by culturing it in a culture medium with the addition of excessive Tween 80 (non-patent document 1). However, the applicant applies this technology to Lactobacillus paracasei, and the result does not see the improvement of bile acid resistance, but confirms the reduction instead.

[0005] Therefore, the present applicant has found that gastric acid and bile acid resistance can be improved by culturing lactic acid bacteria including Lactobacillus paracasei in a medium containing oleic acid or a salt thereof, and has filed a patent application (Patent Document 3).

[0006] However, since the technology for improving the gastric acid and bile acid resistance of lactic acid bacteria including Lactobacillus paracasei is related to various food and beverage products in which live bacteria are treated, it is clear that various technologies are more preferable.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent Application Publication No. 2005-160

[0010] Patent Document 2: Japanese Patent Application Publication No. 2014-139200

[0011] Patent Document 3: International Publication No. WO2021 / 256476

[0012] Non-patent literature

[0013] Non-patent literature 1: Kimoto H, Ohmomo S, Okamoto T. Enhancement of bile tolerance in lactococci by Tween 80. J Appl Microbiol. (2002) 92, 41-46. Summary of the invention

[0014] Therefore, an object of the present invention is to provide a novel method for improving the resistance of Lactobacillus paracasei to gastric acid and bile acid.

[0015] The present inventors have conducted intensive studies to solve the above problems and have found that the above problems can be solved by using lactic acid bacteria having a pH in a specific range after a specific elapsed time when Lactobacillus paracasei and lactic acid bacteria are mixed and cultured, thereby completing the present invention.

[0016] Furthermore, the present inventors have conducted intensive studies to solve the above-mentioned problems and, as a result, have found that the above-mentioned problems can be solved by adjusting the pH at a specific elapsed time in culturing Lactobacillus paracasei to a specific range, thereby completing the present invention.

[0017] That is, the present invention is a method for improving the gastric acid and bile acid tolerance of Lactobacillus paracasei, characterized in that lactic acid bacteria and Lactobacillus paracasei are mixed and cultured, wherein the lactic acid bacteria are inoculated with 0.01% by mass of 10% skim milk culture medium. 7 ~10 9When a pre-cultured bacterial solution of lactic acid bacteria with a concentration of cfu / ml is placed in a constant temperature bath at 37°C for culturing, the pH becomes 6.0-6.5 when the culturing time is 4 hours, the pH becomes 5.0-6.0 when the culturing time is 8 hours, the pH becomes 4.5-5.5 when the culturing time is 12 hours, and the pH becomes 4.0-4.5 when the culturing time is 24 hours.

[0018] In addition, the present invention is a method for improving the gastric acid and bile acid resistance of Lactobacillus paracasei, characterized in that a pre-cultured bacterial liquid of Lactobacillus paracasei is inoculated into a culture medium containing 0.1 to 20% by mass of skim milk, and the pH is adjusted to 5.5 to 6.25 when the culture time is 8 hours, 4.25 to 5.5 when the culture time is 16 hours, and 4.0 to 4.75 when the culture time is 24 hours, and the culture is performed.

[0019] Furthermore, the present invention provides a Lactobacillus paracasei having improved gastric acid and bile acid resistance by the above-mentioned gastric acid and bile acid resistance improving method.

[0020] According to the present invention, the gastric acid and bile acid resistance of Lactobacillus paracasei can be improved.

[0021] Therefore, by utilizing Lactobacillus paracasei with improved gastric acid and bile acid resistance obtained by the present invention, a large number of live bacteria can reach the intestinal tract, and various effects that Lactobacillus paracasei has hitherto been possessed can be more strongly obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph showing the change in pH at each culture time in Example 1.

[0023] Figure 2 This is a graph showing the results of the gastric acid and bile acid continuous tolerance test in Example 1.

[0024] Figure 3 This is a graph showing the survival rate in the gastric acid and bile acid continuous tolerance test in Example 1.

[0025] Figure 4 This is a graph showing the change in pH at each culture time in Example 2.

[0026] Figure 5 This is a graph showing the results of the gastric acid and bile acid continuous tolerance test in Example 3.

[0027] Figure 6 This is a graph showing the change in pH at each culture time in Example 4.

[0028] Figure 7 This is a graph showing the results of the gastric acid and bile acid continuous tolerance test in Example 4.

[0029] Figure 8 This is a graph showing the survival rate in the gastric acid and bile acid continuous tolerance test in Example 4. DETAILED DESCRIPTION

[0030] As the method for improving gastric acid and bile acid resistance of Lactobacillus paracasei of the present invention (hereinafter also referred to as "the improvement method of the present invention"), the following two methods can be mentioned.

[0031] <Improvement method 1 of the present invention>

[0032] A method for improving the gastric acid and bile acid tolerance of Lactobacillus paracasei, characterized in that lactic acid bacteria and Lactobacillus paracasei are mixed and cultured, wherein the lactic acid bacteria are inoculated with 0.01% by mass of 10% skim milk culture medium. 7 ~10 9 When a pre-cultured bacterial solution of lactic acid bacteria with a concentration of 1.5 cfu / ml is placed in a thermostatic chamber at 37°C and cultured, the pH becomes 6.0-6.5 after 4 hours of culture, 5.0-6.0 after 8 hours of culture, 4.5-5.5 after 12 hours of culture, and 4.0-4.5 after 24 hours of culture.

[0033] <Improvement method 2 of the present invention>

[0034] A method for improving gastric acid and bile acid resistance of Lactobacillus paracasei, characterized in that a precultured bacterial solution of Lactobacillus paracasei is inoculated into a culture medium containing 0.1 to 20% by mass of skim milk, and the culture is performed by adjusting the pH so that the pH becomes 5.5 to 6.25 after culturing for 8 hours, the pH becomes 4.25 to 5.5 after culturing for 16 hours, and the pH becomes 4.0 to 4.75 after culturing for 24 hours.

[0035] First, the improvement method 1 of the present invention is described. The lactic acid bacteria used in the improvement method 1 of the present invention are, for example, a pre-cultured bacterial solution of 0.01% by mass of lactic acid bacteria is inoculated in a 10% by mass skim milk medium and statically cultured in a thermostatic bath at 37° C., and the pH becomes 6.0 to 6.5 when the culture time is 4 hours, the pH becomes 5.0 to 6.0 when the culture time is 8 hours, preferably pH 5.4 to 5.9, the pH becomes 4.5 to 5.5 when the culture time is 12 hours, preferably pH 4.6 to 5.2, and the pH becomes 4.0 to 4.5 when the culture time is 24 hours. The pH ranges of these respective culture times can be appropriately combined.

[0036] The skim milk medium may contain, in addition to 10% by mass of skim milk, components used in conventional lactic acid bacteria culture media, for example, vitamins such as vitamin A, vitamin B, vitamin C, vitamin E, salts such as calcium and magnesium, meat extracts, peptones, yeast extracts, nitrogen sources such as amino acids, carbon sources such as glucose, xylose, fructose, and maltose, etc.

[0037] In addition, 10 7 ~10 9 The pre-cultured bacterial solution of lactic acid bacteria of cfu / ml can be prepared by inoculating frozen lactic acid bacteria into skim milk medium and culturing in a thermostatic bath at a temperature suitable for the growth of lactic acid bacteria (preferably 30-37°C) for 24 hours. The thermostatic bath used here is not particularly limited, for example, as long as an incubator, a constant temperature water bath, etc. are used. Here, standing refers to placing it in a static state without stirring. The pH during cultivation can be measured, for example, using a pH meter, etc. at a specified time.

[0038] As such lactic acid bacteria, for example, Streptococcus spp., Lactococcus spp., Lactaseibacillus spp., Lactobacillus spp., Latilactobacillus spp., Ligilactobacillus spp., Limosilactobacillus spp., Lentilactobacillus spp., Levilactobacillus spp., etc. can be enumerated. Among these lactic acid bacteria, lactic acid bacteria of Streptococcus spp. and Lactococcus spp. are preferred. Among these lactic acid bacteria of the genera Streptococcus and Lactococcus, Streptococcus thermophilus and Lactococcus lactis are preferred, and Streptococcus thermophilus YIT 2001, Streptococcus thermophilus YIT 2021, and Lactococcus lactis YIT 2027 are more preferred. These lactic acid bacteria may be used alone or in combination of two or more.

[0039] Among the above lactic acid bacteria, Streptococcus thermophilus YIT 2001 was internationally deposited as Streptococcus thermophilus YIT 2001 (FERM BP-7538, deposit date: April 5, 2001) at the National Institute of Advanced Industrial Science and Technology, International Patent Organisms Depository (IPOD, NITE), which is an international depository institution under the Budapest Treaty (Room 2-5-8120, Kazusa, Kisarazu City, Chiba Prefecture, Japan, 〒292-0818).

[0040] In addition, Streptococcus thermophilus YIT 2021 was internationally deposited in the above-mentioned National Institute of Advanced Industrial Science and Technology, International Patent Organism Depository Center (IPOD, NITE) as Streptococcus thermophilus YIT 2021 (FERM BP-7537, deposit date: April 5, 2001).

[0041] In addition, Lactococcus lactis YIT 2027 was internationally deposited in the above-mentioned National Institute of Advanced Industrial Science and Technology, International Patent Organisms Depository (IPOD, NITE) as Lactococcus lactis YIT 2027 (FERM BP-6224, deposit date: January 16, 1998).

[0042] The present invention improves in method 1, the Lactobacillus paracasei mixed culture with the above-mentioned lactic acid bacteria is not particularly limited, for example, can enumerate Lactobacillus paracasei YIT 0209 (benchmark strain), Lactobacillus paracasei YIT 9029 strain, Lactobacillus paracasei YIT 0180 etc. These Lactobacillus paracasei can use 1 or more than 2 kinds. Among these Lactobacillus paracasei, preferably Lactobacillus paracasei YIT 9029.

[0043] The above-mentioned Lactobacillus paracasei YIT9029 was internationally deposited as Lactobacillus casei YIT9029 (FERM BP-1366, deposit date: May 1, 1981) at the National Institute of Advanced Industrial Science and Technology, International Patent Organisms Depository (IPOD, NITE) as an international depository institution under the Budapest Treaty (Room 120, 2-5-8 Kazusa, Kisarazu City, Chiba Prefecture, Japan, 292-0818). It should be noted that Lactobacillus paracasei YIT 9029 was previously classified as Lactobacillus casei, but in recent years, the reclassification of the genus Lactobacillus has been completed and it has been classified as Lactobacillus paracasei (Zheng et al., Ataxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int. J. Syst. Evol. Microbiol. 2020 Apr; 70 (4): 2782-2858 DOI 10.1099 / ijsem.0.004107). In this specification, for the classification of lactic acid bacteria, the new classification classified based on the above-mentioned document is described.

[0044] The above-mentioned Lactobacillus paracasei YIT 0209 is a reference strain and can be obtained from ATCC or the like.

[0045] The present invention improves the method 1, the method of mixing the above-mentioned lactic acid bacteria and Lactobacillus paracasei is not particularly limited, for example, can be enumerated in a culture medium inoculated with 0.005-0.5 mass %, preferably 0.01-0.1 mass %, Lactobacillus paracasei 0.05-5 mass %, preferably 0.1-1 mass % and cultured at 25-40° C., preferably 30-37° C. for 24-72 hours, preferably 40-50 hours. Cultivation can be standing, shaking.

[0046] The culture medium is not particularly limited, and for example, it is a skim milk culture medium. In addition, the culture medium may contain components used in conventional lactic acid bacteria culture medium, such as vitamins such as vitamin A, vitamin B, vitamin C, and vitamin E, salts such as calcium and magnesium, nitrogen sources such as meat extract, peptone, yeast extract, and amino acids, and carbon sources such as glucose, xylose, fructose, and maltose.

[0047] By carrying out mixed culture of lactic acid bacteria and Lactobacillus paracasei as mentioned above, the gastric acid and bile acid tolerance of Lactobacillus paracasei is improved.Here, the method for confirming the gastric acid and bile acid tolerance of Lactobacillus paracasei is not particularly limited, for example, the gastric acid and bile acid continuous test method using artificial gastric acid and artificial bile acid etc. can be enumerated.The gastric acid and bile acid continuous test method can be carried out by the method described in the embodiment, for example.

[0048] Furthermore, the survival rate of Lactobacillus paracasei with improved gastric acid and bile acid resistance after the gastric acid and bile acid continuous test is, for example, 3 to 100 times higher than that of the case where the improvement method 1 of the present invention is not performed.

[0049] Next, the improvement method 2 of the present invention is described. In the improvement method 2 of the present invention, a pre-culture solution of Lactobacillus paracasei is inoculated into a medium containing 0.1 to 20% by mass of skim milk, and the pH is adjusted so as to be in the following range for the following culture time, and the culture is carried out. The culture conditions are not particularly limited, and for example, the culture can be placed in a thermostatic bath at 37° C. for 24 to 72 hours, preferably 40 to 50 hours.

[0050] In the above-mentioned culture, the method for adjusting the pH to pH 5.5 to 6.25, preferably pH 5.6 to 6.2, when the culture time is 8 hours, pH 4.25 to 5.5, preferably pH 4.4 to 5.4, when the culture time is 16 hours, and pH 4.0 to 4.75 when the culture time is 24 hours is not particularly limited. For example, the pH of the culture medium can be adjusted with an acid such as lactic acid or hydrochloric acid, or a base such as sodium bicarbonate or sodium hydroxide at a predetermined time. In addition, the pH can also be adjusted by culturing specific microorganisms at the same time, adjusting the culture temperature, etc. The range of pH for each of these culture times can be appropriately combined.

[0051] The Lactobacillus paracasei used in the improvement method 2 of the present invention may be the same as the Lactobacillus paracasei used in the improvement method 1 of the present invention.

[0052] The skim milk medium used in the improvement method 2 of the present invention may be the same as the skim milk medium used in the improvement method 1 of the present invention. In addition, the thermostatic bath, the method for preparing the pre-cultured bacterial solution, the standing, and the measurement of pH may be the same as those used in the improvement method 1 of the present invention.

[0053] As described above, the gastric acid and bile acid resistance of Lactobacillus paracasei is improved by culturing Lactobacillus paracasei while adjusting the pH. The method for confirming the gastric acid and bile acid resistance of Lactobacillus paracasei may be the same as that used in the improvement method 1 of the present invention.

[0054] In the improvement method 2 of the present invention, the same lactic acid bacteria as those used in the improvement method 1 of the present invention may be used together with Lactobacillus paracasei during the culture in a range that does not affect Lactobacillus paracasei.

[0055] In addition, the number of surviving bacteria after the gastric acid and bile acid continuous test of Lactobacillus paracasei with improved gastric acid and bile acid resistance is 3 to 19 times higher and the survival rate is 10 to 70 times higher than when the improvement method 2 of the present invention is not performed. Here, improved gastric acid and bile acid resistance means that the survival rate after the gastric acid and bile acid continuous test is increased by 3 times or more.

[0056] According to the improvement method of the present invention described above, Lactobacillus paracasei having improved resistance to gastric acid and bile acid can be obtained.

[0057] The Lactobacillus paracasei can be used in food and beverages such as lactic acid bacteria beverages and fermented milk. In addition, the Lactobacillus paracasei can also be used in preparations such as pharmaceuticals and health foods.

[0058] Specifically, when Lactobacillus paracasei is used in food and beverages, a culture medium containing Lactobacillus paracasei cultured in the enhancement method of the present invention (and lactic acid bacteria cultured in a mixed culture as needed) may be prepared in the same manner as food and beverages using a culture medium containing conventionally known lactic acid bacteria.

[0059] In addition, when Lactobacillus paracasei is used in a preparation, it can be prepared in the same manner as a preparation using a culture medium containing conventionally known lactic acid bacteria by separation, purification, and drying as needed from a culture medium containing Lactobacillus paracasei cultured in the improvement method of the present invention (and lactic acid bacteria cultured in a mixed culture as needed).

[0060] Such food and drink products and preparations contain the Lactobacillus paracasei with improved gastric acid and bile acid tolerance contained in the culture medium, and therefore have higher effects than food and drink products and preparations containing conventional Lactobacillus paracasei. As effects, there are improved bowel movements, activated NK cells, improved sleep quality, etc.

[0061] Example

[0062] Hereinafter, the present invention will be described in detail with reference to examples of the present invention, but the present invention is not limited to these examples.

[0063] Example 1

[0064] Mixed culture test:

[0065] <Preparation of pre-cultured bacterial solution>

[0066] The following pre-cultured bacterial solutions (1) to (5) were prepared.

[0067] (1) Preparation of pre-cultured bacterial solution of Lactobacillus paracasei YIT 9029

[0068] The skim milk medium was inoculated with 0.1% (v / v) of cryopreserved Lactobacillus paracasei YIT 9029 and cultured aerobically at 37°C for 48 hours.

[0069] (2) Preparation of pre-cultured bacterial solution of Streptococcus thermophilus YIT 2001

[0070] The skim milk medium was inoculated with 0.1% (v / v) of cryopreserved Streptococcus thermophilus YIT 2001 and cultured aerobically at 37°C for 24 hours.

[0071] (3) Preparation of pre-cultured bacterial solution of Streptococcus thermophilus YIT 2021

[0072] The skim milk medium was inoculated with 0.1% (v / v) of cryopreserved Streptococcus thermophilus YIT 2021 and cultured aerobically at 37°C for 24 hours.

[0073] (4) Preparation of pre-cultured bacterial solution of Streptococcus thermophilus YIT 2037 (reference strain)

[0074] The skim milk medium was inoculated with 0.1% (v / v) of cryopreserved Streptococcus thermophilus YIT 2037 (reference strain) and cultured aerobically at 37°C for 24 hours.

[0075] (5) Preparation of pre-cultured bacterial solution of Lactococcus lactis YIT 2027

[0076] The skim milk medium was inoculated with 0.1% (v / v) of cryopreserved Lactococcus lactis YIT 2027 and cultured aerobically at 30°C for 24 hours.

[0077] <Mixed culture>

[0078] The following (A) single culture and (B) to (E) mixed culture were performed, and a portable pH meter was immersed in the stirred bacterial solution to measure the pH after 0, 8, 12, 16, 24, and 48 hours of culture. The results are shown in Figure 1 .

[0079] (A) The precultured bacterial solution prepared in (1) was used to inoculate 0.5% by mass of Lactobacillus paracasei YIT 9029 into a 10% by mass skim milk medium, and the culture was placed in a thermostatic chamber at 37°C for culturing.

[0080] (B) Using the precultured bacterial solutions prepared in (1) and (2), 0.5% by mass of Lactobacillus paracasei YIT 9029 and 0.01% by mass of Streptococcus thermophilus YIT 2001 were inoculated into a 10% by mass skim milk medium, respectively, and the medium was placed in a thermostatic chamber at 37°C for culture.

[0081] (C) Using the precultured bacterial solutions prepared in (1) and (3), 0.5% by mass of Lactobacillus paracasei YIT 9029 and 0.01% by mass of Streptococcus thermophilus YIT 2021 were inoculated into a 10% by mass skim milk medium, respectively, and the culture was carried out in a thermostatic chamber at 37°C.

[0082] (D) Using the precultured bacterial solutions prepared in (1) and (4), 0.5% and 0.01% by mass of Lactobacillus paracasei YIT 9029 and Streptococcus thermophilus YIT 2037 (reference strains) were inoculated into a 10% by mass skim milk medium, respectively, and the medium was placed in a thermostatic chamber at 37°C for culture.

[0083] (E) Using the precultured bacterial solutions prepared in (1) and (5), 0.5% by mass of Lactobacillus paracasei YIT9029 and 0.01% by mass of Lactococcus lactis YIT2027 were inoculated into a 10% by mass skim milk medium, respectively, and the culture was carried out in a thermostatic chamber at 37°C.

[0084] <Gastric acid and bile acid continuous tolerance test>

[0085] For the single culture and mixed culture of (A) to (E), a gastric acid bile acid continuous tolerance test was performed after 48 hours of culture. The gastric acid conditions were pH 4.0, 1 hour, and 37°C, and the subsequent bile acid conditions were pH 4.4, 0.3% oxgall, 5 minutes, and 37°C. The bacterial solution sampled before and after gastric acid exposure was appropriately diluted with PBS and smeared on LLV agar medium. Aerobic culture was performed at 37°C for 2 to 3 days and the colonies were measured to determine the number of viable bacteria in the bacterial solution (log 10 cfu / ml) and survival rate (%). The survival rate (%) was calculated by dividing the number of viable bacteria after gastric acid exposure and bile acid exposure by the number of viable bacteria before gastric acid exposure. The same experiment was repeated 4 times, and their results are shown in Figure 2 and Figure 3 .

[0086] <Results>

[0087] These results revealed that gastric acid and bile acid resistance was significantly improved by co-culturing Lactobacillus paracasei YIT 9029 with Streptococcus thermophilus YIT 2001, Streptococcus thermophilus YIT 2021, or Lactococcus lactis YIT 2027.

[0088] Example 2

[0089] Single culture test:

[0090] In order to determine which strain has which property and Lactobacillus paracasei YIT 9029, gastric acid and bile acid tolerance is significantly improved when mixed culture, the strain used in the mixed culture is cultured separately, and the pH after 0, 4, 8, 12, 16, and 24 hours of culture is measured using a pH meter. It should be noted that the culture is the same as in Example 1 except that all strains are used separately. Their results are shown in Figure 4 .

[0091] According to the results, it can be seen that as lactic acid bacteria to be mixed cultured with Lactobacillus paracasei YIT 9029, a strain having a pH of 6.0 to 6.5 at a culture time of 4 hours, a pH of 5.0 to 6.0 at a culture time of 8 hours, a pH of 4.5 to 5.5 at a culture time of 12 hours, and a pH of 4.0 to 4.5 at a culture time of 24 hours, such as Streptococcus thermophilus YIT 2001, Streptococcus thermophilus YIT 2021, or Lactococcus lactis YIT 2027, can be used. On the other hand, Streptococcus thermophilus YIT 2037, which did not improve the gastric acid and bile acid resistance of Lactobacillus paracasei YIT 9029 after mixed culture with Lactobacillus paracasei YIT 9029 in Example 1, does not have such a property.

[0092] Example 3

[0093] Mixed culture test:

[0094] <Preparation of pre-cultured bacterial solution>

[0095] The following pre-cultured bacterial solutions (6) to (10) were prepared.

[0096] (6) Preparation of pre-cultured bacterial solution of Lactobacillus helveticus YIT 0083 (reference strain)

[0097] 0.1% (v / v) of cryopreserved Lactobacillus helveticus YIT 0083 (reference strain) was inoculated into a skim milk medium, and aerobic culture was performed at 37°C for 48 hours.

[0098] (7) Preparation of pre-cultured bacterial solution of Lactobacillus gasseri YIT 0168

[0099] 0.1% (v / v) of frozen Lactobacillus gasseri YIT 0168 was inoculated into skim milk medium and cultured aerobically at 37° C. for 48 hours. It should be noted that Lactobacillus gasseri YIT 0168 was internationally deposited in the aforementioned National Institute of Advanced Industrial Science and Technology, International Patent Organisms Depository (IPOD, NITE) under the old classification of Lactobacillus acidophilus YIT 0168 (FERM BP-7536, deposit date: April 5, 2001).

[0100] (8) Preparation of pre-cultured bacterial solution of Lactobacillus paracasei YIT 0180

[0101] The skim milk medium was inoculated with 0.1% (v / v) of cryopreserved Lactobacillus paracasei YIT 0180 and aerobically cultured at 37°C for 48 hours.

[0102] (9) Preparation of pre-cultured bacterial solution of Lactobacillus paracasei YIT 0209 (reference strain)

[0103] 0.1% (v / v) of cryopreserved Lactobacillus paracasei YIT 0209 (reference strain) was inoculated into a skim milk medium, and aerobic culture was performed at 37°C for 48 hours.

[0104] (10) Preparation of pre-cultured bacterial solution of Lactobacillus paracasei YIT 9029

[0105] The skim milk medium was inoculated with 0.1% (v / v) of cryopreserved Lactobacillus paracasei YIT 9029 and aerobically cultured at 37°C for 48 hours.

[0106] (11) Preparation of pre-cultured bacterial solution of Lactococcus lactis YIT 2027

[0107] The skim milk medium was inoculated with 0.1% (v / v) of cryopreserved Lactococcus lactis YIT 2027, and aerobically cultured at 30°C for 24 hours.

[0108] <Mixed culture>

[0109] The following mixed cultures (F) to (J) and individual cultures of each strain were performed under the same conditions.

[0110] (F) Using the precultured bacterial solutions prepared in (6) and (11), 10% by mass skim milk medium was inoculated with 0.5% by mass of Lactobacillus helveticus YIT0083 (reference strain) and 0.01% by mass of Lactococcus lactis YIT2027, respectively, and cultured in a thermostatic chamber at 37°C.

[0111] (G) Using the precultured bacterial solutions prepared in (7) and (11), 0.5% by mass and 0.01% by mass of Lactobacillus gasseri YIT0168 and Lactococcus lactis YIT2027 were inoculated into a 10% by mass skim milk medium, respectively, and the culture was placed in a thermostatic chamber at 37°C for culturing.

[0112] (H) Using the precultured bacterial solutions prepared in (8) and (11), 10% by mass skim milk medium was inoculated with 0.5% by mass of Lactobacillus paracasei YIT0180 and 0.01% by mass of Lactococcus lactis YIT2027, respectively, and the culture was carried out in a thermostatic chamber at 37°C.

[0113] (I) Using the precultured bacterial solutions prepared in (9) and (11), 10% by mass skim milk medium was inoculated with 0.5% by mass of Lactobacillus paracasei YIT0209 (reference strain) and 0.01% by mass of Lactococcus lactis YIT2027, respectively, and the culture was carried out in a thermostatic chamber at 37°C.

[0114] (J) Using the precultured bacterial solutions prepared in (10) and (11), 10% by mass skim milk medium was inoculated with 0.5% by mass of Lactobacillus paracasei YIT9029 and 0.01% by mass of Lactococcus lactis YIT2027, respectively, and the culture was carried out in a thermostatic chamber at 37°C.

[0115] <Gastric acid and bile acid continuous tolerance test>

[0116] For the mixed culture and single culture of (F) to (J), after 48 hours of culture, the gastric acid bile acid continuous resistance test was performed in the same manner as in Example 1, except that the medium was selected, and the survival rate (%) was calculated. The survival rate (%) was calculated by dividing the number of viable bacteria after gastric acid exposure and after bile acid exposure by the number of viable bacteria before gastric acid exposure. MRS agar medium containing 0.5 μg / ml tetracycline was used as the selective medium in (F) and (G), and MRS agar medium containing 10 μg / ml vancomycin was used as the selective medium in (H) to (J). The same experiment was repeated 4 to 5 times, and their results are shown in Figure 5 In addition, when evaluating the survival rate of Lactobacillus helveticus YIT 0083 (reference strain), the bile acid conditions were pH 4.4, 0.15 mass % of bovine bile acid, 5 minutes, and 37°C.

[0117] <Results>

[0118] From these results, it can be seen that gastric acid and bile acid resistance is significantly improved by co-culturing Lactococcus lactis YIT 2027 with Lactobacillus paracasei YIT 0180, Lactobacillus paracasei YIT 0209 (standard strain) or Lactobacillus paracasei YIT 9029. On the other hand, gastric acid and bile acid resistance of Lactobacillus helveticus and Lactobacillus gasseri, which do not belong to Lactobacillus paracasei, was not improved after co-culturing with Lactococcus lactis YIT 2027.

[0119] Example 4

[0120] pH controlled culture test:

[0121] The pH behavior of Lactobacillus paracasei YIT 9029 and Lactococcus lactis YIT 2027 when mixed culture was matched in the same manner as in Example 1 using a culture apparatus equipped with a pH control device (Bio Jr. 8, manufactured by (Strain) BIOTT), and the pH of Lactobacillus paracasei YIT 9029 when cultured alone was adjusted with lactic acid so that the pH became 5.5 to 6.25 when the culture time was 8 hours, the pH became 4.25 to 5.5 when the culture time was 16 hours, and the pH became 4.0 to 4.75 when the culture time was 24 hours. The results are shown in Figure 6 .

[0122] After 48 hours of culture, the gastric acid bile acid continuous resistance test was carried out in the same manner as in Example 1, and the number of surviving bacteria and the survival rate before gastric acid reaction, after gastric acid reaction, and after bile acid reaction were calculated. The same test was repeated 3 times, and their results are shown in Figure 7 and Figure 8 The conditions for bile acid were pH 4.4, 1.0 mass % bovine bile acid, 5 minutes, and 37°C.

[0123] According to the results, when Lactobacillus paracasei YIT 9029 was cultured alone, the pH was adjusted to 5.5 to 6.25 when the culture time was 8 hours, the pH was adjusted to 4.25 to 5.5 when the culture time was 16 hours, and the pH was adjusted to 4.0 to 4.75 when the culture time was 24 hours. As in the mixed culture as in Example 1, the gastric acid and bile acid resistance of Lactobacillus paracasei YIT 9029 was significantly improved.

[0124] Industrial Applicability

[0125] If the Lactobacillus paracasei having improved resistance to gastric acid and bile acid obtained by the present invention is used, a large amount of live bacteria can be delivered to the intestinal tract, and can be used to improve health.

Claims

1. A method for improving gastric acid and bile acid tolerance of Lactobacillus paracasei, characterized in that: The lactic acid bacteria and Lactobacillus paracasei were mixed and cultured. The lactic acid bacteria were inoculated into a 10% by mass skim milk medium at 0.01% by mass of 10 7 ~10 9 When a pre-cultured bacterial solution of lactic acid bacteria with a concentration of cfu / ml is placed in a constant temperature bath at 37°C for culturing, the pH becomes 6.0-6.5 when the culturing time is 4 hours, the pH becomes 5.0-6.0 when the culturing time is 8 hours, the pH becomes 4.5-5.5 when the culturing time is 12 hours, and the pH becomes 4.0-4.5 when the culturing time is 24 hours.

2. The method for improving gastric acid and bile acid resistance of Lactobacillus paracasei according to claim 1, wherein: The lactic acid bacteria are one or two selected from the group consisting of Streptococcus thermophilus and Lactococcus lactis.

3. The method for improving gastric acid and bile acid resistance of Lactobacillus paracasei according to claim 1, wherein: The Lactobacillus paracasei is Lactobacillus paracasei YIT 9029, ie, FERM BP-1366.

4. A method for improving gastric acid and bile acid tolerance of Lactobacillus paracasei, characterized in that: A preculture solution of Lactobacillus paracasei was inoculated into a medium containing 0.1 to 20% by mass of skim milk, and the culture was performed by adjusting the pH to 5.5 to 6.25 after 8 hours, 4.25 to 5.5 after 16 hours, and 4.0 to 4.75 after 24 hours.

5. The method for improving gastric acid and bile acid resistance of Lactobacillus paracasei according to claim 4, wherein: The Lactobacillus paracasei is Lactobacillus paracasei YIT 9029, ie, FERM BP-1366. 6 . A Lactobacillus paracasei having improved gastric acid and bile acid resistance by the method for improving gastric acid and bile acid resistance according to any one of claims 1 to 5.

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