KR101941645B1 - Lactic acid bacteria strains derived from human body with excellent resistance to environmental stress and Method for producing yogurt using it - Google Patents

Abstract

The present invention relates to a method for producing fermented milk using human lactic acid bacteria having excellent environmental stress resistance, which is isolated from adult feces, has excellent oxidative stress, heavy metals and radiation resistance, has an intestinal harmful enzyme inhibitory activity, Lactobacillus laminosus L3, Enterococcus lassus L54, and Lactobacillus aspirophilus L120 strains, which are excellent in artificial gastric juice and artificial bile fluid resistance, are provided.
A method for producing fermented milk using human-derived lactic acid bacteria having excellent environmental stress resistance comprises the steps of preparing crude oil, sugar, and the strain; Mixing 4 to 6 parts by weight of sugar with respect to 100 parts by weight of crude oil prepared in the preparing step to prepare a mixture; Sterilizing the mixture at a temperature of 85 to 95 DEG C for 5 to 15 minutes; Cooling the sterilized mixture to 40-45 < 0 >C; 100 parts by weight of the cooled mixture is inoculated with 1 part by weight of lactic acid bacteria as a starter; Fermenting the fermented milk by fermenting the mixture to which the starter has been inoculated at a temperature of 35 to 45 DEG C for 6 to 8 hours until the final pH is 4.3 to 4.6; A fermented milk cooling step of cooling the fermented milk to 15 to 20 캜; And a refrigeration step of refrigerating and storing the cooled fermented milk at a temperature of 3 to 5 ° C.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a lactic acid bacterium having excellent resistance to environmental stress and a method for producing fermented milk using the same.

The present invention relates to a human lactic acid bacterium having excellent resistance to environmental stress, and a method for producing fermented milk using the same, which is isolated from adult feces, has excellent oxidative stress, heavy metals and radiation resistance, Lactobacillus lansos L3, Enterococcus lassus L54 and Lactobacillus acidophilus strain L120, which are excellent in artificial gastric juice and artificial bile fluid resistance to be probiotic lactic acid bacteria, and the like.

Most modern people living in a fiercely competitive society are always exposed to excessive stress. All living organisms, including humans, are able to rapidly produce reactive oxygen species (ROS) when exposed to a variety of environmental stress conditions, breaking the balance of antioxidant defenses and inducing oxidative stress, Resulting in chronic degenerative diseases.

In addition, harmful heavy metals such as Cr (Cr), lead (Pb), and cadmium (Cd) ingested through foods are not easily released into the human body and react with human tissues even at low concentrations, Has become a big problem.

Recently, as radiation and radioactive isotopes are widely used in various fields such as medicine, agriculture, and industry, opportunities for radiation exposure of people involved in this field are increased and chemotherapy using radiation is also increasing. In particular, direct exposure of the body to radiation causes DNA damage or induces indirect cell damage by reactive oxygen species.

On the other hand, under stress, the sympathetic nervous system is activated and a lot of corticosteroids, which are stress hormones, are secreted. These corticosteroids interfere with digestive tract exercise and digestive juice secretion and induce the proliferation of intestinal harmful bacteria. Beta-glucosidase, beta-glucuronidase, and tryptophanase enzymes.

Fermented milk, which is a typical long-lived food, contains not only nutritional components of milk, but also organic acids, amino acids, peptides, trace amounts of physiologically active substances and lactic acid bacteria produced by the action of probiotic lactic acid bacteria, It has been reported that it exhibits excellent functions for the prevention of adult diseases and promotion of health in modern people such as improvement of intestinal microflora, stabilization of intestinal microflora, effect of anti-cancer by stimulation of immune system, and lowering of blood cholesterol.

On the other hand, there is no Korean starter for fermented milk production and fermented milk production method using the same to confirm the resistance to various environmental stresses by isolating the lactic acid bacteria living in the intestines of Korean people.

In order to develop a fermented milk product having excellent anti-stress effect, the present invention is to solve the environmental stress resistance by separating the lactic acid bacteria living in the intestines of Korean, .

In addition, the present invention provides a method for effectively removing harmful heavy metal materials which are excellent in oxidative stress resistance due to active oxygen species and absorbed and accumulated in a human body, effectively protecting cells from damage caused by radiation exposure, The present invention also provides a method for producing fermented milk using the human-derived lactic acid bacterium which is capable of effectively improving the intestinal environment by inhibiting the growth of harmful bacteria in the intestines caused by the bacteria.

In order to achieve the above object, the present invention provides a method for producing a probiotic lactic acid bacterium, which is isolated from adult feces, has excellent oxidative stress, heavy metals and radiation resistance, has an inhibitory activity against intestinal harmful enzymes, Superior Lactobacillus laminocus L3, Enterococcus pathum L54, and Lactobacillus acidophilus L120 strains.

The present invention also relates to a method for preparing crude oil, sugar and the strain, Mixing 4 to 6 parts by weight of sugar with respect to 100 parts by weight of crude oil prepared in the preparing step to prepare a mixture; Sterilizing the mixture at a temperature of 85 to 95 DEG C for 5 to 15 minutes; Cooling the sterilized mixture to 40-45 < 0 >C; 100 parts by weight of the cooled mixture is inoculated with 1 part by weight of lactic acid bacteria as a starter; Fermenting the fermented milk by fermenting the mixture to which the starter has been inoculated at a temperature of 35 to 45 DEG C for 6 to 8 hours until the final pH is 4.3 to 4.6; A fermented milk cooling step of cooling the fermented milk to 15 to 20 캜; And cooling the cooled fermented milk at a temperature of 3 to 5 占 폚.

The fermented milk production method using lactic acid bacteria derived from human body having excellent environmental stress resistance according to the present invention is excellent in oxidative stress resistance due to active oxygen species and can effectively remove harmful heavy metal substances to be absorbed and accumulated in human body, It is possible to provide a functional fermented milk which is capable of effectively protecting cells from damage due to stress and inhibiting the growth of harmful bacteria in the intestines due to stress, thereby effectively improving intestinal environment.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph comparing survival rates of viable cell counts after irradiating a screening strain according to an embodiment of the present invention with radiation. FIG.
2 (2a-2c) show the inhibitory activity of beta-glucosidase, beta-glucuronidase, and tryptophanase enzymes on the screening strain according to the embodiment of the present invention Measured graph.
FIG. 3 (3a-3b) is a graph showing artificial gastric juice resistance and artificial bile resistance in a screening strain according to an embodiment of the present invention.
FIG. 4 is a flowchart showing a method for producing fermented milk produced by using a human-derived lactic acid bacterium having excellent resistance to environmental stress according to an embodiment of the present invention. FIG.

Hereinafter, a human-derived lactic acid bacterium having excellent resistance to environmental stress and a method for producing fermented milk using the same will be described in detail as follows.
- Date of deposit of microorganism and deposit number: August 3, 2016 / KCTC18485P for L3
- Date of donation of microorganism and deposit number: August 3, 2016 / KCTC18486P for L54
- Date of deposit of microorganism and deposit number: August 3, 2016 / KCTC18487P for L120

In describing the present invention, the term crude refers to fresh crude oil having an acidity of 0.14 to 0.15% and a pH in the range of 6.6 to 6.8 produced from Holstein-Friesian species for the production of fermented milk.

<Description of operation 1> Selection of a strain having excellent environmental stress resistance

1-1. Separation of lactic acid bacteria from feces of Korean adults

In order to isolate strains resistant to various stresses in the intestinal longevity of elderly people, feces were collected from a predetermined area, for example, 21 elderly people in Sunchang area, and suspended in sterile PBS buffer (10 mM, pH 7.3) And then diluted with sterile physiological saline. The diluted solution was plated on MRS solid medium and anaerobically incubated at 37 ° C for 72 hours. 233 strains were obtained by purely isolating the characteristic colonies on the MRS solid medium. The isolated strains were suspended in 15% glycerol and stored at 70 ℃.

1-2. Selection of lactic acid bacteria with excellent oxidative stress resistance

In order to select lactic acid bacteria resistant to oxidative stress caused by reactive oxygen species (ROS), 233 strains isolated from adult feces were treated with hydrogen peroxide (H ₂ O ₂ ) was treated.

Specifically, the isolate was inoculated 2% in MRS liquid medium, incubated at 37 ° C for 24 hours, washed twice with phosphate buffered saline (PBS), and suspended in the same buffer to a cell number of about 10 ⁷ CFU / ml . The cells were incubated at 37 ° C for 2 hours. Cells were incubated at 37 ° C for 24 hours, and the number of colonies formed was measured. The survival rate (%) was expressed as [(Log index value of experimental group / log index value of control group) × 100], and the control group was set as a hydrogen peroxide treatment group.

As shown in Table 1 below, 20 strains out of 233 strains have a high survival rate of 100% even under the condition of 1 mM of hydrogen peroxide, similar to Lactobacillus lambusus GG (LGG) strains widely used in live bacteria or fermented milk Respectively.

Therefore, the second selection experiment was carried out on 20 strains which showed high tolerance to reactive oxygen species.

Strain
persons survival
rate
(%) Strain
persons survival
rate
(%) Strain
persons survival
rate
(%) Strain
persons survival
rate
(%) Strain
persons survival
rate
(%) Strain
persons survival
rate
(%) L1 64 L40 83 L79 83 L118 48 B7 65 B46 63 L2 91 L41 88 L80 78 L119 95 B8 92 B47 56 L3 100 L42 93 L81 85 L120 100 B9 93 B48 79 L4 95 L43 43 L82 83 L121 67 B10 101 B49 89 L5 65 L44 56 L83 101 L122 65 B11 91 B50 85 L6 72 L45 45 L84 78 L123 68 B12 88 B51 77 L7 54 L46 42 L85 74 L124 63 B13 94 B52 62 L8 57 L47 83 L86 83 L125 80 B14 100 B53 58 L9 83 L48 67 L87 78 L126 73 B15 84 B54 84 L10 68 L49 75 L88 43 L127 66 B16 73 B55 84 L11 85 L50 78 L89 53 L128 77 B17 63 B56 43 L12 100 L51 78 L90 45 L129 89 B18 67 B57 77 L13 82 L52 75 L91 74 L130 87 B19 78 B58 83 L14 63 L53 89 L92 63 L131 95 B20 34 B59 84 L15 73 L54 101 L93 56 L132 100 B21 36 B60 85 L16 66 L55 94 L94 46 L133 88 B22 67 B61 76 L17 74 L56 91 L95 78 L134 78 B23 73 B62 63 L18 46 L57 100 L96 47 L135 87 B24 45 B63 78 L19 67 L58 67 L97 23 L136 73 B25 65 B64 100 L20 62 L59 45 L98 78 L137 76 B26 88 B65 83 L21 53 L60 85 L99 68 L138 73 B27 100 B66 86 L22 58 L61 67 L100 67 L139 76 B28 85 B67 83 L23 84 L62 78 L101 65 L140 72 B29 67 B68 87 L24 77 L63 87 L102 65 L141 68 B30 65 B69 89 L25 84 L64 88 L103 26 L142 77 B31 68 B70 101 L26 57 L65 92 L104 87 L143 101 B32 77 B71 25 L27 42 L66 67 L105 67 L144 95 B33 54 B72 46 L28 32 L67 100 L106 82 L145 101 B34 100 B73 67 L29 44 L68 92 L107 79 L146 92 B35 78 B74 76 L30 77 L69 88 L108 78 L147 42 B36 83 B75 73 L31 67 L70 67 L109 76 L148 74 B37 83 B76 65 L32 56 L71 83 L110 100 L149 75 B38 86 B77 29 L33 78 L72 100 L111 91 L150 50 B39 87 B78 76 L34 78 L73 68 L112 88 B1 52 B40 100 B79 67 L35 54 L74 77 L113 83 B2 77 B41 100 B80 57 L36 88 L75 83 L114 84 B3 91 B42 73 B81 58 L37 45 L76 78 L115 85 B4 88 B43 89 B82 46 L38 67 L77 76 L116 56 B5 89 B44 78 B83 78 L39 68 L78 86 L117 45 B6 78 B45 78 LGG 100

1-3. Selection of lactic acid bacteria with excellent resistance to heavy metals

Harmful heavy metals such as chromium (Cr), lead (Pb), and cadmium (Cd), which are ingested through foods, are not easily released into the human body and react with human tissues even at low concentrations, And to remove harmful heavy metal substances which are absorbed by the human body and accumulated by using lactic acid bacteria.

Specifically, Cr, Pb, and Cd were added to the MRS liquid medium to a final concentration of 0.01%, and then lactic acid bacteria were inoculated and incubated at 37 ° C for 24 hours After incubation, absorbance was measured at 660 nm. The survival rate (%) was expressed as [(absorbance value of the experimental group / absorbance value of the control) × 100], and the control group was set as the heavy metal untreated group.

In order to confirm the concentration of heavy metals adsorbed on the cells, the culture solution of the lactic acid bacteria having heavy metal resistance was centrifuged (3000 rpm, 10 min) to remove the supernatant and the pellet was recovered. 14 ml of aqua regia (10.5 ml of 35% HCl, 3.5 ml of 70% HNO ₃ ) was added to each bacterial cell sample, allowed to stand at room temperature for 16 hours or more, reacted at 60 ° C for 2 hours and cooled at room temperature. Then, the diluted samples were dissolved in 10 ml of 1% nitric acid solution and injected into ICP-OES (iCAP6200DUO, Thermo Fisher Scientific, UK) for quantitative analysis.

As shown in the following Table 2, B40 strain and L3 strain showed high growth rate of 99% and 94% in chromium (Cr) treatment and 20 strains in lead (Pb) treatment, respectively. In the cadmium (Cd) treatment, L72 strain, B40 strain and L3 strain showed growth of 58%, 49% and 48%, respectively. In particular, the weight of heavy metals adsorbed on the cells was measured and the adsorption capacity was confirmed. As a result, the L3 strain showed high heavy metal adsorption capacity of 916 ug / ml, 153 mg / ml and 767 ug / ml in the Cr, Pb and Cd treatments Respectively. Therefore, the L3 strain effectively adsorbs three harmful heavy metals, which may inhibit accumulation of heavy metals in the body.

Strain name Chromium (Cr) Lead (Pb) Cadmium (Cd) Survival rate
(%) Adsorption capacity
(ug / ml) Survival rate
(%) Adsorption capacity
(mg / ml) Survival rate
(%) Adsorption capacity
(ug / ml) L3 94 916 117 153 48 767 L12 77 371 117 130 8 213 L54 79 373 117 132 45 627 L57 63 239 110 148 23 511 L67 74 395 112 142 8 202 L72 46 175 129 107 58 836 L83 64 266 85 67 7 176 L110 65 299 98 71 6 136 L120 69 305 113 138 38 455 L132 66 235 109 81 7 140 L143 75 305 109 87 7 137 L145 79 331 116 99 22 440 B10 71 396 103 85 7 167 B14 71 332 111 94 13 395 B27 70 312 111 93 8 295 B34 74 362 108 87 8 238 B40 99 868 112 121 33 321 B41 57 480 106 81 49 728 B64 70 325 104 81 7 184 B70 78 379 106 84 8 284 LGG 83 388 97 73 45 712

1-4. Selection of lactic acid bacteria with excellent radiation resistance

It is known that exposure to human radiation causes direct DNA damage resulting from this, or indirect cell damage caused by reactive oxygen species. Therefore, in order to protect the cells from damage to the intestinal mucosa by radiation, radiopharmaceuticals were selected for their resistance to radiation.

Specifically, survival rates of the isolates were measured by counting viable cells after irradiation with Co-60 gamma-ray. Gamma irradiation was performed using a source of 11.1 PBq and a Co-60 gamma ray irradiation facility (point source AECL, IR-79, MDS Nordion International Co., Ltd., Canada) at the Korea Atomic Energy Research Institute (Jeongeup, Korea) To obtain the absorbed dose. Absorbed doses were checked using an alanine dosimeter (5 mm, Bruker Instruments, Germany) and the error of the absorbed dose was within ± 5%. 0.9 ml of sterilized saline was added to 0.1 ml of the gamma-irradiated culture, followed by dilution with decidual dilution method, followed by incubation at 37 ° C for 48 hours using a BCP agar plate culture method. The number of generated colonies was then measured and expressed as the survival rate (%) [(log index value of experimental group / Log index value of control group) x 100]. At this time, the control was set as a non-treated gamma ray.

As shown in FIG. 1, most strains including Lactobacillus rhamnosus GG (LGG) showed a survival rate of about 40%, while L120 strains showed the highest survival rate of 67% , Followed by L54 strain with 51% survival rate. Therefore, the L120 strain is considered to be able to effectively protect cells from damage caused by radiation exposure.

1-5. Selection of lactic acid bacteria with excellent inhibitory activity against enteric enzyme activity in intestines

When stressed, the sympathetic nervous system is activated and a lot of corticosteroids, which are stress hormones, are secreted. These corticosteroids interfere with digestive tract exercise and digestive juice secretion, leading to the proliferation of endemic bacteria in the intestines. Therefore, we selected lactic acid bacteria with excellent inhibitory effect on intestinal toxic enzyme activity and tried to improve intestinal environment by suppressing the growth of intestinal harmful bacteria caused by stress.

Specifically, the inhibitory activity of β-glucosidase, β-Glucuronidase, and Tryptophanase enzymes produced by enteropathogenic bacteria in the intestine was measured to compare the inhibitory effect of intestinal toxic enzyme activity Respectively.

0.1 ml of a 10-fold dilution of healthy adult feces was added to the culture solution of lactic acid bacteria, followed by incubation at 37 ° C for 24 hours, followed by centrifugation (3,000 rpm, 5 min) to suspend the cells in sterilized physiological saline.

The inhibitory activity of β-glucosidase was determined by adding 0.2 ml of 2 mM pnitrophenyl-β-D-glucopyranoside and 0.1 ml of enzyme to 0.3 ml of 0.1 M sodium phosphate buffer (pH 7.0) After reacting for 30 minutes, 0.3 ml of 0.5 N NaOH was added to terminate the reaction. The absorbance at 405 nm was then measured using a supernatant centrifuged (13,000 rpm, 5 min).

The inhibitory activity of β-glucuronidase was determined by adding 0.02 ml of 2 mM pnitrophenyl-β-D-glucuronide and 0.1 ml of enzyme solution to 0.38 ml of 0.1 M sodium phosphate buffer (pH 7.0) and incubating the cells in a shaking water bath at 37 ° C. for 30 minutes After the reaction, 0.4 ml of 0.5 N NaOH was added to terminate the reaction. The absorbance at 405 nm was then measured by centrifugation (13,000 rpm, 5 min).

The inhibitory activity of tryptophanase was determined by adding 0.02 to 0.2 ml of a reaction mixture (22.5 ml of 0.05 N potassium phosphate (pH 7.5), 2.75 mg of pyridoxal phosphate, 19.6 mg of disodium EDTA dehydrate, 10 mg of bovine serum albumin, and 87.5 ml of distilled water) M tryptophan and 0.1 ml of the enzyme solution were reacted in a shaking water bath at 37 ° C for 30 minutes, and ₂ ml of a coloring reagent (94.8 ml of 95% EtOH, 5.2 ml of 36N H ₂ SO ₄ and 14.7 g of ρ-diaminobenzaldehyde) And the absorbance at 550 nm was measured using a supernatant centrifuged (13,000 rpm, 5 min).

As shown in Fig. 2 (2a, 2b, 2c), among the isolated strains containing Lactobacillus lambatus GG (LGG) strains, beta-glucosidase, beta-glucosidase Β-Glucuronidase and Tryptophanase were found to be highly effective in inhibiting the enzyme activity. In particular, L54 strain was found to have the highest inhibitory effect on intestinal harmful enzymes. Therefore, L54 strain, which has excellent inhibitory effect on intestinal toxic enzyme activity, can be effectively improved intestinal environment.

1-6. 16S rRNA Gene Sequence Analysis

16S rRNA gene sequencing was performed to identify L3, L54, and L120 strains that were selected as strains resistant to environmental stress. The genomic DNA of the strain was isolated using AccuPrep® Genomic DNA Extraction kit (Bioneer, Daejeon, Korea), and 27F (5'-AGA GTT TGA TCM TGG CTC AG-3 ') and 1492R (5'-TAC GGY TAC CTT GTT ACG ACTT-3 ') polymerase chain reaction (PCR). PCR was performed with My Cycler (BIO-RAD Laboratories, Hercules, Calif., USA) using Accu-PrepR PreMix (Bioneer). 30 sec at 94 占 폚, 1 min at 60 占 폚, 1 min at 72 占 폚, 35 cycles; Followed by reaction at 72 ° C for 7 minutes. The nucleotide sequence of the PCR product was determined using an ABI PRISM 3700 DNA Analyzer (Macrogen, Seoul, Korea).

In addition, the nucleotide sequence of the isolated strain was analyzed by microbial homology using BLAST at National Center for Biotechnology Information (NCBI).

As shown in Table 3 below, 16S rRNA gene sequencing analysis was performed to identify the isolates. As a result, L3 strain showed 99% or more similarity with Lactobacillus lambusus, L54 strain had 99% %, And the strain L120 showed 99% or more similarity with Lactobacillus acidophilus. Finally, the L3 strain was named Lactobacillus laminocus L3, the L54 strain was named Enterococcus lassi L54, and the L120 strain was named Lactobacillus aspirophilus L120.

Strain name 16S rRNA sequence denomination Homology (%) Scientific name L3 99 Lactobacillus rhamnosus Lactobacillus rhamnosus L3 L54 99 Enterococcus faecium Enterococcus faecium L54 L120 99 Lactobacillus acidophilus Lactobacillus acidophilus L120

<Explanation of operation 2> Exploring availability as probiotics

In order for lactobacilli to exert their effects in a probiotic process, their ability to survive in vivo is important and must survive through strong acidic gastric juices and bile fluid secreted from the pancreas and duodenum. Therefore, in order to confirm the survival possibility of Lactobacillus laminosus L3, Enterococcus lassus L54 and Lactobacillus acidophilus L120 strains isolated in <Description of Action 1> described above, the artificial gastric juice and artificial bile resistance test Were carried out using a medium adjusted to pH 2.5 and a medium containing 0.5% and 1.0% of oxgall.

Specifically, the artificial gastric juice resistance test was carried out by adding 1,000 units / ml of pepsin (Sigma Co., USA) to the MRS liquid medium adjusted to pH 2.5 using 1N HCl in an environment similar to that of the digestive tract of the body, And inoculated and cultured at 37 ° C for 2 hours. The cultures were plated on MRS solid medium and anaerobically cultured at 37 ° C for 48 hours. The number of colonies formed was measured and expressed as Log CFU (colony forming unit) / mL. Survival rate (%) was expressed as [(Log index value of experimental group / Log index value of control group) × 100], and control group was set as control group for artificial gastric juice.

In addition, the artificial gastric juice resistance test was carried out at 37 ° C for 24 hours after addition of 0.5% and 1.0% of oxgall (Sigma Co., USA) to the MRS broth. The cultures were plated on MRS solid medium and anaerobically cultured at 37 ° C for 48 hours. The number of colonies formed was measured and expressed as Log CFU (colony forming unit) / mL. Survival rate (%) was expressed as [(Log index value of experimental group / log index value of control group) × 100], and the control group was set as an artificial bile control group.

3, Lactobacillus laminosus L3 strain, Enterococcus lassus L54 strain and Lactobacillus acidophilus strain L120 strain were found to have an 83% (v / v) similarity to Lactobacillus lambosus GG And high survival rate.

Lactobacillus Lymphosus L3 strain, Enterococcus lassus L54 strain, and Lactobacillus acidophilus strain L120 were found to have a high concentration of 0.5% oxgall in the intestinal bile test, similar to Lactobacillus lymphosus GG (LGG) And 1.0% oxgall showed high survival rates of 87% and 57%, respectively. Therefore, the isolated strains were able to survive in the artificial gastric juice and exhibit high resistance to bile, and thus have a high possibility of survival in the intestines. Therefore, it is possible to utilize them as probiotics.

<Description of operation 3> Production of fermented milk

3-1. Manufacture of starter bacteria for the production of fermented milk

Lactobacillus Lymphosus L3, Enterococcus lassus L54, Lactobacillus acidophilus L120, and commercial strains Chr. And 0.8 to 1.2 parts by weight of the Hansen's YF-L812 (Lactobacillus delbrueckii subsp. Bulgaricus, Streptococcus thermophilus) in each oil and sugar mixture to 10mL MRS broth, (1 part by weight of oil and sugar mixture), preferably 1% inoculation Followed by constant culture at 37 占 폚 for 24 hours. After the supernatant was removed by centrifugation, the remaining cells were suspended in sterilized physiological saline, washed, and cultured in 10% sterilized skim milk twice.

3-2. Production method of fermented milk according to starter addition ratio

In the preparation of the fermented milk, 50 g (in 4-6 parts by weight of crude oil) was preferably added to 1000 g of crude oil, and the mixture was sterilized at 90 캜 for 10 minutes and cooled at 43 캜.

Thereafter, a mixed strain (B) in which commercial strains (YF-L812) (A), Lactobacillus lambosus L3, Enterococcus pathum L54, and Lactobacillus acidophilus L120 strains were mixed at a ratio of 1: 0.8-1.2 parts by weight based on 100 parts by weight of a mixture of crude oil and sugar, and preferably 1 part by weight (1%) as an example,

(0.8%) in a proportion of 1.0%: 0.0%, 0.8%: 0.2g%, 0.6g: 0.4g, 0.4g: 0.6g, 0.2g: 0.8g, 0.1g: 0.9g or 0.0: -1.2) and incubated for 6-8 hours at 42 [deg.] C until the pH reached 4.6. Each fermented milk having a pH of 4.6 was then aged while cooling to 15 ° C and refrigerated at 4 ° C. The fermented milk production process is shown in Fig.

In the description of the present invention, the raw material refers to fresh crude oil having an acidity of 0.14 to 0.15% and a pH of 6.6 to 6.8 produced from Holstein-Friesian species for producing fermented milk.

3-3. Quality characteristics during fermented milk manufacturing process

Changes in pH, titratable acidity and number of lactic acid bacteria were measured during the fermentation process. Specifically, a pH meter (UB-10, Denver, USA) was used for pH measurement. 10 mL of sample was mixed with 10 mL of distilled water to prepare a suspension. The suspension was titrated to pH 8.3 by adding NaOH, Was multiplied by the conversion factor of 0.9 of lactic acid, and the weight (g) of the sample was divided to obtain the titratable acidity (%). The change in the number of lactic acid bacteria was obtained by taking 1 mL of the sample, diluting it in sterilized saline by decidual dilution method, and culturing it at 37 ° C for 48 hours in a plate culture method using BCP agar medium. The number of yellow colonies after that was measured and expressed as Log CFU (colony forming unit) / mL.

As shown in the following Table 4, changes in pH, titratable acidity and number of lactic acid bacteria of fermented milk prepared by varying the addition ratio of three kinds of lactic acid bacteria derived from human body and commercial strains (YF-L812) Bacillus lambatus L3, Enterococcus lassus L54, Lactobacillus acidophilus L120 Fermented milk fermented only as a mixture of three strains was not suitable as a starter for fermented milk production due to its low acid production ability. However, the commercial strains (YF-L812) and the mixed strains of three kinds of human-derived lactic acid bacteria were mixed at a ratio of 0.8%: 0.2g%, 0.6g: 0.4g, 0.4g: 0.6g, 0.2g: 0.8g, , The fermented milk reached pH 4.6, the end point of fermentation at 6 hours after fermentation, similar to fermented milk prepared by using the commercial strain alone. The titratable acidity was maintained at 0.85 ~ 0.91% and the number of lactic acid bacteria was maintained at 10 ⁹ CFU / mL. Therefore, in order to develop a lactic acid bacteria starter for producing fermented milk having excellent resistance to environmental stress, a mixed strain of Lactobacillus laminosus L3, Enterococcus lassus L54, and Lactobacillus acidophilus L120 is mixed with lactic acid bacteria having excellent acid producing ability And it is likely to be used commercially in the future.

division sample Fermentation time (h) 0 2 4 6 8

pH

YF 1.0% + Mix 0.0% 6.45 ± 0.00 6.25 ± 0.00 5.05 ± 0.01 4.53 + - 0.01 4.27 ± 0.00 YF 0.8% + Mix 0.2% 6.47 ± 0.02 6.28 ± 0.01 5.08 ± 0.02 4.55 ± 0.01 4.31 ± 0.01 YF 0.6% + Mix 0.4% 6.47 ± 0.01 6.27 ± 0.01 5.11 ± 0.01 4.57 ± 0.02 4.32 ± 0.00 YF 0.4% + Mix 0.6% 6.46 ± 0.00 6.27 ± 0.00 5.14 ± 0.00 4.58 ± 0.02 4.34 ± 0.00 YF 0.2% + Mix 0.8% 6.47 ± 0.02 6.29 ± 0.00 5.18 ± 0.01 4.59 ± 0.01 4.37 ± 0.01 YF 0.1% + Mix 0.9% 6.46 ± 0.01 6.30 ± 0.01 5.18 ± 0.02 4.61 ± 0.01 4.38 ± 0.01 YF 0.0% + Mix 1.0% 6.46 ± 0.01 6.32 ± 0.01 5.88 ± 0.01 5.53 + 0.02 5.38 ± 0.01


Titratable acidity
(%)

YF 1.0% + Mix 0.0% 0.16 + 0.02 0.32 ± 0.02 0.65 ± 0.01 0.91 + - 0.01 1.18 ± 0.01 YF 0.8% + Mix 0.2% 0.16 ± 0.01 0.33 + 0.02 0.64 + 0.02 0.89 + 0.02 1.16 ± 0.01 YF 0.6% + Mix 0.4% 0.15 + 0.02 0.32 ± 0.02 0.64 + 0.02 0.88 + 0.02 1.15 ± 0.02 YF 0.4% + Mix 0.6% 0.16 + 0.02 0.32 ± 0.01 0.62 + 0.02 0.87 ± 0.02 1.14 ± 0.02 YF 0.2% + Mix 0.8% 0.16 + 0.02 0.33 + 0.02 0.63 + 0.02 0.85 ± 0.01 1.12 + 0.02 YF 0.1% + Mix 0.9% 0.16 + 0.02 0.34 + 0.02 0.62 + 0.02 0.85 + 0.02 1.12 + 0.02 YF 0.0% + Mix 1.0% 0.15 + 0.02 0.28 ± 0.02 0.49 ± 0.01 0.53 + 0.02 0.61 ± 0.01

Number of lactic acid bacteria
(log CFU / ml)

YF 1.0% + Mix 0.0% 6.79 + 0.03 7.91 + 0.04 8.88 + 0.03 9.71 + 0.02 10.51 + 0.03 YF 0.8% + Mix 0.2% 6.78 ± 0.05 7.89 ± 0.04 8.90 + 0.04 9.68 + 0.03 10.48 + 0.04 YF 0.6% + Mix 0.4% 6.76 + 0.04 7.90 + 0.02 8.84 ± 0.05 9.66 + 0.03 10.45 ± 0.05 YF 0.4% + Mix 0.6% 6.79 + 0.02 7.87 ± 0.03 8.82 + 0.03 9.69 + 0.02 10.41 + 0.04 YF 0.2% + Mix 0.8% 6.78 + 0.03 7.90 + 0.03 8.83 ± 0.02 9.65 + 0.04 10.34 ± 0.05 YF 0.1% + Mix 0.9% 6.75 + 0.04 7.89 ± 0.02 8.79 ± 0.04 9.66 ± 0.05 10.34 + 0.03 YF 0.0% + Mix 1.0% 6.70 + 0.04 7.80 + 0.03 7.96 ± 0.05 8.34 + 0.02 8.62 ± 0.03

YF: YF-L812 (Lactobacillus delbrueckii subsp bulgaricus, Streptococcus thermophilus.)

Mix: Lactobacillus rhamnosus L3 , Enterococcus faecium L54, Lactobacillus acidophilus L120

3-4. Sensory evaluation of fermented milk

In order to evaluate the sensory characteristics of fermented milk, 10 sensory evaluation personnel of the institute were selected as the sensory evaluation panel. The panel explained the purpose of the experiment and educated about each measurement. Then, 9 point scale method was used and color, flavor, taste, texture, and overall acceptability were measured. All measurements were expressed as mean ± standard deviation.

As shown in Table 5 below, the fermented milk produced from three kinds of human-derived lactic acid bacteria having excellent resistance to environmental stress was subjected to sensory evaluation. As a result, fermented milk prepared by using the commercial strain (YF-L812) (YF-L812) and fermented milk prepared by mixing three kinds of human-derived lactic acid bacteria without addition of commercial fermented milk and commercial strain (YF-L812) Did not show any difference. However, a commercial strain (YF-L812) containing 5 kinds of Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus laminosus, Enterococcus pathum and Lactobacillus acidophilus lactic acid bacteria and a mixture of three kinds of human-derived lactic acid bacteria Fermented milk showed a high preference. Especially, it was confirmed that the overall preference was increased as the degree of preference for the flavor increased as the amount of Lactobacillus laminosus L3, Enterococcus lassus L54, and Lactobacillus acidophilus L150 was increased there was.

Therefore, the addition amount of the lactic acid bacteria starter to produce the fermented milk having excellent resistance to environmental stress is 0.1 to 10 times that of the commercial strain (YF-L812), Lactobacillus laminosus L3, Enterococcus lassi L54, and Lactobacillus acidophilus L120 %: 0.9% mixture ratio (1: 9) would be most suitable.

sample Sensory evaluation color incense flavor Texture Overall likelihood YF 1.0% + Mix 0.0% 6.1 ± 0.97 5.8 ± 1.15 5.9 ± 1.43 6.2 ± 1.34 6.5 ± 1.14 YF 0.8% + Mix 0.2% 6.1 ± 0.98 6.1 ± 1.23 5.9 ± 1.21 6.2 ± 1.31 6.8 ± 1.22 YF 0.6% + Mix 0.4% 6.2 ± 0.96 6.3 ± 1.14 6.1 ± 1.45 6.2 ± 1.24 6.8 ± 1.43 YF 0.4% + Mix 0.6% 6.1 ± 0.96 6.5 ± 1.24 6.1 ± 1.36 6.1 ± 1.12 7.1 ± 1.54 YF 0.2% + Mix 0.8% 6.1 ± 0.96 6.9 ± 1.26 6.2 ± 1.16 6.1 ± 1.22 7.4 ± 1.24 YF 0.1% + Mix 0.9% 6.1 ± 0.97 7.2 ± 1.08 6.2 ± 1.32 6.1 ± 1.15 7.5 ± 1.16 YF 0.0% + Mix 1.0% 6.0 ± 0.98 5.7 ± 1.35 5.8 ± 0.98 6.1 ± 0.97 6.2 ± 1.43

YF: YF-L812 (Lactobacillus delbrueckii subsp bulgaricus, Streptococcus thermophilus.)

Mix: Lactobacillus rhamnosus L3 , Enterococcus faecium L54, Lactobacillus acidophilus L120

The technical idea of the present invention is as follows.

According to the method described above, the following three strains are newly isolated.

Lactobacillus Lymphosus L3, Enterococcus pathum L54, and Lactobacillus acidophilus L120.

And the fermented milk is produced by inoculation using the newly isolated strain and two commercial strains.

As a detailed operation, in the fermented milk production method,

Mixing crude oil and sugar to produce a first mixture;

Sterilizing the first mixture;

Cooling the sterilized first mixture;

Inoculating the cooled first mixture with probiotic lactic acid bacteria as a starter;

Culturing a second mixture in which the probiotic lactic acid bacteria has been inoculated with the starter;

Cooling the cultured second mixture; And

And fermenting the fermented milk using the lactic acid bacterium derived from human body, which is excellent in resistance to environmental stress.

Preparing a crude oil and sugar, Lactobacillus lambosus L3, Enterococcus lassi L54 and Lactobacillus acidophilus L120, Lactobacillus bulgaricus and Streptococcus thermophilus strain;

Mixing 4 to 6 parts by weight, preferably 5 parts by weight (5% based on crude oil) of sugar with respect to 100 parts by weight of the crude oil prepared in the preparation step to prepare a first mixture;

Sterilizing the first mixture at a temperature of 85 to 95 DEG C for 5 to 15 minutes;

Cooling the sterilized first mixture to 40 to 45 캜;

An inoculation step of inoculating 0.8-1.2 parts by weight of lactic acid bacteria as a starter with respect to 100 parts by weight of the cooled first mixture;

Fermenting the fermented milk by fermenting the second mixture in which lactic acid bacteria have been inoculated with the starter at a temperature of 35 to 45 캜 for 6 to 8 hours until the final pH is 4.3 to 4.6;

Cooling the fermented milk to 15 to 20 캜; And

And cooling the cooled fermented milk at a temperature of 3 to 5 DEG C to produce fermented milk using the human lactic acid bacteria having excellent environmental stress resistance.

The inoculated lactic acid bacteria are composed of Lactobacillus laminosus L3, Enterococcus lassus L54, Lactobacillus acidophilus L120, Lactobacillus bulgaricus, and Streptococcus thermophilus.

The inoculated lactic acid bacterium was prepared by mixing 100 parts by weight of a mixture of crude oil and sugar with a mixture of Lactobacillus laminosus L3, Enterococcus lassus L54, Lactobacillus acidophilus L120 mixed bacterium (A), Lactobacillus bulgaricus and Streptococcus thermo 0.8-1.2 parts by weight of the mixed microorganism (B) of Phyllis is inoculated, and the above A and B are inoculated at a ratio of 9: 1.

100 parts by weight of Lactobacillus laminocus L3 is composed of 100 parts by weight of Enterococcus passum L54 and 100 parts by weight of Lactobacillus acidophilus L120.

As described above, the present invention relates to a human-derived lactic acid bacterium having excellent resistance to environmental stress, and a method for producing fermented milk using the same, wherein the human lactic acid bacterium is isolated from adult feces and has excellent oxidative stress, heavy metals and radiation resistance, Lactobacillus lambatus L3, Enterococcus lassus L54 and Lactobacillus acidophilus strain L120, which are excellent in artificial gastric juice and artificial bile fluid resistance to be probiotic lactic acid bacteria, and the like, .

Claims (6)

A method for producing a fermented milk,
Mixing crude oil and sugar to produce a first mixture;
Sterilizing the first mixture;
Cooling the sterilized first mixture;
Inoculating the cooled first mixture with probiotic lactic acid bacteria as a starter;
Culturing a second mixture in which the probiotic lactic acid bacteria has been inoculated with the starter;
Cooling the cultured second mixture; And
Wherein the inoculated lactic acid bacterium is selected from the group consisting of Lactobacillus laminosus L3 deposit number KCTC18485P, Enterococcus pathum L54 deposit number KCTC18486P, Lactobacillus acidophilus L120 A lactic acid bacteria belonging to the genus Lactobacillus, a lactic acid bacteria belonging to the genus Lactobacillus, a lactic acid bacteria belonging to the genus Lactobacillus, and a Streptococcus thermophilus. Crude oil and sugar, Lactobacillus Lymphosus L3 Accession No. KCTC18485P, Enterococcus lassi L54 Accession No. KCTC18486P and Lactobacillus acidophilus L120 Accession No. KCTC18487P strain, Lactobacillus bulgaricus and Streptococcus thermophilus strain step;
Mixing 4 to 6 parts by weight of sugar with 100 parts by weight of crude oil prepared in the preparation step to prepare a first mixture;
Sterilizing the first mixture at a temperature of 85 to 95 DEG C for 5 to 15 minutes;
Cooling the sterilized first mixture to 40 to 45 캜;
An inoculation step of inoculating 0.8-1.2 parts by weight of lactic acid bacteria as a starter with respect to 100 parts by weight of the cooled first mixture;
Fermenting the fermented milk by fermenting the second mixture inoculated with 0.8-1.2 parts by weight of lactic acid bacteria with the starter at a temperature of 35 to 45 ° C for 6 to 8 hours until the final pH is 4.3 to 4.6;
Cooling the fermented milk to 15 to 20 캜; And
And cooling the cooled fermented milk at a temperature of 3 to 5 占 폚. The method for producing fermented milk using the human-derived lactic acid bacterium according to claim 1,
delete 3. The method according to claim 1 or 2,
The inoculated lactic acid bacteria were mixed with 100 parts by weight of a mixture of crude oil and sugar with 100 parts by weight of lactobacillus laminosis L3 deposit number KCTC18485P, enterococcus pathum L54 deposit number KCTC18486P, Lactobacillus acidophilus L120 deposit number KCTC18487P mixed bacteria (A) , 0.8-1.2 parts by weight of a mixed microorganism (B) of Lactobacillus bulgaricus and Streptococcus thermophilus, and said A and B are inoculated at a ratio of 9: 1. For the production of fermented milk using
5. The method of claim 4,
100 parts by weight of Enterococcus pathum L54 deposit number KCTC18486P and 100 parts by weight of Lactobacillus acidophilus L120 deposit number KCTC18487P relative to 100 parts by weight of Lactobacillus laminocus L3 deposit number KCTC18485P. A method for producing fermented milk using lactic acid bacteria derived from human body. delete

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