CN114634901B - Lactobacillus casei LC16 for promoting bone health and culture method and application thereof - Google Patents
Lactobacillus casei LC16 for promoting bone health and culture method and application thereof Download PDFInfo
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- CN114634901B CN114634901B CN202210537666.5A CN202210537666A CN114634901B CN 114634901 B CN114634901 B CN 114634901B CN 202210537666 A CN202210537666 A CN 202210537666A CN 114634901 B CN114634901 B CN 114634901B
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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Abstract
The invention relates to lactobacillus casei LC16 for promoting bone health, a culture method and application thereof, wherein the lactobacillus casei LC16 for promoting bone health is named as lactobacillus caseiLactobacillus caseiThe LC16 strain has a preservation number of CGMCC No. 24110. The strain can promote bone health and reduce the risk of osteoporosis, and is specifically embodied in that: can obviously increase bone density and promote calcium absorption; can promote osteoblast proliferation, inhibit osteoclast, and regulate bone metabolism.
Description
Technical Field
The invention belongs to the technical field of microbial culture, relates to lactobacillus casei LC16 for promoting bone health, a culture method and application thereof, and has the functions of promoting bone health and reducing the risk of osteoporosis, and lactobacillus casei LC16 for promoting bone health and a culture method and application thereof.
Background
The bone health is one of the important basic stones for human body health, and is also a characteristic of human body longevity, and all actions of the human body can not leave the support of the bone. With the increasing aging speed of the population in China, bone health problems such as osteoporosis threaten the health of the old, particularly postmenopausal women, and become a serious social problem. It is a systemic skeletal disease characterized by low bone mass and degeneration of bone tissue microstructure, accompanied by increased bone fragility and easy fracture, belonging to common diseases of middle-aged and elderly people, and seriously damaging body health and life quality.
With the growing concern of research on bone health, it has been found that intestinal microorganisms are closely related to bone metabolism. Research shows that the proliferation of beneficial bacteria in intestinal microorganisms in the intestines can not only inhibit the growth of pathogenic bacteria in the intestines and prevent and treat various intestinal diseases, but also is closely related to probiotics in the health of organs and tissues far away from the intestines, such as skin, arteries and bones. Particularly, the conservation of the bone health can produce short-chain fatty acid through fibers in fermented foods of beneficial bacteria in the intestinal tract, such as lactobacillus and the like, reduce the local pH value of the intestinal tract, and reduce the formation of a compound of calcium ions and phosphorus in the intestinal tract, thereby promoting the calcium absorption; beneficial bacteria can change the immune state of the whole body and bone marrow, regulate osteoclast generation and further influence bone metabolism; the beneficial flora can stimulate intestinal tract cells to secrete the incretin, including glucose-dependent glucagon peptide (GIP) and glucagon-like peptide-1 (GLP-1), promote bone formation, inhibit bone absorption, promote bone health and reduce the risk of osteoporosis. Therefore, intestinal beneficial bacteria, such as lactobacillus casei, lactobacillus paracasei, bifidobacterium lactis and the like, may become new therapeutic targets for treating osteoporosis and preventing fracture.
Therefore, it is an urgent problem to provide a microbial preparation which can effectively improve/treat bone health problems such as osteoporosis, and a user can try to use a health product or a medicine containing specific probiotics to solve the problem of poor life experience of a patient due to osteoporosis or other bone health problems.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide lactobacillus casei LC16 for promoting bone health and a culture method and application thereof, and provides lactobacillus casei LC16 for promoting bone health and reducing the risk of osteoporosis and a culture method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a lactobacillus casei LC16 for promoting bone health, wherein the lactobacillus casei LC16 for promoting bone health is named as lactobacillus caseiLactobacillus caseiThe LC16 strain has a preservation number of CGMCC No.24110 and a preservation date of 2021, 12 months and 15 days.
The invention discloses a new lactobacillus casei capable of promoting bone health, which is obtained and preserved by separating and preserving a lactobacillus casei from a milk curd sample of a Zhenglan flag of Sinomeng of inner Mongolia and Xiliangyen province, and is named as lactobacillus caseiLactobacillus caseiThe LC16 strain can promote bone health and reduce the risk of osteoporosis, and is specifically represented by the following components: (1) can obviously increase the bone mineral density and promote the calcium absorption; (2) can promote osteoblast proliferation, inhibit osteoclast, and regulate bone metabolism. Thus, the Lactobacillus caseiLactobacillus caseiThe LC16 strain is used in preparing products (such as medicines, health products, etc.) for improving or treating osteoporosis.
In addition, the lactobacillus casei is a probiotic, so the safety is high when the lactobacillus casei LC16 screened by the invention is used in products (such as medicines, health products and the like) for improving or treating osteoporosis; and the drug resistance can not be generated, and the drug can be used for improving or treating osteoporosis for a long time.
The screening steps of the lactobacillus casei related by the invention are as follows:
(1) selecting milk tofu samples separated from YunyiRi Hippo Hoyle on the blue flag of Xilei Meng of inner Mongolia, performing 10-fold gradient dilution by using normal saline with the mass concentration of 0.9%, diluting for 3 times, coating the milk tofu samples on a solid culture medium, culturing for 48 hours at 38 ℃, selecting 3 strains with different forms, streaking and purifying the strains on the surface of an improved MRS solid culture medium, selecting single strains, performing enlarged culture by using a liquid culture medium at 37 ℃, and then preserving by using glycerol with the mass concentration of 40%.
(2) In-vitro physiological property test is carried out on the single strains from the preserved 3 strains of the milk curd, and a single strain with the most growth capacity, acid resistance and cholate resistance (artificial simulation) and SCFA (short chain fatty acid) production is screened out and identified.
Preferably, the lactobacillus caseiLactobacillus caseiThe culture method of the LC16 strain comprises the following steps: lactobacillus caseiLactobacillus caseiInoculating LC16 strain in culture medium, and culturing at 35-38 deg.C (such as 35 deg.C, 36 deg.C, 37 deg.C, 38 deg.C, etc.) for 18-22h (such as 18h, 19 h, 20h, 21 h, 22h, etc.); other specific values within the range can be selected, and are not further described herein.
The formula of the culture medium comprises: peptone, beef extract, glucose, lactose, yeast extract, diammonium hydrogen citrate, and K 2 PO 4 ·3H 2 O、MgSO 4 ·7H 2 O、MnSO 4 And L-cysteine.
Specifically, the formula composition of the culture medium (liquid) comprises: 10g of peptone, 10g of beef extract, 20g of glucose, 10g of lactose, 5g of yeast extract, 2g of diammonium hydrogen citrate and K 2 PO 4 ·3H 2 O 2g、MgSO 4 ·7H 2 O 0.6g、MnSO 4 0.01g and 1g of L-cysteine.
The invention preferably selects the culture conditions, namely lactobacillus caseiLactobacillus caseiThe LC16 strain can reach a growth stationary phase and has more excellent utilization capability of carbon sources (glucose, maltose, lactose, galactose, melezitose, sucrose and the like).
In a second aspect, the present invention provides the use of a lactobacillus casei for promoting bone health as described in the first aspect in the manufacture of a product for the amelioration or treatment of osteoporosis; the product comprises health products or medicines.
The invention relates to lactobacillus caseiLactobacillus caseiThe LC16 strain can be used in the related products independently, and can also be combined with other strains.
In a third aspect, the invention provides a probiotic preparation with effect of improving or treating osteoporosis, wherein the probiotic preparation with effect of improving or treating osteoporosis comprises lactobacillus casei of the first aspectLactobacillus caseiThe strain LC 16.
Preferably, in the probiotic agent, the lactobacillus caseiLactobacillus caseiThe viable count of LC16 strain is not less than 1 × 10 8 CFU/mL or 1X 10 8 CFU/g, e.g. 1X 10 8 CFU/mL、2×10 8 CFU/mL、5×10 8 CFU/mL、8×10 8 CFU/mL、1×10 9 CFU/mL、5×10 9 CFU/mL、1×10 10 CFU/mL, etc., and other specific values within the range can be selected, which are not described in detail herein.
Preferably, the probiotic agent can be in the form of freeze-dried powder, and the freeze-dried powder can be further prepared into capsules, tablets and other dosage forms.
Preferably, the probiotic with the effect of improving or treating osteoporosis further comprises a protective agent and/or a prebiotic;
the protective agent comprises deslagged soybean powder;
the prebiotics comprise one or a combination of at least two of inulin, fructo-oligosaccharide, galacto-oligosaccharide, mannose oligomer, trehalose, soybean oligosaccharide, resistant dextrin, spirulina, polydextrose, alpha-lactoalbumin or lactoferrin.
In a fourth aspect, the present invention provides a composite probiotic having an effect of improving or treating osteoporosis, comprisingIn one aspect, the Lactobacillus caseiLactobacillus caseiLC16 strain and Lactobacillus paracaseiLactobacillus paracaseiLC86 strain; the lactobacillus paracaseiLactobacillus paracaseiThe preservation number of the LC86 strain is CGMCC number 1.12731, and the preservation date is 2020, 7 and 20 days.
The invention also creatively discovers the lactobacillus caseiLactobacillus caseiThe LC16 strain can be combined with lactobacillus paracaseiLactobacillus paracaseiThe LC86 strain is compounded for use to promote bone health, has excellent effects, and compared with a single microbial inoculum, the compound microbial inoculum has more remarkable effects of promoting bone health and reducing osteoporosis risk, which shows that the LC16 strain and the LC86 strain have synergistic effects in promoting bone health and reducing osteoporosis risk.
Preferably, the lactobacillus caseiLactobacillus caseiLC16 strain and Lactobacillus paracaseiLactobacillus paracaseiThe mass ratio of the LC86 strain is (1-4): (1-4), such as 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, etc., and other specific point values in the value range can be selected, which is not described in detail herein.
In the composite probiotic preparation, the two strains have better synergistic effect when meeting the specific mass proportion relation.
Preferably, the complex probiotic with the effect of improving or treating osteoporosis further comprises a protective agent and/or a prebiotic;
the protective agent comprises deslagged soybean powder;
the prebiotics comprise one or a combination of at least two of inulin, fructo-oligosaccharide, galacto-oligosaccharide, mannose oligomer, trehalose, soybean oligosaccharide, resistant dextrin, spirulina, polydextrose, alpha-lactoalbumin or lactoferrin.
Further preferably, the composite probiotic with the effect of improving or treating osteoporosis also comprises lactobacillus reuteriLactobacillus reuteriLR08 strain; the Lactobacillus reuteri strainLactobacillus reuteriThe preservation number of the LR08 strain is CGMCC number 1.12733The preservation date is 2020, 7 and 20.
The invention also creatively discovers that lactobacillus reuteri is added into the composite probioticLactobacillus reuteriThe composite probiotic obtained from the LR08 strain has a further optimized effect, and compared with a single microbial inoculum or a compound mode of the two, the composite microbial inocula of the three strains have more remarkable effects of promoting bone health and reducing osteoporosis risk, which shows that the LC16 strain, the LC86 strain and the LR08 strain have synergistic effects in promoting bone health and reducing osteoporosis risk.
The Lactobacillus reuteri strainLactobacillus reuteriLR08 strain and Lactobacillus caseiLactobacillus caseiLC16 strain and Lactobacillus paracaseiLactobacillus paracaseiThe mass ratio of the LC86 strain is (1-4): (1-4): (1-4), such as 1:4:1, 1:3:1, 1:2:1, 1:1:1, 2:1:1, 3:1:1, 4:1:1, 1:4:2, 1:3:2, 1:2:2, 1:1:2, 2:1:2, 3:1:2, 4:1:2, 1:4:4, 1:3:4, 1:2:4, 1:1:4, 2:1:4, 3:1:4, 4:1:4, etc., and other specific points in the value range can be selected, and are not described in detail herein.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a new lactobacillus casei capable of promoting bone health, which is obtained and preserved by separating and preserving a lactobacillus casei from a milk curd sample of a Zhenglan flag of Sinomeng of inner Mongolia and Xiliangyen province, and is named as lactobacillus caseiLactobacillus caseiThe LC16 strain can promote bone health and reduce the risk of osteoporosis, and is specifically represented by the following components: (1) can obviously increase the bone mineral density and promote the calcium absorption; (2) can promote osteoblast proliferation, inhibit osteoclast, and regulate bone metabolism. Thus, the Lactobacillus caseiLactobacillus caseiThe LC16 strain is used in preparing products (such as medicines, health products, etc.) for improving or treating osteoporosis.
The invention also creatively discovers the lactobacillus caseiLactobacillus caseiThe LC16 strain can be combined with lactobacillus paracaseiLactobacillus paracaseiThe LC86 strain is compounded for use to promote bone health and has excellent propertyCompared with a single microbial inoculum, the compound microbial inoculum has more remarkable effects of promoting bone health and reducing osteoporosis risk, and shows that the LC16 strain and the LC86 strain have synergistic effects on promoting bone health and reducing osteoporosis risk. And lactobacillus reuteri is added into the composite probioticLactobacillus reuteriThe composite probiotic agent obtained from the LR08 strain has a further optimized effect, and compared with a single agent or a compound mode of the two, the composite probiotic agent of the three strains has more remarkable effects of promoting bone health and reducing osteoporosis risk.
Detailed Description
In order to further illustrate the technical means and effects of the present invention, the technical solutions of the present invention are further described below with reference to the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.
The lactobacillus casei is lactobacillus caseiLactobacillus caseiThe LC16 strain has a preservation unit of China general microbiological culture Collection center (CGMCC), a preservation number of CGMCC No.24110, a preservation date of 2021, 12 and 15 days, and a preservation address of No. 3 of Xilu No. 1 of Beijing, Chaoyang, North Cheng, China.
The lactobacillus paracasei is lactobacillus paracaseiLactobacillus paracaseiThe LC86 strain has a preservation unit of China general microbiological culture Collection center (CGMCC), a preservation number of CGMCC 1.12731, a preservation date of 2020, 7 and 20 days, and a preservation address of No. 3 of Xilu No. 1 Beichen of Chaoyang district in Beijing.
The following related Lactobacillus reuteri is Lactobacillus reuteriLactobacillus reuteriThe LR08 strain has a preservation unit of China general microbiological culture Collection center (CGMCC), a preservation number of CGMCC 1.12733, a preservation date of 2020, 7 and 20 days, and a preservation address of No. 3 of Xilu No. 1 of Beijing Kogyo-Yang district, Beijing.
The following related MRS solid culture media: weighing 10g of peptone, 10g of beef extract, 20g of glucose, 10g of lactose, 5g of yeast extract, 2g of diammonium hydrogen citrate and K 2 PO 4 ·3H 2 O 2 g、MgSO 4 ·7H 2 O 0.6 g、MnSO 4 Dissolving 0.01g of agar, 20g of agar and 1g of L-cysteine by using deionized water, adding 1 mL of Tween 80, diluting to a constant volume of 1L, sterilizing, cooling, and pouring into a sterilized culture dish for later use.
The following related MRS liquid culture media: weighing 10g of peptone, 10g of beef extract, 20g of glucose, 10g of lactose, 5g of yeast extract, 2g of diammonium hydrogen citrate and K 2 PO 4 ·3H 2 O 2 g、MgSO 4 ·7H 2 O 0.6 g、MnSO 4 0.01g and 1g of L-cysteine, dissolving with deionized water, adding 1 mL of Tween 80, diluting to 1L, sterilizing, cooling, and pouring into a sterilized culture dish for later use.
The following related Lactobacillus casei LC16 bacterial suspensions: inoculating lactobacillus casei in MRS liquid culture medium, culturing at 38 deg.C for 18 hr for activation, and continuously activating for 2 times to obtain activation solution; inoculating the activated solution into an MRS liquid culture medium according to the inoculation amount of 2% (v/v), and culturing at 38 ℃ for 18h to obtain a bacterial solution; centrifuging the bacterial liquid at 8000g for 10min, and filtering the supernatant with 0.22 μm sterile filter membrane to obtain Lactobacillus casei supernatant; and (4) resuspending the thallus by using PBS to obtain the bacillus subtilis.
The following related bacterial suspension of lactobacillus paracasei LC 86: inoculating lactobacillus paracasei LC86 in MRS liquid culture medium, culturing at 38 deg.C for 18h for activation, and continuously activating for 2 times to obtain activated solution; inoculating the activated liquid into an MRS liquid culture medium according to the inoculation amount of 2% (v/v), and culturing at 38 ℃ for 18h to obtain a bacterial liquid; centrifuging the bacterial liquid at 8000g for 10min, and filtering the supernatant with 0.22 μm sterile filter membrane to obtain Lactobacillus paracasei supernatant; and (5) resuspending the thallus by using PBS.
The following related lactobacillus reuteri LR08 bacterial suspensions: inoculating lactobacillus reuteri LR08 in MRS liquid culture medium, culturing at 38 deg.C for 20h for activation, and continuously activating for 2 times to obtain activation solution; inoculating the activated liquid into an MRS liquid culture medium according to the inoculation amount of 2% (v/v), and culturing at 38 ℃ for 20h to obtain a bacterial liquid; centrifuging the bacterial liquid at 8000g for 10min, and filtering the supernatant with 0.22 μm sterile filter membrane to obtain Lactobacillus reuteri supernatant; and (4) resuspending the thallus by using PBS to obtain the bacillus subtilis.
Example 1
In this example, a lactobacillus casei strain for promoting bone health was selected, and the steps were as follows:
(1) selecting milk tofu samples separated from YunyiRi Hippo Hosiella on the blue flag of Xilei Meng of inner Mongolia, performing 10-fold gradient dilution by using normal saline with the mass concentration of 0.9%, diluting for 3 times, coating the milk tofu samples on a solid culture medium, culturing for 48 hours at 38 ℃, picking out different forms of fungus, streaking and purifying the fungus on the surface of an improved MRS solid culture medium, picking out single fungus colonies, performing expanded culture by using a liquid culture medium at 37 ℃, and preserving by using glycerol with the mass concentration of 40%.
(2) The method comprises the following steps of carrying out in-vitro physiological property test on a single strain from preserved milk curd, and screening out a single strain with the highest growth capacity, acid and bile salt resistance (artificial simulation) and SCFA (short chain fatty acid) production quantity, wherein the specific steps are as follows:
A. acid and bile salt resistance test:
MRS liquid culture medium, MRS solid culture medium and separation culture medium (0.5 percent CaCO is added to solid CMRS) 3 ) The main reagents include pepsin, trypsin, sodium taurocholate and CaCO 3 And the like. Adjusting pH of MRS culture medium to 3.0, sterilizing at 121 deg.C for 15min, inoculating the activated two-generation liquid culture spread culture medium at an inoculum size of 2%, culturing at 37 deg.C for 24h, and measuring the absorbance change Δ OD in 24h process 600 A value; adding 0.3% of ox bile salt into MRS culture medium, sterilizing at 121 deg.C for 15min, inoculating the activated liquid culture at 2%, culturing at 37 deg.C for 24h, and measuring the change of absorbance (Δ OD) in 24h process 600 The last two Δ OD 600 About 10 strains with relatively large values were subjected to the next experiment.
Acid resistance test: adjusting pH to 3.0 with 37% hydrochloric acid based on PBS buffer solution of pH7.0, sterilizing at 121 deg.C for 15min, inoculating the activated liquid culture of two generations at 10% inoculum size, culturing at 37 deg.C, and sampling for 0min, 30min, 60min, 90min and 120min respectively to determine viable count.
Bile salt resistance test: the liquid culture after two generations of strain activation is inoculated into MRS culture media with different cholate concentrations (the culture media respectively contain 0.1%, 0.2%, 0.3%, 0.5% and 2% cholate) by the inoculation amount of 2%, and meanwhile, the MRS culture media without cholate are used as a reference, sampling is carried out after constant temperature culture is carried out for 24 hours at 37 ℃ to determine the viable count, and the excellent strains with acid resistance and cholate resistance are screened by combining the previous experimental result.
B. Acid-producing capability test: the acid production capacity of the strain is determined by a titration method. Activating the strain preserved by the glycerinum pipe, inoculating the activated strain into an MRS liquid culture medium according to the inoculation amount of 2%, culturing at the constant temperature of 37 ℃ for 24h, putting 10mL of fermentation liquor of each strain into 50mL of sterile water, dropwise adding 2-3 drops of 1g/L phenolphthalein serving as an indicator, titrating by using 0.1mol/L NaOH standard solution, and taking pink of the solution and no fading after 30s as a titration end point, wherein each sample is parallel for 3 times. The non-inoculated MRS liquid culture medium is used as a blank control, and the calculation formula is as follows: total acidity/(g. L) -1 ) = [(V1-V2)·c·100]V0 (V1 is the volume of NaOH solution consumed by the sample, mL; V2 is the volume of NaOH solution consumed by the blank control, mL; V0 is the total volume of the diluent, mL; c is the concentration of standard NaOH, mol/L). The probiotics can produce short chain fatty acids such as butyric acid, propionic acid, acetic acid and the like while rapidly growing nutrients in a metabolic culture medium. And (3) comparing the acid production rate of single bacteria, and screening the lactobacillus casei LC16 with excellent performance by combining a tolerance experiment.
Example 2
In this example, the strain obtained by screening in example 1 was subjected to morphological identification and 16S rRNA molecular biology identification, which comprises the following steps:
(1) and (3) morphological identification:
the strain is inoculated in MRS solid medium, cultured for 48h at 38 ℃, and observed under a microscope. The bacterial colony is milky white, round and bright. The strain in logarithmic growth phase is selected and detected by an optical microscope, and is observed by microscopic examination after smear and gram staining: gram staining is positive, the strain is rod-shaped, and spores and flagella do not exist.
(2) Molecular biological identification of 16S rRNA:
taking out the strain preserved at-80 deg.C, inoculating into a centrifuge tube containing 20mL MRS liquid culture medium at a ratio of 2%, culturing at 38 deg.C for 18h, centrifuging at 8000 rpm for 10min, removing supernatant, and collecting thallus. Extracting genome of the strain, adding a bacterial universal primer for PCR amplification, and delivering an amplification product to Shanghai biological engineering GmbH for sequencing identification. The 16S rDNA sequence of the strain is shown in SEQ ID No. 1 through sequencing analysis. The sequence obtained by sequencing is compared with the nucleic acid sequence in GeneBank, and the result shows that the strain is really lactobacillus casei.
SEQ ID No:1：
GAGCATCGTTCGTCACCTTAGACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTCAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCGCTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCACGCCGACAACAGTTACTCTGCCGACCATTCTTCTCCAACAACAGAGTTTTACGACCCGAAAGCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATCATCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATACGCCGCGGGTCCATCCAAAAGCGATAGCTTGCGCCATCTTTCAGCCAAGAACCATGCGGTTCTTGGATTTATGCGGTATTAGCATCTGTTTCCAAATGTTATCCCCCACTTAAGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCACTCGTTTTAAGTCGAATCTCAGTGCAAGCACCGTTCATCGACCAAAACTCGTTCGACTGCATGTATAGGCACCCCGCCCCCCG。
Based on the results of the molecular biological identification and morphological identification of 16S rRNA in example 2, it was confirmed that the strain belongs to Lactobacillus casei, which was named Lactobacillus caseiLactobacillus caseiThe strain LC 16.
Example 3
In this example, the physiological and biochemical characteristics of the strain screened in example 1 were characterized, and the results are shown in table 1:
TABLE 1
Example 4
This example is for Lactobacillus caseiLactobacillus caseiThe culture conditions of the LC16 strain are optimized, and the steps are as follows:
inoculating Lactobacillus casei LC16 in MRS liquid culture medium, culturing at 10-50 deg.C for 48 hr, and measuring OD of culture solution with microplate reader at intervals during culture 600 Numerical values. The results are shown in table 2:
TABLE 2
The result shows that the lactobacillus casei LC16 grows optimally at 35-38 ℃ and pH of about 6.86, and the growth stationary phase can be reached after culturing for 18-22 h.
The carbon source utilization capacity of the strain Lactobacillus casei LC16 is judged and detected by performing sugar fermentation reaction on a detection strain, namely Lactobacillus casei LC16 by using an API 50CHL culture medium (a basic culture medium consisting of API 50CH test strips of 48 fermentable carbohydrates) and the API 50CH test strips according to API bacteria identification standards. The principle of the method is that a strain to be measured is made into a suspension to be inoculated into each test strip small tube, and after cultivation, the carbon source tube which can be utilized produces acid due to fermentation, and the pH value is reduced, so that the indicator is discolored.
As a result, the strain LC16 can utilize various sugar sources such as glucose, maltose, lactose, galactose, melezitose and sucrose as carbon sources.
Example 5
This example demonstrates the gastric acid resistance of lactobacillus casei LC16 by the following steps:
(1) preparing artificial gastric juice:
the artificial gastric juice contains 0.20% of NaCl and 0.30% of pepsin by mass fraction, the pH is respectively adjusted to 2.0, 2.5 and 3.0 by using HCl, and the artificial gastric juice is filtered and sterilized for standby.
(2) And (3) testing gastric acid resistance:
1.0 mL of Lactobacillus casei LC16 bacterial suspension (concentration 1X 10) 9 CFU/mL, the method of national standard GB4789.35-2016 food safety national standard food microbiology detection lactic acid bacteria detection is adopted to measure the concentration of bacteria liquid), the bacteria liquid is respectively mixed with 9.0 mL of artificial gastric juice with pH of 2.0, 2.5 and 3.0, anaerobic static culture is carried out at 37 ℃, samples are respectively taken after starting (0 h) and processing for 3h, the viable count is determined by a pouring culture method, and the survival rate is calculated, the formula is as follows:
survival (%) = N1/N0 × 100%,
wherein, N1: viable count after 3h of artificial gastric juice treatment; n0: viable count of 0 h. The test results are shown in Table 3.
TABLE 3
As can be seen from Table 3, the Lactobacillus casei LC16 has good gastric acid resistance, and the survival rate can reach more than 75.0 percent after the Lactobacillus casei LC16 is incubated in artificial gastric juice with the pH of 2.0 for 3 hours; the survival rate can reach more than 93.4 percent after the artificial gastric juice with the pH value of 2.5 is incubated for 3 hours; the survival rate can reach more than 95.1 percent after the artificial gastric juice with the pH value of 3.0 is incubated for 3 hours. The good acid resistance creates conditions for preparing products for improving or treating bone health problems such as osteoporosis and the like.
Example 6
This example explores the effect of lactobacillus casei LC16 on bone density in rats by the following steps:
(1) construction of a test rat model:
72 clean grade SD female rats weighing 230 g + -20 g, provided by the Shanghai laboratory animal research center, were subject to ethical approval by the animal ethical Committee (ethical No.: 2021082003) of the Shanghai laboratory animal research center. According to the conventional method, rats were anesthetized with a 30mg/kg · bw sodium pentobarbital solution by intraperitoneal injection, and both ovaries (control group and intervention group) were excised in a sterile manner, and one group was performed in the same manner without excision of both ovaries of rats (sham operation group). After 5 days of ovariectomy, vaginal smear examination was performed, and rats with incomplete ovariectomy were removed to complete the modeling of experimental rats.
(2) Grouping and administration:
the test was divided into 9 groups of 8 rats each, namely, a sham operation group, a model control group (MC group, purified water was given), a positive control group (PC group, 1.0 mg/(kg. bw. d) of estradiol, purified water was given), a calcium carbonate control group (75 mg/kg. bw. d), and a probiotic LC16 dry control group (bacterial suspension 10) 8 CFU/day), probiotic LC86 dried group (bacterial suspension 10) 8 CFU/day), probiotic LR08 dried group (bacterial suspension 10) 8 CFU/day), probiotic LC16+ LC86 combination intervention group (bacterial suspension (5 × 10) 7 ) CFU/day + bacterial suspension (5X 10) 7 ) CFU/day), probiotic LC16+ LC86+ LR08 combination intervention group (bacterial suspension (4 × 10) 7 ) CFU/day + bacterial suspension (4X 10) 7 ) CFU/day + bacterial suspension (2X 10) 7 ) CFU/day). Gavage was performed at a rate of 10mL/kg once a day for 12 weeks, and body weights were recorded weekly and compared to the differences in body weight change in rats of each group, with the results shown in Table 4:
TABLE 4
From the data in table 4, it can be seen that: the weight change of the MC group is more obvious compared with that of a sham operation group rat, the weight of the MC group increases by about (134 +/-6.1) g in 12 weeks, the weight of the sham operation control group increases by (94 +/-5.5) g in 12 weeks, and hormone level disorder caused by female reduction possibly occurs after the operation of the MC group rat, so that abnormal weight rising can prove that the modeling is successful.
(3) Measuring the bone density of the femur:
the rats were dissected, the left femur was dissected and baked to constant weight. The results of measuring the bone density at the midpoint of the femur and at the distal end of the femur of a rat using a discovery-wi type bone densitometer and measuring the bone calcium content by the atomic absorption method are shown in table 5.
TABLE 5
As can be seen from table 5, the distal bone density, bone calcium content and center point bone density were significantly higher in the PC group and the sham group than in the MC group. The distal bone density and the bone calcium content of the LC16 intervention group, the LC86 intervention group, the LR08 intervention group and the calcium carbonate control group are obviously increased, and the effects of the LC16+ LC86 combination intervention group and the LC16+ LC86+ LR08 combination intervention group are more obvious.
Example 7
This example explores the effect of lactobacillus casei LC16 on the calcium absorption capacity of rats by the following steps:
(1) test rats:
56 weaned rats born at about 4 weeks, weighing 75 g. + -.5 g, were provided by the laboratory animal research center of Shanghai city and were subject to ethical approval by the animal ethical Committee (ethical No. 2021082003) of the laboratory animal research center of Shanghai.
(2) Grouping and administration:
preparing freeze-dried fungus powder: pre-culturing the bacterial suspension at 38 deg.C for 2 hr, lyophilizing to obtain lyophilized bacterial powder, and mixing with milk mineral salt (0525 BG milk calcium, milk white powder, available from Alla food raw materials trade (Beijing) Co., Ltd.) under stirring.
The experiment was divided into 7 groups of 8 rats each, each of which was:
(2.1) a low calcium control group (administered 500mg/(kg bw d));
(2.2) calcium carbonate control group (calcium carbonate was administered at 3000mg/(kg bw d));
(2.3) probiotic LC16 drying group (lyophilized powder containing LC16 Strain 10) 8 CFU/day, calcium content 1000mg/(kg bw d));
(2.4) probiotic LC86 drying group (lyophilized powder containing LC86 Strain 10) 8 CFU/day, calcium content 1000mg/(kg bw d));
(2.5) probiotic LR08 Dry Pre-group (lyophilized powder containing LR08 Strain 10) 8 CFU/day, calcium content 1000mg/(kg bw d));
(2.6) probiotic LC16+ LC86 combined drying group (lyophilized bacterial powder containing LC16 strain (5X 10) 7 ) CFU/day + strain containing LC86 (5X 10) 7 ) CFU/day + calcium content 1000mg/(kg bw d));
(2.7) probiotic LC16+ LC86+ LR08 combined drying group (lyophilized bacteria powder containing LC16 (4 x 10) 7 ) CFU/day + strain containing LC86 (4X 10) 7 ) CFU/day + LR 08-containing strain (2X 10) 7 ) CFU/day + calcium content 1000mg/(kg bw d)).
(3) Determination of bone calcium content and apparent absorption rate of calcium:
gavage was performed at a dose of 10mL/kg once a day for 4 weeks, the length and weight of the rats were recorded weekly, 3 weeks later, calcium metabolism test was performed for 3 days, food intake was recorded, 72h feces were collected and calcium content in the feces was measured. The rat feces are dried in an oven at 75 ℃, cooled and ground to be fine (the whole process is carried out in a sterile room).
And (3) determination of bone calcium content: the measurement is carried out by an atomic absorption method.
Apparent absorption rate of calcium:
calcium intake (mg) = feed calcium content (mg/g) × 3 days feed consumption + sample calcium intake content
Fecal calcium (mg) = fecal calcium content (mg/g) × 3 days fecal excretion
Apparent absorption rate of calcium (%) = (calcium intake-fecal calcium)/calcium intake × 100%
The results are shown in Table 6:
TABLE 6
From the data in table 6, it can be seen that: compared with the low-calcium control group, the weight and the body length of the weaning rats in each group have no obvious difference, but the apparent absorption rate of calcium in the intervention group is obviously higher than that of the low-calcium control group, namely, the function of promoting the calcium absorption of the body can be achieved by using the probiotics under the condition of not influencing the basic physiology.
Example 8
This example explores the effect of lactobacillus casei LC16 on mouse osteoblast proliferation by the following steps:
(1) firstly, the prepared bacterial suspensions or compound bacterial suspensions (LC 16+ LC86 (1: 1), LC16+ LR08 (2: 1), LC86+ LR08 (2: 1), LC16+ LC86+ LR08 (2: 2: 1)) and the commercial bacteria (Lactobacillus paracasei LP-33, purchased from Jingyue biotechnology limited) (the total bacterial concentration of each group is 10) 9 CFU/mL) and osteoblast (mouse osteoblast precursor cell MC3T3-E1, obtained from research center for laboratory animals in Shanghai) induction culture medium at a volume ratio of 1:50 or 1:100, respectively, to obtain culture solution with bacteria: taking out the cryopreservation tube filled with the mouse osteoblast, quickly thawing at 37 ℃ under constant temperature and aseptic conditions (the step is completed in an aseptic chamber), sucking the cell suspension into a high-temperature sterilized centrifuge tube by using a pipettor, and slowly dropwise adding the cell suspension into the centrifuge tube
MEM complete medium (purchased from Wuhan Pronosus Life technologies, Ltd.), mixing, centrifuging at 5000g for 5min, removing supernatant, resuspending the precipitated fraction with probiotic medium of different concentrations, mixing, transferring to 100mm cell culture dish with pipette, and placing in CO 2 Constant temperature incubator 37 deg.C, 5% CO 2 Culturing under the condition, and periodically replacing the bacterial supernatant culture solution (about 48 h/time).
(2) The method of using MTT (also called MTT tetrazolium salt colorimetry) to accomplish the dry prognosis is a method for detecting the survival and growth of cellsSuccinate dehydrogenase in the mitochondria of living cells can be reduced to purplish crystal) to determine the growth rate of osteoblasts: extracting the 3 rd generation osteoblast in logarithmic growth phase in a sterile operating platform to
-MEM complete medium, diluted to 2.5X 10 4 Osteoblasts were seeded in a 96-well plate at a concentration of/mL, and at the time of measurement, 20. mu.L of a 5 mg/mL MTT solution was added to each well and placed in CO 2 Constant temperature incubator 37 deg.C, 5% CO 2 Incubate for 4h under conditions. Sucking out the culture solution, adding dimethyl sulfoxide (DMSO) 100 μ L, mixing for 10min, dissolving the crystal completely, and measuring absorbance (OD) 490nm ). In addition, sterilized sodium butyrate (purchased from Merck Sigma-Aldrich brand) was added to the induction medium in a sterile operating room at a concentration of 0.5mmol/L (butyric acid as a short chain fatty acid has been shown to produce cell morphology and cell differentiation inducing effects in different cell cultures) and was performed as described above.
The results are shown in Table 7:
TABLE 7
From the data in table 7, it can be seen that: each bacterial suspension group can stimulate the proliferation, differentiation and maturation of osteoblasts very significantly compared to the control group. At the same time, the results of 0.5mmol/L butyric acid group are combined, so that the SCFA which can directly act on osteoblasts exists in metabolites of LC16, LC86 and LR08, and the action effect is highlighted along with the increase of concentration and the prolongation of action time (from 24h to 48 h). No obvious effect was found in the medium with commercial bacterial intervention, indicating that there were no SCFAs among the metabolites that could produce direct effects on osteoblasts.
Example 9
This example explores the effect of lactobacillus casei LC16 on osteoclast inhibition in mice, by the following steps:
(1) firstly, the prepared bacterial suspensions or compound bacterial suspensions (LC 16+ LC86 (1: 1), LC16+ LR08 (2: 1), LC86+ LR08 (2: 1), LC16+ LC86+ LR08 (2: 2: 1)) and the commercial bacteria (Lactobacillus paracasei LP-33, purchased from Jingyue biotechnology limited) (the total bacterial concentration of each group is 10) 9 CFU/mL) and osteoclast (mouse mononuclear macrophage RAW 264.7, obtained from research center of laboratory animals in Shanghai) induction culture medium at a volume ratio of 1:50 or 1:100, respectively, to obtain culture solution with bacteria: taking out the cryopreservation tube filled with RAW 264.7 cells, rapidly thawing at 37 deg.C under constant temperature and aseptic condition (the step is completed in aseptic chamber), sucking cell suspension into high temperature sterilized centrifuge tube by using pipettor, and slowly dripping into centrifuge tube
MEM complete medium (purchased from Wuhan Pronosus Life technologies, Ltd.), mixing, centrifuging at 5000g for 5min, removing supernatant, resuspending the precipitated fraction with probiotic medium of different concentrations, mixing, transferring to 100mm cell culture dish with pipette, and placing in CO 2 Constant temperature incubator 37 deg.C, 5% CO 2 Culturing under the condition, and periodically replacing the bacterial supernatant culture solution (about 48 h/time).
(2) The dry prognosis was performed by measuring the osteoclast growth inhibition using MTT, as in example 7, and the results are shown in Table 8:
TABLE 8
From the data in table 8: with the time, the proliferation and differentiation of osteoclast in the control group are obvious. Compared with a control group, the bacterial suspensions of each group can remarkably inhibit the proliferation, differentiation and maturation of osteoclast cells. There was no difference among the commercial bacterial supernatants in each group, and the results were similar to those of the control group. And the combination of the results of 0.5mmol/L butyric acid group can prove that the butyric acid-containing culture medium can inhibit the proliferation and differentiation of osteoclast to a certain extent. That is, LC16, LC86, LR08 can directly inhibit the proliferation and differentiation of osteoclasts using their metabolites, such as SCFA, butyrate, lactate, etc.
Combining with examples 7 and 8, osteoblasts and osteoclasts are jointly involved in bone metabolism to complete the replacement of bones, and lactobacillus casei can directly act on the two cells through substances such as exercise fatty acid and the like generated by the lactobacillus casei to regulate the balance of the two cells so as to improve bone quality.
The applicant states that the invention is described by the above examples to describe a bone health promoting lactobacillus casei of the invention and the culturing method and application thereof, but the invention is not limited to the above examples, i.e. it does not mean that the invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Sequence listing
<110> Mikang Probiotics (Suzhou) GmbH
<120> Lactobacillus casei LC16 for promoting bone health, and culture method and application thereof
<130> 2022
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1480
<212> DNA
<213> Lactobacillus casei LC16 Strain
<400> 1
gagcatcgtt cgtcacctta gacggctcgc tccctaaaag ggttacgcca ccggcttcgg 60
gtgttacaaa ctctcatggt gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc 120
gcggcgtgct gatccgcgat tactagcgat tccgacttcg tgtaggcgag ttgcagccta 180
cagtccgaac tgagaatggc tttaagagat tagcttgacc tcgcggtctc gcaactcgtt 240
gtaccatcca ttgtagcacg tgtgtagccc aggtcataag gggcatgatg atttgacgtc 300
atccccacct tcctccggtt tgtcaccggc agtcttacta gagtgcccaa ctcaatgctg 360
gcaactagtc ataagggttg cgctcgttgc gggacttaac ccaacatctc acgacacgag 420
ctgacgacaa ccatgcacca cctgtcattt tgcccccgaa ggggaaacct gatctctcag 480
gtgatcaaaa gatgtcaaga cctggtaagg ttcttcgcgt tgcttcgaat taaaccacat 540
gctccaccgc ttgtgcgggc ccccgtcaat tcctttgagt ttcaaccttg cggtcgtact 600
ccccaggcgg aatgcttaat gcgttagctg cggcactgaa gggcggaaac cctccaacac 660
ctagcattca tcgtttacgg catggactac cagggtatct aatcctgttc gctacccatg 720
ctttcgagcc tcagcgtcag ttacagacca gacagccgcc ttcgccactg gtgttcttcc 780
atatatctac gcatttcacc gctacacatg gagttccact gtcctcttct gcactcaagt 840
ttcccagttt ccgatgcgct tcctcggtta agccgagggc tttcacatca gacttaaaaa 900
accgcctgcg ctcgctttac gcccaataaa tccggataac gcttgccacc tacgtattac 960
cgcggctgct ggcacgtagt tagccgtggc tttctggttg gataccgtca cgccgacaac 1020
agttactctg ccgaccattc ttctccaaca acagagtttt acgacccgaa agccttcttc 1080
actcacgcgg cgttgctcca tcagacttgc gtccattgtg gaagattccc tactgctgcc 1140
tcccgtagga gtttgggccg tgtctcagtc ccaatgtggc cgatcaacct ctcagttcgg 1200
ctacgtatca tcgccttggt gagccgttac ctcaccaact agctaatacg ccgcgggtcc 1260
atccaaaagc gatagcttgc gccatctttc agccaagaac catgcggttc ttggatttat 1320
gcggtattag catctgtttc caaatgttat cccccactta agggcaggtt acccacgtgt 1380
tactcacccg tccgccactc gttttaagtc gaatctcagt gcaagcaccg ttcatcgacc 1440
aaaactcgtt cgactgcatg tataggcacc ccgccccccg 1480
Claims (2)
1. The composite probiotic preparation with the effect of improving or treating osteoporosis is characterized in that strains in the composite probiotic preparation with the effect of improving or treating osteoporosis consist of lactobacillus casei with the mass ratio of (1-4) to (1-4)Lactobacillus caseiLC16 strain and Lactobacillus paracaseiLactobacillus paracaseiLC86 strain composition; the lactobacillus paracaseiLactobacillus paracaseiThe preservation number of the LC86 strain is CGMCC number 1.12731, and the preservation date is 2020, 7 months and 20 days; the lactobacillus caseiLactobacillus caseiThe preservation number of the LC16 strain is CGMCC No.24110, and the preservation date is 12 months and 15 days in 2021.
2. The composite probiotic preparation with the effect of improving or treating osteoporosis is characterized in that strains in the composite probiotic preparation with the effect of improving or treating osteoporosis are lactobacillus casei with the mass ratio of (1-4): 1-4Lactobacillus caseiLC16 strain and lactobacillus paracaseiLactobacillus paracaseiLC86 strain and Lactobacillus reuteriLactobacillus reuteriLR08 strain composition; the lactobacillus paracaseiLactobacillus paracaseiThe preservation number of the LC86 strain is CGMCC number 1.12731, and the preservation date is 2020, 7 months and 20 days; the lactobacillus caseiLactobacillus caseiThe preservation number of the LC16 strain is CGMCC No.24110, and the preservation date is 12 months and 15 days in 2021; the Lactobacillus reuteri strainLactobacillus reuteriThe accession number of the LR08 strain is CGMCC number 1.12733, and the preservation date is 2020, 7 and 20 days.
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