Application of culture of lactobacillus plantarum PL-02 in improving calcium absorption

Abstract

The invention relates to an application of a culture of lactobacillus plantarum PL-02 in improving calcium absorption. In particular, the invention relates to an application of a culture of lactobacillus plantarum PL-02 (CGMCC (China General Microbiological Culture Collection Center) NO.20485) in the improvement of calcium absorption.

Description

Technical Field

[0001] The present invention relates to using a culture of Lactobacillus plantarum PL-02 (CGMCC NO. 20485) to enhance calcium absorption in an individual. Background Art

[0002] Calcium is an essential element that the human body needs in large quantities. It plays an important role in blood coagulation, muscle contraction, bone construction, and the maintenance of normal heart rhythm and nerve function. When the human body lacks calcium, it will lead to various calcium deficiency-related conditions.

[0003] Common methods for delaying or improving calcium deficiency-related conditions include increasing exercise or providing calcium supplements (such as calcium carbonate, calcium oxide, calcium phosphate, and calcium lactate gluconate) to increase calcium intake. However, the calcium in calcium supplements may not be effectively absorbed by the human body. On the other hand, excessive calcium supplements may increase urinary calcium levels and lead to the formation of calcium-containing kidney stones. Therefore, researchers in this field are committed to developing methods that can effectively increase calcium absorption without producing undesirable side effects.

[0004] In Huang WC et al., (2019), Nutrients, 19; 11(11): 2836, doi: 10.3390 / nu11112836., Huang WC et al. administered Lactobacillus plantarum TWK10 to healthy subjects and found that Lactobacillus plantarum TWK10 can effectively improve the subjects' athletic performance. However, in Hsu HY et al., (2022), Microorganisms, 7; 10(4): 784, doi: 10.3390 / microorganisms10040784., Hsu HY et al. administered Lactobacillus plantarum TWK10 to rats to evaluate the safety of Lactobacillus plantarum TWK10 and analyzed various biochemical parameters in serum. The results showed that Lactobacillus plantarum TWK10 had no effect on the calcium content in serum. Summary of the invention

[0005] In the present invention, the applicant unexpectedly discovered that the culture of Lactobacillus plantarum PL-02 (CGMCC NO.20485) is significantly better than other Lactobacillus plantarum strains in enhancing the gene expression of calcium channel protein TRPV6 in intestinal cells and calcium ion transport, and is therefore expected to be used to enhance calcium absorption in an individual.

[0006] Thus, in a first aspect, the present invention provides a use of a culture supply of Lactobacillus plantarum PL-02 for the preparation of a composition for enhancing calcium absorption in an individual.

[0007] Preferably, the composition further comprises cultures of Lactococcus lactis LY-66 (CGMCC NO. 21838) and Bifidobacterium longum subsp. longum OLP-01 (CGMCC NO. 17345).

[0008] Preferably, the culture is a liquid culture.

[0009] More preferably, the liquid culture contains substantially no bacterial cells.

[0010] Preferably, the culture contains only bacterial cells.

[0011] More preferably, the subject is a subject with a condition associated with calcium deficiency.

[0012] Preferably, the composition further comprises a calcium supplement.

[0013] Preferably, the composition is a food composition.

[0014] Preferably, the composition is a pharmaceutical composition.

[0015] More preferably, the pharmaceutical composition is in a dosage form for oral administration, topical administration or parenteral administration.

[0016] In a second aspect, the present invention provides a method for increasing calcium absorption in a subject, comprising administering a culture of Lactobacillus plantarum PL-02 to a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above and other objects, features and advantages of the present invention will become apparent after referring to the following detailed description and preferred embodiments and accompanying drawings, in which:

[0018] Figure 1 The relative multiples of calcium ion transport measured in each group after treatment with the culture supernatant of lactic acid bacteria in Example 1 are shown, wherein "*" and "**" respectively indicate p<0.05 and p<0.01 when compared with the Ca control group;

[0019] Figure 2 The relative multiples of calcium ion transport measured in each group in Example 1 after being treated with culture supernatants containing different volume percentages of lactic acid bacteria are shown, wherein "**" indicates that p < 0.01 when compared with the Ca control group;

[0020] Figure 3 The relative multiples of calcium ion transport measured in each group after the treatment with the lactic acid bacteria suspension in Example 1 are shown, wherein "*" and "***" respectively indicate p<0.05 and p<0.001 when compared with the Ca control group; and

[0021] Figure 4 The relative expression multiples of the TRPV6 gene measured in each group in Example 2 are shown. DETAILED DESCRIPTION

[0022] For the purposes of this specification, it will be expressly understood that the word "comprising" means "including, but not limited to," and that the word "comprises" has a corresponding meaning.

[0023] Unless otherwise defined, all technical and scientific terms used in this article have the meaning commonly understood by those skilled in the art. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used to implement the present invention. Of course, the present invention is in no way limited to the methods and materials described.

[0024] The present invention provides a use of a culture of Lactobacillus plantarum PL-02 (CGMCCNO.20485) for preparing a composition for improving calcium absorption in an individual.

[0025] According to the present invention, the culture can be obtained by culturing Lactobacillus plantarum PL-02 in a medium suitable for growth.

[0026] The culture medium suitable for the present invention can be prepared by those skilled in the art, or can be a commercially available product, including, but not limited to: MRS broth, MRS broth supplemented with cysteine, mineral medium supplemented with glucose and soy peptone.

[0027] As used herein, the terms "culturing," "fermentation," and "cultivation" are used interchangeably.

[0028] The operation process and parameter conditions of the cultivation fall within the professional accomplishment and routine technical scope of those skilled in the art. In this regard, reference may be made to, for example, Hsieh P Set al. (2013), New Microbiol., 36: 167-179.

[0029] According to the present invention, the culturing may be performed at a temperature ranging from 25° C. to 40° C. for 20 to 40 hours. In a preferred embodiment of the present invention, the culturing is performed at 37° C. for 24 hours.

[0030] According to the present invention, the culture may have a range of 10 6 Up to 10 12 The bacterial concentration in CFU / mL is preferably 10 6 Up to 10 10 In a preferred embodiment of the present invention, the bacterial concentration of the culture is 9×10 6 In another preferred embodiment of the present invention, the bacterial concentration of the culture is 9×107 CFU / mL.

[0031] According to the present invention, the culture may be a liquid culture.

[0032] According to the present invention, the liquid culture can be substantially free of cells by solid-liquid separation.

[0033] According to the present invention, the solid-liquid separation treatment can be selected from the group consisting of: centrifugation treatment, filtration treatment, concentration treatment, and combinations thereof. In a preferred embodiment of the present invention, the solid-liquid separation treatment is centrifugation treatment.

[0034] According to the present invention, the liquid culture may be sterilized to be substantially free of viable cells.

[0035] As used herein, the term "substantially free of" means that the specified component lacks a meaningful amount. Preferably, the amount of the component has no measurable effect on the properties of the liquid culture. More preferably, the liquid culture is completely free of the component.

[0036] According to the present invention, the culture may contain only bacterial cells.

[0037] According to the present invention, the culture may be concentrated or non-concentrated, liquid, paste, semi-solid, or solid [e.g., pellets, granules, or powder], and may be heat-inactivated, frozen, dried, or freeze-dried [e.g., in freeze-dried form or spray / fluid bed dried form].

[0038] According to the present invention, the composition may further contain a culture of a lactic acid bacteria strain selected from the group consisting of: Lactococcus lactis LY-66 (CGMCC NO.21838), Bifidobacterium longum subsp. longum OLP-01 (CGMCC NO.17345), and combinations thereof.

[0039] According to the present invention, based on the total amount of the composition, the composition may include: 10%-90% of Lactobacillus plantarum PL-02, 5%-45% of Lactococcus lactis LY-66, and 5%-45% of Bifidobacterium longum bifidum OLP-01. Preferably, based on the total amount of the composition, the composition may include: 50%-90% of Lactobacillus plantarum PL-02, 5%-25% of Lactococcus lactis LY-66, and 5%-25% of Bifidobacterium longum bifidum OLP-01.

[0040] According to the present invention, the composition contains Lactobacillus plantarum PL-02, Lactococcus lactis LY-66 and Bifidobacterium longum bifidum OLP-01 in a relative ratio (e.g., bacterial number ratio or volume ratio) of 1:0.2:0.2 to 1:5:5. Preferably, the composition contains a liquid culture of Lactobacillus plantarum PL-02, Lactococcus lactis LY-66 and Bifidobacterium longum bifidum OLP-01 in a volume ratio of 1:0.5:0.5, which is substantially free of bacterial cells.

[0041] As used herein, the term "subject" means any mammal of interest, such as humans, monkeys, cows, sheep, horses, pigs, goats, dogs, cats, mice, and rats.

[0042] According to the present invention, the individual may be an individual with a calcium deficiency-related condition. Preferably, the calcium deficiency-related condition may be selected from the group consisting of osteopenia, rickets, joint pain, muscle cramps, hypertension, palpitations, anxiety, obesity, skin inflammation, and hypocalcemia.

[0043] Alternatively, the subject may also be a subject with dysmenorrhea, or a subject that is perimenopausal, menopausal, or postmenopausal.

[0044] In some embodiments, the subject is a subject without osteoporosis.

[0045] According to the present invention, the composition may further comprise calcium supplements.

[0046] According to the present invention, the calcium supplement includes, but is not limited to: calcium chloride, calcium carbonate, calcium oxide, calcium dihydrogenphosphate, dicalcium phosphate, tricalcium bis(phosphate), calcium lactate gluconate, calcium L-threonate, calcium citrate, calcium lactate, and combinations thereof. In a preferred embodiment of the present invention, the calcium supplement may be calcium chloride.

[0047] According to the present invention, the composition can be a food composition, for example, in the form of a food additive, which can be added to an edible material to prepare a food product for human or animal consumption. According to the present invention, the types of the food product can include, but are not limited to: milk powder, fermented milk, yogurt, butter, beverages (e.g., tea, coffee), functional beverages, flour products, baked foods, confectionery, candies, fermented foods, animal feeds, health foods, infant food, and dietary supplements.

[0048] According to the present invention, the composition may be a pharmaceutical composition.

[0049] According to the present invention, the pharmaceutical composition is in a dosage form for oral administration, topical administration or parenteral administration.

[0050] According to the present invention, the pharmaceutical composition may further include a pharmaceutically acceptable carrier widely used in drug manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more agents selected from the following: solvent, buffer, emulsifier, suspending agent, decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizer, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome and the like. The selection and amount of these agents fall within the professional qualities and routine technical scope of those skilled in the art.

[0051] According to the present invention, the pharmaceutical composition can be manufactured into a dosage form suitable for oral administration using techniques well known to those skilled in the art, including, but not limited to: sterile powder, tablet, troche, lozenge, pellet, capsule, dispersible powder or granule, solution, suspension, emulsion, syrup, elixir, slurry and the like.

[0052] According to the present invention, the pharmaceutical composition can also be manufactured into an external preparation suitable for topical application to the skin using techniques well known to those skilled in the art, including, but not limited to: emulsion, gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion, serum, paste, foam, drop, suspension, salve and bandage.

[0053] According to the present invention, the pharmaceutical composition can be manufactured into a dosage form suitable for parenteral administration [including injections, for example, sterile aqueous solutions or dispersions] using techniques well known to those skilled in the art, and administered by a route selected from the group consisting of: intraperitoneal injection, intrapleural injection, intramuscular injection, intravenous injection, intraarterial injection, intraarticular injection, intrasynovial injection, intraepidermal injection, subcutaneous injection, intradermal injection, intralesional injection, and sublingual administration.

[0054] The present invention also provides a method for increasing calcium absorption in a subject, comprising administering the culture described above to a subject in need thereof.

[0055] As used herein, the terms "administering" and "administration" are used interchangeably and mean introducing, providing or delivering a predetermined active ingredient to a subject by any appropriate route to perform its intended effect.

[0056] According to the present invention, the dosage and frequency of administration of the culture will vary depending on the severity of the disease to be improved, the route of administration, and the weight, age, physical condition and response of the individual to be improved. Generally speaking, the culture can be administered orally, topically or parenterally in a single dose or divided into multiple doses.

[0057] Detailed description of preferred embodiments

[0058] The present invention will be further described with reference to the following examples, but it should be understood that these examples are only for illustration and should not be construed as limitations on the implementation of the present invention.

[0059] <Example>

[0060] General experimental materials:

[0061] 1. Source and culture of human colon adenocarcinoma cell line Caco-2:

[0062] The human colon adenocarcinoma cell line Caco-2 ( HTB-37 TM ) was purchased from the American Type Culture Collection (ATCC).

[0063] Caco-2 cells were cultured in a 10 cm petri dish containing Dulbecco's Modified Eagle's Medium (DMEM) (Cytiva) [supplemented with 10% fetal bovine serum (FBS) (Gibco, Cat. no. 26140079) and 1% penicillin-streptomycin (Cytiva)], and cultured in an incubator set at 37°C and 5% CO2. Thereafter, fresh medium was replaced approximately every 2-3 days. Subculture was performed when the cell density reached about 80-90% confluence.

[0064] 2. Lactic acid bacteria strains:

[0065] The lactobacillus strains used for efficacy evaluation in the following examples have been deposited in the Bioresource Collection and Research Center (BCRC) (300 No. 331, Food Road, Hsinchu City, Taiwan) of the Food Industry Research and Development Institute (FIRDI), China Taiwan, and are publicly available. In addition, these lactobacillus strains also have been deposited in the China General Microbiological Culture Collection Center (CGMCC) of the China Microbiological Culture Collection Committee according to the provisions of the Budapest Treaty (the Budapest Treaty), and are publicly available. For clarity, the relevant information of these lactobacillus strains (including: relevant source, deposit number and preservation date, etc.) has been integrated in Table 1 below.

[0066] Table 1. Information about each lactic acid bacteria strain

[0067]

[0068] For comparison, the following Lactobacillus plantarum strains were also used: 299v strain purchased from JAPAN ACE INTERNATIONALMERCHANDISE CO., LTD., TWK10 strain purchased from GenMont Biotech Inc., GMNL-662 strain purchased from SYNBIO TECH Inc., and gL-299 strain isolated by the applicant from commercially available sauerkraut.

[0069] 3. Preparation of lactic acid bacteria suspension and culture supernatant containing 5 mM calcium chloride:

[0070] The lactic acid bacteria strains described in item 2 above were inoculated into MRS broth (Difco, Cat. no. 288130) and cultured in an incubator (37°C, 5% CO2) under facultative anaerobic conditions for 24 hours to activate the strains. Next, the activated strains were inoculated into MRS broth at an inoculum volume of 2% (v / v) and cultured in an incubator (37°C, 5% CO2) under facultative anaerobic conditions for 24 hours. Thereafter, the resulting cultures were incubated at 4°C at 3,000 °C.

[0071] The cells were centrifuged at 400 rpm for 10 minutes to precipitate the bacteria, and the supernatant was collected. An appropriate amount of calcium chloride (Sigma-Aldrich, Cat. no. V900269) was then added to obtain culture supernatants of lactic acid bacteria strains containing 5 mM calcium chloride. As for the pellets, an appropriate amount of DMEM medium supplemented with 5 mM calcium chloride was used to suspend the bacteria and adjust the bacterial concentration to a desired level (the bacterial count was performed using a plate counting medium), thereby obtaining suspensions of lactic acid bacteria strains containing 5 mM calcium chloride.

[0072] General experimental methods:

[0073] 1. Statistical analysis:

[0074] In the following examples, the experimental data obtained are expressed as "mean ±

[0075] The difference between the experimental data of each group was evaluated by Student's t-test. If the analysis result was p < 0.05, it was statistically significant.

[0076] Example 1. Evaluation of the efficacy of Lactobacillus plantarum PL-02 in improving intestinal calcium ion transport

[0077] First, the Caco-2 cells that were subcultured according to item 1 of “General Experimental Materials” were divided into 13 groups, including 1 blank control group, 1 Ca control group, 1 single-bacteria experimental group, 4 single-bacteria comparison groups (i.e., single-bacteria comparison groups 1 to 4), 1 compound bacteria experimental group, and 5 compound bacteria comparison groups (i.e., compound bacteria comparison groups 1 to 5). 5 The number of cells / well was cultured in 2.5 mL of DMEM medium. Embedded dish ( The Caco-2 cells were attached to the bottom of the inserts to form a Caco-2 cell monolayer, and the culture medium was replaced every 3 days. Next, the liquid in each embedded dish was removed and washed twice with an appropriate amount of phosphate buffered saline (PBS), and then the culture supernatant of lactic acid bacteria containing 5mM calcium chloride obtained in accordance with item 3 of "General Experimental Materials" was added to the corresponding single bacteria group according to Table 2 below, and mixed with each other according to the volume percentage shown in Table 3 below and added to the corresponding composite bacteria group. In addition, DMEM medium supplemented with 5mM calcium chloride was added to the Ca control group, and DMEM medium was added to the blank control group, and then cultured in an incubator (37°C, 5% CO2) for 24 hours. At 30 minutes and 24 hours after the treatment with the culture supernatant of lactic acid bacteria, the liquid in each well was collected and the calcium ion concentration was measured using a calcium colorimetric assay kit (Calcium Colorimetric Assay Kit) (BioVision, K380-250).

[0078] Table 2. Lactic acid bacteria strains treated in each single bacteria group

[0079] Group Lactobacillus plantarum strains Single bacteria experimental group PL-02 Single bacteria comparison group 1 GMNL-662 Single bacteria comparison group 2 299v Single bacteria comparison group 3 TWK10 Single bacteria comparison group 4 gL-299

[0080] Table 3. Lactic acid bacteria strains and their culture contents treated by each composite bacteria group

[0081]

[0082] The calcium ion concentration increase rate was calculated by substituting the calcium ion concentration measured after 30 minutes and 24 hours of treatment into the following formula (1):

[0083] Formula (1): A = B / C

[0084] Where: A = calcium ion concentration increase rate

[0085] B = Calcium ion concentration measured in each group after 24 hours of treatment

[0086] C = Calcium ion concentration of each group measured after 30 minutes of treatment

[0087] The relative multiple of calcium ion transport is calculated by substituting the obtained calcium ion concentration increase rate into the following formula (2):

[0088] Formula (2): D=(EF) / (GF)

[0089] Where: D = relative multiple of calcium ion transport

[0090] E = calcium ion concentration increase rate of each group

[0091] F = calcium ion concentration increase rate of the blank control group

[0092] G=Calcium ion concentration increase rate of the Ca control group

[0093] Afterwards, the obtained experimental data were analyzed according to the method described in item 1 of the above “General Experimental Methods”.

[0094] In addition, in order to further evaluate the effect of changes in the culture content of the three lactic acid bacteria strains contained in the above-mentioned composite bacteria experimental group on the efficacy, the experiment was generally carried out with reference to the above-mentioned operating steps, except that: the Caco-2 cells were divided into 8 groups, including 1 blank control group, 1 Ca control group, 1 composite bacteria comparison group and 5 composite bacteria experimental groups (that is, composite bacteria experimental groups 1 to 5), and the culture supernatant of lactic acid bacteria containing 5mM calcium chloride obtained in accordance with item 3 of the "General Experimental Materials" was mixed with each other and added to the corresponding groups according to the volume percentage shown in Table 4 below.

[0095] Table 4. Lactic acid bacteria strains and their culture contents treated by each composite bacteria group

[0096]

[0097] In addition, in order to evaluate the effectiveness of the suspension of lactic acid bacteria strains in improving intestinal calcium ion transport, the experiment was carried out in accordance with the above-mentioned operating procedures, except that the Caco-2 cells were divided into 7 groups, including 1 blank control group, 1 Ca control group, 1 single-bacteria experimental group, 4 single-bacteria comparison groups (i.e., single-bacteria comparison groups 1 to 4), and the suspension of lactic acid bacteria containing 5 mM calcium chloride obtained in accordance with item 3 of "General Experimental Materials" (bacterial concentration of 9×10 6 CFU / mL) were added to the corresponding single bacteria groups according to Table 2 above.

[0098] result:

[0099] Figure 1 The relative multiples of calcium ion transport measured by each group of Caco-2 cell monolayers after treatment with culture supernatants of different lactic acid bacteria strains are shown. Figure 1 It can be seen that compared with the Ca control group, the relative multiples of calcium ion transport in the single-bacteria experimental group and single-bacteria comparison groups 1, 2 and 4 all showed a significant increase, among which the relative multiples of calcium ion transport in the single-bacteria experimental group were significantly higher than those in single-bacteria comparison groups 1, 2 and 4, while the single-bacteria comparison group 3 showed a slight decrease, which means that the culture supernatant of Lactobacillus plantarum PL-02 can effectively enhance intestinal calcium ion transport, and this effect is significantly better than other Lactobacillus plantarum strains.

[0100] Furthermore, compared with the single bacteria experimental group and the composite bacteria comparison group 1, the relative multiple of calcium ion transport in the composite bacteria experimental group showed a significant increase, while the relative multiples of calcium ion transport in the composite bacteria comparison groups 2 to 4 were between those in the corresponding single bacteria comparison group and the composite bacteria comparison group 1, respectively. As for the relative multiple of calcium ion transport in the composite bacteria comparison group 5, there was no significant difference between the corresponding single bacteria comparison groups, which indicates that the further combination of Lactobacillus plantarum PL-02 with Bifidobacterium longum subsp. longum OLP-01 and Lactococcus lactis LY-66 can unexpectedly show a synergistic effect.

[0101] Figure 2 The relative multiples of calcium ion transport measured by each group of Caco-2 cell monolayers after being treated with culture supernatants containing different volume percentages of lactic acid bacteria strains are shown. Figure 2It can be seen that compared with the composite bacteria comparison group, the relative multiples of calcium ion transport in each composite bacteria experimental group showed a significant increase, which means that: under the condition of equal amounts of culture supernatants of Lactobacillus longum Bifidobacterium longum subsp. longum OLP-01 and Lactococcus lactis LY-66, replacing part of the culture supernatant (from 10 to 90 vol%) with the culture supernatant of Lactobacillus plantarum PL-02 can show a better effect in improving intestinal calcium ion transport.

[0102] Figure 3 The relative multiples of calcium ion transport measured by each group of Caco-2 cell monolayers after treatment with suspensions of different lactic acid bacteria strains are shown. Figure 3 It can be seen that compared with the Ca control group, the relative multiples of calcium ion transport in single bacteria comparison groups 2 to 4 only showed a slight increase, while the single bacteria experimental group and single bacteria comparison group 1 both showed a significant increase, among which the increase in the relative multiples of calcium ion transport in the single bacteria experimental group was the most significant and higher than the sum of the single bacteria comparison groups 1 to 4. This indicates that the suspension of Lactobacillus plantarum PL-02 also has a similar excellent effect in improving intestinal calcium ion transport.

[0103] Example 2. Evaluation of the efficacy of Lactobacillus plantarum PL-02 in increasing the expression of calcium channel protein in intestinal cells

[0104] Experimental methods:

[0105] First, the Caco-2 cells that were subcultured according to item 1 of “General Experimental Materials” were divided into 7 groups, including 1 blank control group, 1 Ca control group, 1 experimental group, and 4 comparison groups (i.e., comparison groups 1 to 4). 5 The number of cells / well was cultured in each well of a 6-well culture plate containing 3 mL of DMEM medium (supplemented with 10% FBS and 1% penicillin-streptomycin) and cultured in an incubator (37°C, 5% CO2) for 11 days, during which fresh medium was replaced every 3 days. After that, the liquid in each well was removed and the suspension of lactic acid bacteria strains containing 5 mM calcium chloride obtained in accordance with item 3 of "General Experimental Materials" (bacterial concentration of 9×10 7 CFU / mL) were added to the experimental group and each comparison group according to the following Table 5, DMEM medium supplemented with 5 mM calcium chloride was added to the Ca control group, and DMEM medium was added to the blank control group, followed by culture in an incubator (37°C, 5% CO2) for 6 hours.

[0106] Table 5. Lactic acid bacteria strains treated in each group

[0107] Group Lactobacillus plantarum strains Experimental Group PL-02 Comparison Group 1 GMNL-662 Comparison Group 2 299v Comparison Group 3 TWK10 Comparison Group 4 gL-299

[0108] Afterwards, the liquid in each well was removed and the total RNAs (total RNAs) of each group of cell culture were extracted using the Total RNA Extraction Miniprep System (Viogene, Cat. no. GR1001) according to the manufacturer's instructions. The total RNAs of each group were respectively transcribed with cDNA reverse transcription reagent (GoScript TM Reverse Transcriptase (Promega, Cat. no. A5003) was used to perform reverse transcription reaction according to the manufacturer's instructions to synthesize first-strand cDNA.

[0109] Next, the obtained first-strand cDNA was used as a template and a specific primer pair designed for the TRPV6 gene was used to perform quantitative real-time polymerase chain reaction (hereinafter referred to as quantitative real-time PCR). In addition, the gene expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The relevant information about these primer pairs (including: target gene, nucleotide sequence of each primer and the size of the amplified PCR product) has been integrated in the following Table 6.

[0110] Table 6. Primers used for quantitative real-time PCR

[0111]

[0112] Quantitative real-time PCR was performed using StepOnePlus TM Real-time PCR system (StepOnePlus TM Real-Time PCRSystem) (Applied Biosystems TM ) and were performed according to the manufacturer's operating instructions, and the operating conditions and reaction conditions of quantitative real-time PCR are shown in Table 7 below.

[0113] Table 7. Reaction conditions for quantitative real-time PCR

[0114]

[0115] Each PCR product thus obtained was detected by fluorescence of SYBR Green (double-stranded DNA binding dye), and the cycle threshold (C t The expression level of TRPV6 gene in each group was compared using the comparative C t Method (comparative C t The TRPV6 gene cycle threshold values ​​obtained were normalized with the GAPDH gene cycle threshold values ​​obtained using the same method.

[0116] The relative expression fold of the TRPV6 gene was calculated by substituting the obtained TRPV6 gene expression level into the following formula (3):

[0117] Formula (3): H = (IJ) / (KJ)

[0118] Where: H = relative expression fold of TRPV6 gene

[0119] I = TRPV6 gene expression level in each group

[0120] J = TRPV6 gene expression level in the blank control group

[0121] TRPV6 gene expression level in K=Ca control group

[0122] Afterwards, the obtained experimental data were analyzed according to the method described in item 1 of the above “General Experimental Methods”.

[0123] result:

[0124] Figure 4 The relative expression fold of TRPV6 gene in each group of Caco-2 cells after being treated with different strains of Lactobacillus plantarum is shown. Figure 4 It can be seen that compared with the Ca control group, the relative expression multiples of TRPV6 gene in comparison groups 1, 2 and 4 only showed a slight increase, while comparison group 3 showed a slight decrease, and the experimental group showed a significant increase, which was more than three times that of comparison group 1. This shows that Lactobacillus plantarum PL-02 can effectively increase the expression of TRPV6 in intestinal cells, and this effect is significantly better than other Lactobacillus plantarum strains.

[0125] Based on the above experimental results, it can be seen that compared with other strains of Lactobacillus plantarum, the culture of Lactobacillus plantarum PL-02 (including the bacterial part and the fluid part without bacterial part) has significantly excellent effects in enhancing TRPV6 expression and calcium ion transport in intestinal cells, and is therefore expected to be used to promote calcium absorption.

[0126] All patents and literature cited in this specification are incorporated herein by reference in their entirety. In the event of a conflict, the detailed description of the present application (including limitations) will prevail.

[0127] Although the present invention has been described with reference to the specific embodiments above, it is apparent that many modifications and variations can be made without departing from the scope and spirit of the invention. It is therefore intended that the present invention be limited only as indicated by the appended claims.

[0128] Information on the deposit of biological materials

[0129] Deposit number: CGMCC No.20485

[0130] Classification: Lactobacillus plantarum

[0131] Date of deposit: August 6, 2020

[0132] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0133] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China

[0134] Deposit number: CGMCC No.21838

[0135] Classification and nomenclature: Lactococcus lactis

[0136] Date of deposit: February 26, 2021

[0137] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0138] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China

[0139] Deposit number: CGMCC No.17345

[0140] Classification and nomenclature: Bifidobacterium longum subsp. longum

[0141] Date of deposit: March 18, 2019

[0142] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0143] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

Claims

1. A culture of Lactobacillus plantarum PL-02 (CGMCC NO. 20485) for use in preparing a composition for improving calcium absorption in an individual.

2. The use according to claim 1, characterized in that: The composition further comprises cultures of Lactococcus lactis LY-66 (CGMCC No. 21838) and Bifidobacterium longum subsp. longum OLP-01 (CGMCC No. 17345).

3. The use according to claim 1 or 2, characterized in that: The culture is a liquid culture.

4. The use according to claim 3, characterized in that: The liquid culture contains substantially no bacterial cells.

5. The use according to claim 1 or 2, characterized in that: The culture contained only bacterial cells.

6. The use according to claim 1, characterized in that: The subject is a subject with a calcium deficiency-related condition.

7. The use according to claim 1, characterized in that: The composition further comprises a calcium supplement.

8. The use according to claim 1, characterized in that: The composition is a food composition.

9. The use according to claim 1, characterized in that: The composition is a pharmaceutical composition.

10. The use according to claim 9, characterized in that: The pharmaceutical composition is in a dosage form for oral administration, topical administration or parenteral administration.

Images