TWI780620B - Lactobacillus senioris and use for manufacturing compositions for anti-prostate cancer - Google Patents

Abstract

The present invention discloses a Lactobacillus seniorisWu/NPU-1 strain and its use for manufacturing compositions for anti-prostate cancer. A substrate comprising a Caulerpa lentilliferamaterial is fermented by the Lactobacillus seniorisWu/NPU-1 strain in an anaerobic environment for 1-10 days, and the Lactobacillus seniorisWu/NPU-1 strain is removed to obtain a Caulerpa lentilliferaferment. The Caulerpa lentilliferaferment inhibits the growth of prostate cancer cells and induces apoptosis of the prostate cancer cells. The Lactobacillus seniorisWu/NPU-1 strain is deposited at Food Industry Research and Development Institute under the accession number BCRC 911030.

Description

Lactobacillus argentica and its use in the preparation of anti-prostate cancer composition

The present invention relates to a Lactobacillus argentosa and its use in preparing an anti-prostate cancer composition. The Lactobacillus argentosa is fermented with sea grapes to obtain an anti-prostate cancer composition.

Sea grape ( Caulerpa lentillifera ) is a plant belonging to the phylum Chlorophyta, Pleurophyta, Pleurophyceae, and Pteridaceae. It mainly grows in tropical and some temperate sea areas; the algae of sea grapes can be divided into erect stems and stolons And rhizomes, the erect stems grow many spherical twigs, which look like bunches of grapes, so they are called sea grapes, and are listed as raw materials for food by the Ministry of Health and Welfare of China. According to previous studies, such as Taiwan Patent No. TW I558408(B), sea grape extract has the effect of suppressing allergies; however, other effects of sea grape have not been extensively studied at present.

Today, in view of the fact that the existing efficacy of sea grapes has not been extensively studied, the inventor is a tireless spirit, and with the help of his rich professional knowledge and many years of practical experience, he improves it and develops it accordingly. this invention.

The present invention discloses a strain of Lactobacillus senioris Wu/NPU-1, which is deposited in the Food Industry Development Research Institute with a registration number of BCRC 911030.

The present invention also discloses a sea grape ferment that has the effect of inhibiting prostate cells, and the use of a sea grape ferment in the preparation of an anti-prostate cancer composition; the preparation method of the sea grape ferment is to use Lactobacillus argentii Wu/ NPU-1 strain, fermented a fermented substrate containing sea grape ( Caulerpa lentillifera ), fermented in an anaerobic environment for 1-10 days, and then removed the thalline of Lactobacillus argentilis, to obtain the inhibitory A ferment for the function of prostate cells.

In one embodiment of the present invention, the fermented base comprising sea grapes further comprises skim milk.

In one embodiment of the present invention, the fermented base containing sea grapes comprises 1-30 wt% sea grapes and 1-30 wt% skimmed milk.

In one embodiment of the present invention, the Lactobacillus algigum is fermented in anaerobic conditions for 2-5 days, and the fermentation temperature is between 25-40°C.

In one embodiment of the present invention, the fermented product having the effect of inhibiting prostate cells further includes at least one of excipients, preservatives, diluents, fillers, absorption enhancers, and sweeteners.

Thus, the Lactobacillus argentitidis Wu/NPU-1 strain of the present invention can ferment the fermentation substrate containing sea grape, and the obtained sea grape ferment can inhibit the growth of prostate cancer cells to achieve the effect of anti-prostate cancer .

In order to enable the technical means of the present invention to achieve a more complete and clear disclosure, the following specific examples are used to describe the scope of practical application of the present invention in detail, but it is not intended to limit the scope of the present invention in any form. For the scope of the invention, please also refer to the disclosed drawings.

1. Strain isolation, purification and preservation

First, isolate lactic acid bacteria from kimchi, sauerkraut, and seaweed drift. The isolation method is briefly described as follows: Take 1 gram of pickle, sauerkraut, or seaweed drift sample, add 4 grams of protein water (peptone water), mix well, and use Obtain a mixed solution; serially dilute the mixed solution 10 times to obtain a 0.1-fold diluted mixed solution, a 0.01-fold diluted mixed solution and a 0.001-fold diluted mixed solution; and take 0.1 mL of the above-mentioned diluted mixed solution and apply it to the 1% calcium carbonate (CaCO ₃ ) MRS agaric gum culture plate, cultured in an anaerobic environment, 30 ^o C environment for 3-5 days; after 5 days of culture, select from the MRS agaric gum culture plate with transparent Then these colonies were separated and cultured by drawing lines, and then cultivated for 3 days under the aforementioned environment; again, the colonies with transparent rings were also picked out to obtain multiple isolated (isolated) colonies; in this test A total of 88 strains with acid-producing properties were isolated, and these strains were further analyzed by Gram staining and catalase reaction tests. The test results showed that these strains were all Gram-positive and catalase-negative. All of them were lactic acid bacteria strains, and these strains were stored in glycerol tubes at -80 ^o C.

2. Screen the strains that can ferment the fermentation substrate containing sea grapes

First, the MRS culture solution containing 10 wt% sea grape powder (hereinafter referred to as Cl-MRS culture solution) was prepared, and then the 88 strains of lactic acid bacteria isolated above were inoculated into the Cl-MRS culture solution, and dried at 30 ^o C. After culturing in an oxygen environment for 2 days, the Cl-MRS culture solution was spread on the MRS agarose gum culture plate to analyze its growth; in addition, the total phenol content in the Cl-MRS culture solution after 2 days of culture was also detected Finally, the potential strains with fermentation substrates containing sea grapes were selected by polyphenol content index (phenolic content index). Among them, the sea grape powder used in this test was obtained by drying the sea grape algae and then pulverizing the algae with a pulverizer.

The calculation formula of polyphenol content index is:

(Total phenol content in Cl-MRS culture fluid/MRS agaric gum culture plate colony number) X 100%

Please refer to the first figure to the fifth figure, which are the analysis results of the polyphenol content index of the 88 strains screened above, according to the third figure (D), the fourth figure (A) and (B), in which No. 057, No. The strains No. 062, No. 063, No. 064 and No. 066 have a higher polyphenol content index, which means that they can ferment sea grapes; and, in the fifth figure (D), the strain marked 12251 is purchased Lactobacillus plantarum ( L. plantarum , number BCRC 10069) from the Biological Resource Conservation and Research Center, and the strain marked 10357 was Lactobacillus plantarum ( L. plantarum , number BCRC 12944) purchased from the Biological Resource Conservation and Research Center. In addition, although No. 062 strain has the highest polyphenol content index, but the number of cultured colonies is low, it is considered that No. 062 strain may have poor growth or survival in the fermentation environment, so subsequent selection also has a high polyphenol content index. The No. 064 strain with phenol content index but better growth or survival was used as the strain for subsequent production of sea grape ferment.

3. Strain identification

The identification of lactic acid bacteria strains was entrusted to the Institute of Food Industry Development for analysis, which used a reference method to sequence the 16S rRNA of the strains to be identified, and compared its sequence with the database; the No. 064 strain selected in this case, After sequencing, its 16S rRNA sequence is 100% similar to the 16S rRNA sequence of Lactobacillus senioris ; in addition, the No. 064 strain in this case is a Gram-positive bacillus without catalase activity , oxidase (Oxidase) and motility, and will not produce endospores, and can grow in both aerobic and anaerobic environments; further, use the API 50 CHL lactic acid bacteria identification kit for physiological and biochemical tests. The test results are shown in Table 1 , where "-" means that the strain cannot use this carbon source to produce acid, and "+" means that the strain can use this carbon source to produce acid; 12364T), can use D-ribose (D-Ribose), D-xylose (D-Xylose), D-glucose (D-Glucose), N-acetylglucosamine (N-Acetylglucosamine) and potassium gluconate (Potassium Gluconate) carbon source for acid production. In addition, among the 49 physiological and biochemical test items, 47 items of the No. 064 strain in this case are the same as the standard strain of Lactobacillus argentii (number YIT 12364T), so the No. 064 strain can be confirmed It is Lactobacillus senioris ( Lactobacillus senioris ), and No. 064 strain is named Wu/NPU-1 strain.

Table I Strain 064 Lactobacillus argentii standard strain Strain 064 Lactobacillus argentii standard strain Glycerol - - Salicin - - Erythritol - - D-Cellobiose - - D-Arabinose - - D-Maltose - - L-Arabinose - + D-Melibiose - - D-Ribose + + D-Saccharose - - D-Xylose + + D-Trehalose - - L-Xylose - - Inulin - - A-Adonitol - - D-Melezitose - - Methyl-D-Xylopyranoside - - D-Raffinose - - D-Galactose + + Amidon - - D-Glucose + + Glycogens - - D-Fructose - - Xylitol - - D-Mannose - - Gentiobiose - - L-Sorbose - - D-Turanose - - L-Rhamnose - - D-Lyxose - - Dulcitol - - D-Tagatose - - Inositol - - D-Fucose - - Methyl-D-mannopyranoside - - L-Fucose - - Methyl-D-glucopyranoside - - D-Arabitol - - N-Acetylglucosamine + W L-Arabitol - - Amygdalin - - Potassium Gluconate + + Arbutin - - Pottassium 2-Ketogluconate - - Esculin - -

4. Preparation of sea grape ferment

The Wu/NPU-1 strain mentioned above was inoculated into 10 mL fermentation medium at a density of 1×10 ⁹ CFU/mL; the fermentation medium contained 10 wt% sea grape powder, 10 wt% skimmed milk , and the remaining percentage of pure water; culture the fermented substrate with Wu/NPU-1 strain at 37°C for 3 days in an anaerobic environment, then centrifuge the fermented substrate to collect the supernatant; then The supernatant is filtered through a 0.22 μm filter membrane to remove bacteria to obtain a filtrate; the filtrate is made into a powder by freeze-drying to obtain the sea grape ferment in this case, and it is stored at -20 ℃ refrigerator for later use.

5. Anti-prostate cancer efficacy test of sea grape ferment

(1) Cell Growth Inhibition Test

In this experiment, the prostate cancer cell lines used included androgen-dependent LNCaP cell lines, and androgen-independent 22Rv1 cell lines and PC-3 cell lines; androgen-dependent cell lines, in the absence of androgen Under stimulation, its growth will be affected; non-androgen-dependent cell lines, because their androgen receptors (Androgen receptor) are mutated (such as 22Rv1 cell lines), or do not have androgen receptors (such as PC-3 cell line), so in the absence of androgen, the cells can still continue to activate and maintain their physiological functions. Among them, PC-3 cell line is considered to have the characteristics of cancer stem cells in recent studies

After the three cell lines used in this case were cultured overnight, 0.005-10 mg/mL sea grape ferment was added to the cell culture medium, and after 2 days of continuous culture, the cell viability was observed by thiazolium blue (MTT) cell survival test method. The growth situation of the three cell lines, the thiazole blue cell survival test method is a living cell staining method, the principle is to use the succinate dehydrogenase (succinate dehydrogenase) in the mitochondria of the cell to convert the tetrazolium ring of thiazole blue Cut off to form formazan, turn the yellow thiazole blue into dark blue, and then use the depth of the generated color to understand the situation of cell growth; the test method steps are briefly described as follows: take sea grape ferment and 1×10 ⁵ cells/mL prostate cancer cells were cultured together in an incubator with 5% CO ₂ at 37 ^o C for 48 hours, and the concentrations of sea grape extract were 0.05, 0.5, 1, 5, 10 mg/mL ; After culturing for 48 hours, add thiazolium blue reagent (1 mg/mL) to the cells at an amount of 100 μL/well, then place the cells at 37 ^o C for 2 hours, remove the supernatant, and add 200 μL / dimethyl sulfide (DMSO) in cell culture wells, shake for 2 minutes, and then detect the absorbance value at a wavelength of 570 nm, as an indicator of cell survival rate, the higher the absorbance value, the better the cell survival of the group good.

Please refer to Figure 6 (A), which is the growth result of the LNCaP cell line. It can be observed that the sea grape ferment can significantly inhibit the growth of the LNCaP cell, and it is dose-dependent (dose dependent), and it is higher than the sea grape ferment. When the concentration is 10 mg/mL, the cell growth inhibition rate will reach 56.5±2.8%.

Please refer to Figure 6 (B) again, it can be observed that the inhibition of sea grape ferment on 22Rv1 cells is weak, and the growth of 22Rv1 cells has no effect on the group added with sea grape ferment at a concentration lower than 5 mg/mL Significantly affected; when the added concentration of sea grape ferment was 10 mg/mL, the growth of 22Rv1 cells would be inhibited, and the cell growth inhibition rate was 30.4±5.0%.

Please refer to Figure 6 (C) again, even if the sea grape ferment was added at a concentration of 10 mg/mL, it still could not effectively inhibit the growth activity of the PC-3 cell line.

Therefore, according to the above results, it is shown that the sea grape ferment prepared in this case has a better growth inhibitory effect on the androgen-dependent LNCaP prostate cancer cell line, but it has a better growth inhibitory effect on the non-androgen-dependent prostate cancer cell line (22Rv1 cell line and PC -3 cell line), the sea grape ferment prepared in this case could not effectively inhibit its growth.

(2) Apoptosis test

In this experiment, the applicant of this case stained the nucleus of the LNCaP cell line with Hoechst 33258 fluorescent dye to observe the apoptosis of the LNCaP cells after administration of sea grape ferment, and calculate the apoptosis rate; please refer to the seventh Figure, in the group treated with sea grape ferment, the nucleus of LNCaP cells will swell, and the generation of apoptotic body can be observed, and the phenomenon of sea grape ferment-induced apoptosis of LNCaP cells has dose Dependence; further collect proteins from LNCaP cells, and analyze the expression of apoptosis-related proteins in cells by Western's blotting, please refer to Figure 8, the expression of Bax protein in cells treated with sea grape ferment There was a significant increase, and the expression of Bcl-x _L and Bcl-2 proteins also decreased, so the mitochondria of the cell released cytochrome C into the cytoplasm; in addition, please refer to Figure 9, sea grape ferment treatment In LNCaP cells, the caspase-3 and caspase-8 proteins will also be activated, which will reduce the expression of proform caspase-3 and caspase-8, and make the cleaved caspase-3 and caspase The expression level of -8 increases, and further hydrolyzes PARP protein and fragments PARP protein, thereby increasing the expression of cleaved PARP protein.

According to the above experimental results, it can be seen that the sea grape ferment obtained by fermenting the fermentation substrate containing sea grape with the Wu/NPU-1 strain in this case can inhibit the growth of prostate cancer cell lines and destroy the function of mitochondria and Activation of the caspase series of enzymes will eventually lead to the apoptosis of prostate cancer cells, thereby achieving the effect of anti-prostate cancer.

To sum up, the Lactobacillus argentii of the present invention and its use in the preparation of anti-prostate cancer composition can indeed achieve the expected use effect through the above disclosed embodiments, and the present invention has not been disclosed before the application. Sincerely, it has fully complied with the provisions and requirements of the Patent Law. ￠It is really convenient to file an application for a patent for invention according to the law, and ask for the review and approval of the patent.

However, the above-mentioned descriptions are only preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention; those who are familiar with the art generally do other things based on the characteristics and scope of the present invention. Any effect change or modification should be regarded as not departing from the design scope of the present invention.

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The first picture: the analysis picture of strains with high polyphenol content index screened in this case (1).

The second picture: the analysis picture of strains with high polyphenol content index screened in this case (2).

Figure 3: The analysis chart of screening strains with high polyphenol content index in this case (3).

Figure 4: Analysis of strains with high polyphenol content index screened in this case (4).

Figure 5: The analysis chart of strains with high polyphenol content index screened in this case (5).

Figure 6: Analysis of inhibition of prostate cancer cell growth by sea grape ferment in this case.

Figure 7: Analysis of apoptosis of prostate cancer cells induced by sea grape ferment in this case.

Figure 8: Analysis of proteins related to cell apoptosis regulated by sea grape ferment (1).

Figure 9: Analysis of proteins related to cell apoptosis regulated by sea grape ferment (2).

Domestic storage information

Food Industry Development Research Institute

December 25, 109

Deposit number: BCRC 911030

Claims (10)

A Lactobacillus senioris ( Lactobacillus senioris ) Wu/NPU-1 strain is deposited in the Food Industry Development Institute of the Foundation, and its deposit number is BCRC 911030. A sea grape ferment that has the effect of inhibiting prostate cancer cells is fermented by using a Lactobacillus senioris ( Lactobacillus senioris ) Wu/NPU-1 strain to ferment a fermentation substrate containing sea grape ( Caulerpa lentillifera ) in an anaerobic environment Ferment for 1-10 days, and then remove the bacteria of the Lactobacillus argentiliae Wu/NPU-1 strain to obtain the fermented product that has the effect of inhibiting prostate cells, wherein the Lactobacillus argentiliae Wu/NPU-1 strain is It is deposited with the Food Industry Development Research Institute of the Foundation, and its deposit number is BCRC 911030. The sea grape ferment having the effect of inhibiting prostate cancer cells according to claim 2, wherein the ferment base containing sea grape further contains skim milk. The sea grape ferment having the effect of inhibiting prostate cancer cells as described in claim 3, wherein the fermentation substrate containing sea grapes contains 1-30wt% sea grapes and 1-30wt% skimmed milk. The sea grape ferment having the effect of inhibiting prostate cancer cells as described in claim 2, wherein the Lactobacillus algigum is fermented in anaerobic conditions for 2-5 days, and the fermenting temperature is between 25-40°C. The sea grape ferment having the effect of inhibiting prostate cancer cells according to claim 2, further comprising at least one of excipients, preservatives, diluents, fillers, absorption promoters, and sweeteners. Use of a sea grape ferment ( Caulerpa lentillifera ) in the preparation of an anti-prostate cancer composition, wherein the sea grape ferment is prepared by adding a Lactobacillus senioris to a ferment substrate containing sea grape The Wu/NPU-1 strain was fermented in an anaerobic environment for 1-10 days, and then the Lactobacillus argentiliae cells were removed to obtain the fermented product that has the function of inhibiting prostate cells, wherein the Lactobacillus argentilis is stored In the Food Industry Development Research Institute of the Foundation, and the registration number is BCRC 911030. The use as described in claim 7, wherein the fermented base containing sea grapes further includes skim milk. The use as described in claim 8, wherein the fermented base containing sea grapes comprises 1-30wt% sea grapes and 1-30wt% skimmed milk. The use as described in claim item 7, wherein the Lactobacillus argentilis is fermented in anaerobic conditions for 2-5 days, and the fermenting temperature is between 25-40°C.

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