CN114917281A - Morinda officinalis fermented product and application thereof - Google Patents
Morinda officinalis fermented product and application thereof Download PDFInfo
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- CN114917281A CN114917281A CN202210679913.5A CN202210679913A CN114917281A CN 114917281 A CN114917281 A CN 114917281A CN 202210679913 A CN202210679913 A CN 202210679913A CN 114917281 A CN114917281 A CN 114917281A
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- fermentation
- lactobacillus rhamnosus
- fermentation broth
- morinda officinalis
- morinda
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/74—Rubiaceae (Madder family)
- A61K36/746—Morinda
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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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
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Diabetes (AREA)
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Abstract
The invention belongs to the technical field of biological fermentation, and provides lactobacillus rhamnosus (Lactobacillus rhamnosus)Lactobacillus rhamnosus) Y5 with preservation number of CGMCC No. 24886. The lactobacillus rhamnosus Y5 can be inoculated into fermentation culture medium containing radix Morindae officinalis, and fermented to obtain fermentation liquid and water extract of the fermentation liquid. The fermentation liquid or water extract can be used for preparing medicine or health food for promoting nerve injury regeneration and improving diabetic erectile dysfunction. The morinda officinalis fermented product is a biological fermented product obtained by a modern fermentation process, the production condition is controllable, and the product stability is good.
Description
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to microbial fermentation of morinda officinalis and application thereof.
Background
Morinda officinalis is derived from Morinda officinalis (Morinda officinalis L.) of Morinda of RubiaceaeMorinda officinalisHow) is mainly distributed in tropical and subtropical areas of China, such as Guangxi, Guangdong, Fujian, Hainan and the like, has sweet and pungent properties and tastes, is slightly warm and nontoxic, and has the effects of tonifying kidney yang and strengthening kidney yangThe efficacies of muscles and bones and dispelling wind-damp are one of the four famous south Chinese herbs. The processing of radix Morindae officinalis is carried out before the radix Morindae officinalis is used as medicine, and mainly comprises salt processing of radix Morindae officinalis, steaming of radix Morindae officinalis, water processing of radix Glycyrrhizae to radix Morindae officinalis (processed radix Morindae officinalis), and boiling of radix Morindae officinalis. Research shows that morinda officinalis contains various chemical components, and part of active components have the effects of resisting depression, dementia, aging and inflammation, promoting angiogenesis, resisting osteoporosis and the like.
The fermentation method for processing the Chinese herbal medicine is one of the traditional Chinese medicine processing methods, and by means of the action of enzyme and microorganism, the biological performance of the Chinese herbal medicine is converted by the microorganism fermentation under the conditions of proper temperature, proper humidity, ventilation, proper moisture and the like, so that the original property and the efficacy of the Chinese herbal medicine are changed, the original efficacy is enhanced or a new efficacy is generated, the toxicity is reduced, and the original medicine using variety of the Chinese herbal medicine is expanded. The fermentation processing of the morinda officinalis is significant for improving the drug effect, reducing the toxic and side effects and expanding the application range of the morinda officinalis.
Schwann cells are glial cells of the peripheral nervous system, participate in the formation of myelin sheaths of the peripheral nervous system, and provide essential nutritional support to the encapsulated neurons. As a skeleton cell of the nerve, Schwann cells play a vital role in nerve injury regeneration, and can secrete various neurotrophic factors to play a key role in the induction and regeneration of the damaged nerve; erectile Dysfunction (ED) is a common disease, the cavernous smooth muscle cells (CCSMC) of the penis are at the core of normal erection, and reduction of apoptosis of CCSMC after cavernous injury is especially important for restoration of erectile function.
According to the literature report, after the morinda officinalis is fermented and processed, the effective ingredients are improved to a certain extent, but the research on the effects of the morinda officinalis fermented extract on nerve injury regeneration, reduction of oxidative stress of corpus cavernosum penis, increase of the level of cGMP of corpus cavernosum penis, improvement of the smooth muscle content and angiogenesis of corpus cavernosum penis, improvement of the erectile function of a mouse and the like is less.
Disclosure of Invention
Aiming at the fermentation processing technology and the application of the morinda officinalis in the prior art, the invention provides a fermentation processing method of morinda officinalis and the application of the fermentation processing method of morinda officinalis in promoting nerve injury regeneration, reducing oxidative stress of corpus cavernosum penis, increasing cGMP level of corpus cavernosum penis, improving smooth muscle content and angiogenesis of corpus cavernosum penis and improving erectile dysfunction caused by diabetes.
In order to achieve the purpose, the invention adopts the following technical scheme.
Lactobacillus rhamnosus (A) and (B)Lactobacillus rhamnosus) Y5 with preservation number of CGMCC number 24886.
A method for fermenting Morinda citrifolia comprising the steps of: inoculating the lactobacillus rhamnosus Y5 seed liquid into a fermentation culture medium containing morinda officinalis, and fermenting to obtain a fermentation liquid.
Preferably, the bacterium content of the lactobacillus rhamnosus Y5 seed liquid is 10 8 -10 9 cfu/mL; the inoculation amount is 1-5% (volume ratio).
Preferably, the fermentation conditions are at a temperature of 35 ℃.
Preferably, the fermentation medium containing morinda citrifolia comprises per liter: 10-20g of glucose and 80-150g of morinda officinalis.
A fermentation broth and an aqueous extract of the fermentation broth obtained by the above method.
The water extract of the fermentation liquor is liquid obtained by filtering the fermentation liquor through a 20-40 mesh filter screen or centrifuging at 1000-2000rpm after heating and refluxing, or further concentrated or dried.
An application of the above fermentation liquid or water extract in preparing medicine or health food for promoting nerve injury regeneration and improving erectile dysfunction due to diabetes is provided.
A health food or medicine containing the above fermentation liquid or water extract of the fermentation liquid is provided.
Preferably, the health food or the medicine also comprises auxiliary materials or other effective components which are acceptable in the food or the medicine.
The invention has the following advantages:
the morinda officinalis fermented product is a biological fermented product obtained by a modern fermentation process, the production condition is controllable, the product stability is good, and the morinda officinalis fermented product is protectedH 2 O 2 The induced CCSMC and RSC-96 cell apoptosis is superior to the unfermented morinda officinalis extract in the aspects of reducing the oxidative stress of the corpus cavernosum, increasing the cGMP level of the corpus cavernosum, improving the smooth muscle content and angiogenesis of the corpus cavernosum, and improving the erectile function of a diabetic erectile dysfunction mouse.
Biological preservation information
Lactobacillus rhamnosus (A), (B)Lactobacillus rhamnosus) Y5, deposited in China general microbiological culture Collection center (CGMCC) at 12.05.2022 with the preservation number of CGMCC 24886, with the preservation address of China Beijing, the institute of microbiology, China academy of sciences, No. 3, West Lu 1, Beijing, Chaoyang.
Drawings
FIG. 1 shows Morinda citrifolia extract (MO) and Morinda citrifolia fermented product (Y5 MO) vs. H 2 O 2 The protective effect of damaging CCSMC and Schwann cell apoptosis;
FIG. 2 is a graph showing the effect of MO and Y5MO on ICP (intracavernosal pressure) in diabetic rats;
FIG. 3 is a graph of the effect of MO and Y5MO on MDA, SOD and cGMP levels in diabetic rats;
FIG. 4 is a graph showing the effect of Y5MO on apoptotic PI3K/Akt/eNOS pathway-related protein expression;
FIG. 5 is a graph of the effect of MO and Y5MO on the smooth muscle content and angiogenesis of the corpus cavernosum of diabetic rats.
Detailed Description
The present invention will be further described with reference to the following examples and the accompanying drawings, but the present invention is not limited by the following examples.
EXAMPLE 1 isolation and identification of fermentation Strain
Dissolving 1g of experimental material (probiotic powder) in 9 mL of sterile distilled water, placing in a constant temperature oscillator at 120 rpm for 30 min, standing after the operation is finished, and performing gradient dilution on a sample solution; respectively take 10 -5 -10 -7 200. mu.L of the diluted solution was coated on a coating film containing 5% CaCO 3 The MRS solid culture medium is placed in a constant temperature incubator at 37 ℃ and cultured for 24-48 h; selecting the appearance according to the size of the calcium dissolving ring after the single fungus grows out of the solid culture mediumAnd colonies with different morphologies are purified for 3-4 times on a new MRS solid culture medium. All the obtained strains are used for fermenting morinda officinalis (fermentation culture medium contains 10 g/L glucose and 100 g/L morinda officinalis), preferably the strains capable of improving the content of active ingredients in fermentation extract, so that strain Y5 with good effect is obtained, and the strain is stored in glycerol at-80 ℃ for later use.
Through DNA extraction, sequencing and comparison, Y5 belongs to Lactobacillus rhamnosus (L.) (Lactobacillus rhamnosus). Is preserved in China general microbiological culture Collection center (CGMCC) at 2022, 05 and 12 months, with the preservation number of CGMCC number 24886.
Example 2 preparation of Morinda citrifolia extract
1. Fermented extract of Morinda officinalis
(1) Cleaning dried Morinda officinalis with clear water, removing soil, drying and pulverizing into 60 mesh powder;
weighing 10 g of glucose and 100 g of morinda officinalis, adding 1000mL of distilled water, subpackaging, and sterilizing at 121 ℃ for 20 min to obtain a fermentation culture medium;
(2) activating strain Y5, inoculating to seed culture medium (containing yeast extract 5 g/L, tryptone 10 g/L, and sodium chloride 10 g/L) respectively, culturing at 35 deg.C and 160 rpm under shaking for 48 hr, adjusting concentration to 10 8 Obtaining a seed solution by cfu/mL;
inoculating 30 mL of seed liquid into each liter of fermentation medium, and performing oscillation fermentation at 35 ℃ and 160 rpm for 6 days to obtain fermentation liquor;
(3) heating and refluxing the fermentation liquor obtained in the step (2) at 100 ℃ for 1h, and filtering by a 40-mesh filter screen to obtain a morinda officinalis fermentation extract YBMO.
2. Morinda officinalis extract
The content of morinda officinalis crude drug in each liter of fermentation liquor is 100 g according to the conversion of morinda officinalis fermentation extract. According to the content, 20g of morinda officinalis powder is weighed and added into 200 mL of distilled water, heated and refluxed for 1h at 100 ℃, filtered and centrifuged to obtain morinda officinalis extract, which is named as MO.
Example 3 Morinda citrifolia extract pairs H 2 O 2 Protective effects of induced CCSMC and RSC-96 cell apoptosis
The penis seaCotton Smooth Muscle Cells (CCSMC) or Schwann cells (RSC-96) at 3X 10 5 one/mL (100. mu.L/well) density seeded in 96-well plates, 37C 5% CO 2 The cells were cultured in the incubator of (1) for 24 hours. Setting blank control group, control group and experimental group, pretreating cells with extract for 6 hr in experimental group, adding 50 μ M H in control group and experimental group 2 O 2 Cells were stimulated for 12 hours. Subsequently, 10. mu.L of MTT solution was added to each well, incubated at 37 ℃ for 4 hours, the medium was removed, 150. mu.L of DMSO was added to dissolve formaldehyde crystals, and the Optical Density (OD) was measured at 490 nm.
The results are shown in FIG. 1: y5MO and MO treatment, then for H 2 O 2 The induced CCSMCs and Schwann cells have certain protection effect, and the protection effect of Y5MO is superior to that of MO.
Example 4 Effect of Morinda citrifolia fermentates on erectile function in diabetic rats
Establishment of diabetic ED rat model: 48 male Sprague-Dawley rats, 8 weeks old, were selected. All experimental procedures were approved by the university of Shandong committee for animal protection and use. Animals were kept in a Specific Pathogen Free (SPF) environment at 22-24 ℃, with a 12 hour light/12 hour dark cycle weekly, with regular dosing of water and food. To establish a diabetic rat model, 40 rats were intraperitoneally injected with streptozotocin after fasting, and the remaining 8 rats were given an equal volume of 0.1 mol/L citrate phosphate buffer (pH = 4.2) subcutaneously. Rats whose blood glucose levels continued to exceed 16.7 mM after 72h of monitoring were identified as diabetic. After 12 weeks, the presence or absence of ED was identified by observing penile congestion, length and circumference changes using apomorphine test, and rats without changes were considered to be DMED rats. 40 DMED rats were divided into 5 groups by APO assay: DMED group, n = 8; DMED + 100 mg/kg MO, n = 8; DMED + 300 mg/kg MO, n = 8; DMED + 100 mg/kg Y5MO, n = 8; DMED + 300 mg/kg Y5MO, n = 8.
Evaluation of erectile function: after 8 weeks of treatment, intracavernosal pressure (ICP) and mean systemic arterial pressure (MAP) were measured in different groups of male rats. After anesthesia with 2.5% sodium pentobarbital (intraperitoneal, 70 mg/kg), the male rats were placed face up. The left carotid artery was cannulated with PE-50 tubing and systemic arterial blood pressure was recorded with a pressure transducer. A needle (23 gauge) filled with heparin solution (250U/mL) was attached to the sensor and inserted into the corpus cavernosum to record ICP. The cavernous nerve was isolated and stimulated with a hook-shaped bipolar electrode at a voltage of 5V, frequency of 25 Hz, duration of 60 s, pulse width of 5 ms. Changes in ICP and MAP were recorded by the BL-420V pressure sensor system and the data visualized using software.
The results are shown in fig. 2, the treatment with morinda citrifolia fermented product can improve ICP level in diabetic rats, and the improvement effect of morinda citrifolia fermented product is significantly better than that of unfermented morinda citrifolia extract.
Measurement of the level of oxidative stress of the cavernous body: the level of oxidative stress of the sponge cells was assessed by measuring Malondialdehyde (MDA) and superoxide dismutase (SOD) levels. The content of MDA in the sponge is determined according to the technical manual of a detection kit (Nanjing Biochemical Co., Ltd., Nanjing, China). Total SOD activity was detected using a spectrophotometric kit with the maximum absorbance at 525 nm and the results expressed as U/mg protein. The level of cGMP in the sea corpus was determined using a commercial cGMP enzyme linked immunoassay kit (R & D Systems, inc., Minneapolis, MN, USA).
The results are shown in fig. 3, and the Morinda citrifolia ferment (Y5 MO) treatment reduced the level of oxidative stress in the penis of DMED rats and increased the level of sea body cGMP. Compared with normal rats, the DM rats have obviously raised MDA level and obviously lowered SOD level. The non-fermented MO group had a certain reduction in MDA content (P < 0.05) compared to DMED, while the morinda officinalis fermented group had a significantly reduced MDA content (P < 0.05) compared to the morinda officinalis extract (MO) treated group. Compared with diabetic rats, the SOD level was partially restored by MO treatment (P < 0.05), while the SOD level was significantly higher in the Y5MO treated group than in the MO treated group (P < 0.01).
PI3K/Akt/eNOS pathway-related protein expression: the expression condition of the apoptosis PI3K/Akt/eNOS pathway related protein is detected by a protein immunoblotting technology.
The results are shown in FIG. 4, Y5MO reduced apoptosis by up-regulating PI3K/Akt/eNOS pathway expression.
And (3) immunofluorescence staining: penile tissue was fixed by soaking in 4% paraformaldehyde and then dehydrated overnight. The samples were embedded and cut into 5 μm sections. After 1h of blocking, the cells were incubated with anti-alpha-smooth muscle actin (alpha-SMA, 1:1000; Abcam), endothelial nitric oxide synthase (eNOS; 1:50; Abcam). Slides were washed and incubated with Alexa fluor-594 conjugated secondary antibody (Invitrogen, Carlsbad, Calif., USA). Nuclei were stained with DAPI reagent (Thermo Fisher, MA, USA) for 5 min. Finally, the slides were examined under a fluorescence microscope (Leica, Heidelberg, Germany).
Masson staining: expression of smooth muscle cells and collagen fibers in the sponge was determined using the Masson trichrome staining kit (Dako Sciences, Glostrup, Denmark). Collagen fibers were dyed blue, elastic fibers were dyed brown, muscle fibers were dyed red, and nuclei were counterdyed dark blue. Smooth muscle to collagen ratios were analyzed using Image-Pro Plus 5.0 software (Media Cybernetics, inc., Bethesda, MD, USA).
The results are shown in fig. 5, and the morinda citrifolia fermented product treatment can increase cavernous smooth muscle content and angiogenesis, and the effect is better than that of the morinda citrifolia extract.
EXAMPLE 5 preparation of tablets containing Morinda citrifolia ferment
The strain Y5 was inoculated into a seed medium (containing 5 g/L yeast extract, 10 g/L tryptone, 10 g/L sodium chloride) and cultured with shaking at 35 ℃ and 160 rpm for 48 hours, the concentration was adjusted to 10 8 cfu/mL to obtain a seed solution;
inoculating the seed liquid into a fermentation medium (containing 15 g/L glucose and 150 g/L radix Morindae officinalis) according to 5% of the volume of the fermentation medium, and performing shaking fermentation at 35 deg.C and 200 rpm for 7 days to obtain a fermentation liquid;
heating and refluxing the fermentation liquid at 100 ℃ for 1h, filtering by a 40-mesh filter screen, concentrating and drying the filtrate to obtain a solid morinda officinalis fermented product;
mixing the morinda officinalis fermented product with auxiliary materials such as microcrystalline cellulose, starch, dextrin and the like according to the addition amount of 1% (w/w), and tabletting, wherein each tablet is 1g, so as to obtain the morinda officinalis fermented product-containing tablet.
Example 6 preparation of an oral liquid containing fermented Morinda officinalis
The strain Y5 was inoculated into a seed medium (containing 5 g/L yeast extract, 10 g/L tryptone, 10 g/L sodium chloride) and cultured with shaking at 35 ℃ and 160 rpm for 48 hours, the concentration was adjusted to 10 9 cfu/mL to obtain a seed solution;
inoculating the seed solution into a fermentation medium (containing 20 g/L glucose and 80 g/L radix Morindae officinalis) according to 1% of the volume of the fermentation medium, and performing shaking fermentation at 160 rpm at 35 deg.C for 5 days to obtain a fermentation solution;
heating and refluxing the fermentation liquid at 100 ℃ for 1h, centrifuging at 1500rpm, and concentrating the supernatant by 5 times to obtain a radix Morindae officinalis fermentation concentrated solution;
mixing the radix Morindae officinalis fermented concentrated solution with adjuvants such as Mel, antiseptic, Tween-80, purified water, etc. according to the addition amount of 5% (w/v), homogenizing, filling 10 mL/brown penicillin bottles, and pasteurizing to obtain oral liquid containing radix Morindae officinalis fermented product.
Claims (10)
1. Lactobacillus rhamnosus (A) strainLactobacillus rhamnosus) Y5, wherein the accession number is CGMCC number 24886.
2. A method for fermenting Morinda citrifolia comprising the steps of: lactobacillus rhamnosus (A), (B), (C) and (C)Lactobacillus rhamnosus) Inoculating the Y5 seed solution into a fermentation culture medium containing morinda officinalis, and fermenting to obtain fermentation broth of fermented morinda officinalis;
the Lactobacillus rhamnosus (A), (B), (C) and (C)Lactobacillus rhamnosus) The preservation number of Y5 is CGMCC number 24886.
3. The method of claim 2, wherein the lactobacillus rhamnosus Y5 seed liquid has a bacteria content of 10 8 -10 9 cfu/mL; the inoculation amount is 1-5%.
4. The method of claim 2, wherein the fermentation conditions are at a temperature of 35 ℃.
5. The method of claim 2, wherein the Morinda citrifolia-containing fermentation medium comprises, per liter: 10-20g of glucose and 80-150g of morinda officinalis.
6. A fermentation broth and an aqueous extract of the fermentation broth obtained by the method of any one of claims 2 to 5.
7. The aqueous extract of fermentation broth as claimed in claim 6, wherein the liquid obtained by centrifugation at 1000-2000rpm or the further concentrated or dried product after heating the fermentation broth for reflux is obtained by filtration through a 20-40 mesh screen.
8. Use of the fermentation broth or aqueous extract of claim 6 for the preparation of a medicament or health food for promoting nerve injury regeneration and improving erectile dysfunction in diabetes.
9. A health food or pharmaceutical comprising the fermentation broth or aqueous extract of fermentation broth of claim 6.
10. The health food or medicine of claim 9, further comprising an adjuvant or other effective ingredient acceptable in food or medicine.
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