CN112553115A - Application of lactobacillus plantarum ZDY2013 in preparation of products for relieving kidney injury - Google Patents
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Images
Classifications
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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
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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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
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/38—Other non-alcoholic beverages
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- A—HUMAN NECESSITIES
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- 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
- A23L21/00—Marmalades, jams, jellies or the like; Products from apiculture; Preparation or treatment thereof
- A23L21/10—Marmalades; Jams; Jellies; Other similar fruit or vegetable compositions; Simulated fruit products
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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
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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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
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Abstract
The invention discloses application of lactobacillus plantarum ZDY2013 in preparation of a product for relieving kidney injury, and provides application in preparation of a medicine or a health-care product or food for relieving kidney injury, a health-care product or food for relieving high-salt diet-induced kidney injury, a probiotic microbial inoculum for relieving high-salt induced kidney injury and a preparation method of the probiotic microbial inoculum. The lactobacillus plantarum ZDY2013 is extremely acid-resistant and bile salt-resistant, can keep higher survival rate in gastrointestinal fluids, and can inhibit the growth of food-borne pathogenic bacteria. In addition, the intestinal flora diversity can be adjusted, so that the gastrointestinal health of a host can be improved. Has the effects of relieving renal fibrosis, improving inflammation appearance symptoms, regulating oxidative stress, regulating and controlling the expression of inflammatory factors and achieving the effect of effectively preventing and treating high-salt diet-induced renal injury.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a novel application of lactobacillus plantarum ZDY 2013.
Background
High-salt diet (HSD) causes various diseases to gain more and more attention, however, the high-salt diet is related to diseases such as hypertension, kidney tissue injury, autoimmune diseases, diabetes, osteoporosis and the like. Studies have shown that the feces of mice in the high-salt diet group can cause intestinal leakage and kidney damage in recipient mice after transplantation, and in addition, there is a strong correlation between high-salt intake and oxidative stress, which can lead to tubular dopamine oxidation, and further lysosomal and mitochondrial dysfunction and inflammatory renal reactions. At present, the medicines for treating kidney injury mainly comprise heparin, codina, rituximab, adefovir, tenofovir and the like, and have high medicine price and uncontrollable side effect.
The lactobacillus plantarum is used as intestinal gram-positive probiotics, can adjust the balance of intestinal flora and inhibit the growth of harmful bacteria, and plays an important role in intestinal immunity.
The lactobacillus plantarum ZDY2013 is preserved in China Center for Type Culture Collection (China Center for Type Culture Collection, CCTCC M for short) in 2015 28 days in Wuhan City, the address of eight-path Wuhan university school in Wuchang area in Wuhan city, Hubei, postfix: 430072) and the preservation number is CCTCC M2014170. The strain is disclosed in Chinese patent 'high acid-resistant lactobacillus plantarum ZDY 2013' (patent number ZL 201510552202.1), and any individual can be obtained from China center for type culture collection as long as the individual is not in commercial use.
Disclosure of Invention
The invention aims to provide application of lactobacillus plantarum ZDY2013 in preparation of a medicine or a health-care product or food for relieving kidney injury. The preservation number of the lactobacillus plantarum ZDY2013 is CCTCC M2014170.
Preferably, the kidney injury is caused by a high salt diet.
Preferably, the kidney injury is inflammation and/or fibrosis of kidney tissue.
The second purpose of the invention is to provide a health care product or food for relieving high-salt diet-induced kidney injury. The active ingredients of the lactobacillus plantarum strain comprise lactobacillus plantarum ZDY2013, and the preservation number of the lactobacillus plantarum ZDY2013 is CCTCC M2014170.
Preferably, the food is a food which can be survived by a plurality of lactobacillus, such as beverage, yoghourt, jelly and the like, and can be survived by lactobacillus plantarum, particularly according to actual needs.
Preferably, the dosage of the lactobacillus plantarum bacterial preparation for relieving the kidney injury induced by the high-salt diet used by the invention is 1 × 108cfu/kg, calculated according to specific conditions, that is, each kg of microbial inoculum used by animals and human bodies contains 10 lactobacillus plantarum ZDY20138cfu。
The third purpose of the invention is to provide a probiotic microbial agent for relieving high-salt induced kidney injury, which is prepared from lactobacillus plantarum ZDY2013 and exists in the form of fresh viable bacteria in a stable period.
The preparation method of the lactobacillus plantarum microbial inoculum for relieving kidney injury comprises the following steps.
(1) The lactobacillus plantarum ZDY2013 MRS liquid culture medium is anaerobically cultured for 24 +/-2 h at 37 +/-1 ℃ until the lactobacillus plantarum reaches the stationary phase, wherein the preservation number of the lactobacillus plantarum ZDY2013 is CCTCC M2014170.
(2) The cells obtained in step (1) were collected by centrifugation and washed 2 to 4 times with sterile 1 XPBS.
(3) Adjusting the concentration of the cells obtained in step (2) to 1-2X 10 with sterile 1X PBS8 cfu/mL。
(4) Adding appropriate adjuvant into the obtained thallus to make into medicine, food or health product.
The lactobacillus plantarum ZDY2013 disclosed by the invention is extremely resistant to acid and bile salts, can keep a higher survival rate in gastrointestinal fluids, and can inhibit the growth of food-borne pathogenic bacteria. In addition, the intestinal flora diversity can be adjusted, so that the gastrointestinal health of a host can be improved. Has the effects of relieving renal fibrosis, improving inflammation appearance symptoms, regulating oxidative stress, regulating and controlling the expression of inflammatory factors and achieving the effect of effectively preventing and treating high-salt diet-induced renal injury.
Drawings
FIG. 1 shows the body weight change of mice in different groups.
FIG. 2 is a HE stained section of kidney tissue from different groups of mice. Compared with the NSD blank group, the HSD group has fuzzy submucosa edges at the position indicated by the short arrow, obvious inflammatory cell infiltration at the position indicated by the long arrow, and the HHL, HHZ and HLZ groups are all relieved after the probiotics are relieved, wherein the colonic muscle layer is smooth, and the inflammation phenomenon is obviously reduced.
FIG. 3 shows Masson stained sections of kidney tissues of different groups of mice. And observing the tissue fibrosis degree, wherein compared with the NSD blank group, the kidney tissue fibrosis at the position indicated by the arrow of the HSD group is obvious, and the kidney tissue fibrosis of the HHL, HHZ and HLZ groups is relieved after the probiotics are relieved.
FIG. 4 is a graph of kidney tissue fibrosis quantification in different groups of mice.
FIG. 5 shows the difference of colon tissue inflammatory factor and antioxidant factor expression in different groups of mice.
FIG. 6 shows the difference of the expression of the inflammatory factor and antioxidant factor in the kidney tissue of different groups of mice.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Example 1.
Activation of the cells: uniformly mixing lactobacillus plantarum ZDY2013 preserved in glycerol in a vortex mode, sucking 10 mu L of the mixture, coating the mixture on an MRS solid culture medium, carrying out anaerobic culture at 37 ℃ for 24h, selecting a single colony, inoculating the single colony in 4mL of liquid MRS culture medium, carrying out anaerobic culture at 37 ℃ for 24h, inoculating the single colony in 10mL of MRS liquid culture medium according to the inoculation amount of 1%, and carrying out anaerobic culture at 37 ℃ for 24h to reach a stationary phase.
Preparing an MRS culture medium: 5g/L of peptone, 5g/L of beef extract, 10g/L of tryptone, 5g/L of yeast powder, 20g/L of glucose, 5g/L of anhydrous sodium acetate, 2g/L of diamine hydrogen citrate, 2g/L of dipotassium hydrogen phosphate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate, 801 m L/L of Tween-E, pH6.3 +/-0.1, 121 ℃ and 15 minutes of sterilization.
Example 2.
Preparing a lactobacillus plantarum microbial inoculum: centrifuging activated Lactobacillus plantarum ZDY2013 in stationary phase, collecting fresh thallus, washing with sterile 1 × PBS for 3 times, and adjusting thallus concentration to 10 with sterile 1 × PBS8cfu/mL, preparing the microbial inoculum required by the experiment for later use.
Example 3.
And (3) constructing a high-salt diet mouse model and designing an experiment.
After C57BL/6J mice were acclimated in a sterile mouse room for one week, normal daily ration and normal drinking water were given to NSD group (n = 10) mice, 4% NaCl high-salt daily ration and normal drinking water were given to experimental group mice, the experimental group mice were divided into high-salt group, HSD group (n = 7), namely, gavage 200 μ L1 xPBS per day as a control, HHZ group (n = 8), namely, gavage 200 μ L1 x 10 per day10CFU/mL Lactobacillus plantarum ZDY2013, HLZ group (n = 8), i.e. gavage 200 μ L1 x 10 per day8CFU/mL plant milkBacillus ZDY 2013. HHL group (n = 8), gavage 200 μ L1 x 10 daily10CFU/mL Lactobacillus rhamnosus (GG). (Lactobacillus rhamnosus (GG) is used as a probiotic strain with a completely-developed function and effect which is recognized by International biologies and plays a role of a control with Lactobacillus plantarum ZDY 2013.) in the experimental process, the weight of the mouse is measured once every two days, the weight change of the mouse is observed, the mouse is euthanized by ethyl ether after 2w gastric lavage, and serum, colon and kidney tissues of the mouse are taken for later use in a sterile environment. And taking colon and kidney tissues, quickly washing the colon and the kidney tissues by using normal saline, soaking the sample in 10% formalin solution, and performing HE staining. And (5) experimental results.
(1) The invention obviously improves the weight of the mouse.
After the mice in the treated group are treated by the lactobacillus plantarum ZDY2013 microbial inoculum for 5 days, the weight of the mice in the higher-salt diet group of the mice in the treated group is obviously improved, and as shown in figure 1, after the gastric lavage treatment is finished, the weight of the mice in the probiotic microbial inoculum treated group can be obviously recovered to be close to the weight of a normal diet group. The invention can effectively relieve the weight loss caused by high-salt diet and improve the weight of mice.
(2) The invention obviously improves the pathological condition of the mouse.
The kidney tissues of different groups of mice are prepared into pathological sections, the kidney tissues of the high-salt diet group of mice are compared with a natural control group, and as can be seen from figure 2, the glomeruli of the high-salt diet group have obvious atrophy, fibrosis and renal tubular cavity expansion. Inflammation occurs in the interstitium. The glomerulus part of the lactobacillus plantarum microbial inoculum treatment group is fibrous, has no atrophy phenomenon, and is infiltrated by inflammatory cells. The microbial inoculum can effectively relieve the conditions of kidney tissue inflammation and fibrosis of high-salt diet mice, and has the effect of repairing high-salt induced kidney injury.
(3) The microbial inoculum of the invention can obviously regulate the expression of the colon tissue inflammatory factor induced by high salt.
Extracting RNA from colon tissue, reverse transcription, and detecting inflammatory factor TGF-beta 1，Expression levels of IFN-. gamma.FN-1.
As can be seen from FIG. 3, the expression level of the inflammatory factor of the mice in the Lactobacillus plantarum mice in the higher salt diet group is significantly regulated, the expression level of the proinflammatory factor IFN-gamma is down-regulated, and the expression level of the inflammation-inhibiting factor TGF-beta 1 is up-regulated. The microbial inoculum of the invention can achieve the aim of protecting the organism against inflammation by regulating the expression level of the cell factor.
(4) The microbial inoculum of the invention can obviously regulate the expression of the kidney-related factors induced by high salt.
Extracting kidney tissue with RNA, reverse transcription, and detecting inflammatory factor TGF-beta 1，The expression level of CCL4, and the expression levels of antioxidant factors SOD1 and SOD2 are detected.
As can be seen from FIG. 4, the expression level of the inflammatory factor of the mice in the higher salt diet group of the mice in the invention group (Lactobacillus plantarum group) is remarkably regulated, the anti-inflammatory factor TGF-beta 1 is down-regulated, and the oxidative stress factors SOD1, SOD2 and the chemotactic factor CCL4 are down-regulated. The microbial inoculum can be used for protecting the kidney injury induced by high salt by regulating the expression level of cell factors and oxidative stress.
The above are merely specific embodiments of the present invention, and the scope of the present invention is not limited thereby; any alterations and modifications without departing from the spirit of the invention are within the scope of the invention.
Claims (8)
1. The lactobacillus plantarum ZDY2013 is applied to preparation of medicines, health-care products or foods for relieving kidney injury, and the preservation number of the lactobacillus plantarum ZDY2013 is CCTCC M2014170.
2. The use of Lactobacillus plantarum ZDY2013 in the preparation of a medicament or health care product or food for alleviating kidney injury according to claim 1, characterized in that said kidney injury is caused by a high-salt diet.
3. The use of lactobacillus plantarum ZDY2013 in the preparation of a medicament or health care product or food for alleviating kidney injury according to claim 1, wherein the kidney injury is kidney tissue inflammation and/or fibrosis.
4. A health-care product or food for relieving high-salt diet-induced kidney injury is characterized in that the active ingredients of the health-care product or food comprise lactobacillus plantarum ZDY2013, and the preservation number of the lactobacillus plantarum ZDY2013 is CCTCC M2014170.
5. The health product or food for relieving the high-salt diet-induced kidney injury according to claim 4, wherein the food is a beverage, yogurt or jelly.
6. The health product or food for relieving kidney injury induced by high-salt diet as claimed in claim 4, characterized in that the lactobacillus plantarum bacterial agent dosage is 1 x 108 cfu/kg。
7. A probiotic microbial agent for relieving high-salt induced kidney injury is characterized by being prepared from lactobacillus plantarum ZDY2013 and existing in the form of fresh live bacteria.
8. The method for preparing a probiotic preparation for alleviating kidney injury as claimed in claim 7, wherein the method comprises the following steps:
(1) carrying out anaerobic culture on lactobacillus plantarum ZDY2013 MRS liquid culture medium at 37 +/-1 ℃ for 24 +/-2 h until the lactobacillus plantarum reaches a thallus in a stationary phase;
(2) centrifugally collecting the thalli in the step (1), and washing the thalli for 2-4 times by using sterile 1 multiplied by PBS;
(3) adjusting the concentration of the cells obtained in step (2) to 1-2X 10 with sterile 1X PBS8 cfu/mL；
(4) Adding appropriate adjuvant into the obtained thallus, and making into medicine, food or health product.
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