CN110771708A - Preparation method and application of poria cocos mycelium lithocarpus litseifolius fermented tea - Google Patents
Preparation method and application of poria cocos mycelium lithocarpus litseifolius fermented tea Download PDFInfo
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Abstract
The invention provides a preparation method of poria cocos mycelium Litsea litsea Litsea: preparing a poria cocos mycelium agent; fermenting Poria cocos mycelium Litsea pungens leaf; and (5) preparing fermented tea. The invention also provides application of the poria cocos mycelium Lithocarpus litseifolius fermented tea extract in alleviating the GDM model of a high-fat-fed C57BL6J mouse. The invention has the following beneficial effects: the soup color is orange red and bright, the aroma is changed from the original slight grass smell to mellow aroma, and the taste is more mellow; the content of essential amino acids required by human body is increased; the tea extract is used for adjuvant treatment of Gestational Diabetes Mellitus (GDM) model mice, the anxiety phenotype of the mice disappears, the glucose tolerance level gradually recovers to normal, and the fetal and placental indexes recover to the normal range.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to a preparation method and application of poria cocos mycelium Litsea litsea Litsea.
[ background of the invention ]
The incidence rate of Gestational Diabetes (GDM) is high, and the disease shows a trend of increasing year by year, and seriously harms the pregnancy period of mothers and children and the postpartum health. The control of GDM is mainly used to prevent and treat diabetes. Although great progress is made in the prevention and treatment of diabetes, a great deal of uncertainty still exists in the medicinal prevention and treatment of GDM, such as administration routes, toxic and side effects of medicaments and the like, so that the development of GDM prevention and treatment preparations from medicinal and edible resources is very necessary. In view of the abundant resources and low utilization rate of Lithocarpus litseifolius leaves, the experiment takes Lithocarpus litseifolius crude and old leaves as raw materials, poria cocos mycelium is inoculated, Lithocarpus litseifolius fermented tea is prepared through fermentation, and then the alleviation effect of the Lithocarpus litseifolius fermented tea on a high-fat-fed C57BL6J mouse GDM model is detected, so that a theoretical and practical basis is provided for effectively and reasonably developing and utilizing the Lithocarpus litseifolius crude and old leaves.
Lithocarpus litseifolius (Lithocarpus litseifolius) also called sweet tea (commonly known as hydrangea glauca and Lithocarpus polystachyus (hook. f.) Rehd), which are plants of Lithocarpus in Fagaceae, are sporadically distributed in the low mountain dense forest in south province of Yangtze river in China in a wild manner, wherein more snow mountain veins are distributed in the middle and west of Hunan province. According to records in the Chinese herbal medicine assembly, the Lithocarpus polystachyus rehd tea can prevent and treat hypertension, damp-heat dysentery, skin itch and other symptoms, and has the effects of nourishing liver and kidney, harmonizing stomach, lowering adverse qi, moistening lung, relieving cough, relieving sleepiness, sobering and the like. The existing scientific research shows that the chemical components of Lithocarpus litseifolius are mainly flavonoid and triterpenoid components, wherein dihydrochalcones are the main sweet components and effective components, and have effects in resisting inflammation, relieving pain, regulating immunity, enhancing anti-stress capability, reducing blood lipid, resisting fatty liver, resisting type II diabetes and diabetic complications, and synergistically enhancing the curative effect of antitumor drugs. Due to the fact that lithocarpus litseifolius has high sweetness, low calorie and good taste, and the lithocarpus litseifolius can be used as a new resource food after being approved and approved by the national health council in 2017 in addition to the medicinal efficacy of the lithocarpus litseifolius.
Poria cocos is a dry sclerotium of a fungus in the family of Polyporaceae, and is first recorded in Shen nong's herbal Jing in China. As a traditional edible and medicinal homologous fungus in China, poria cocos is rich in various active ingredients such as polysaccharide, triterpene, fatty acid, sterol, enzyme and mineral elements, wherein the pachyman has pharmacological activities of resisting tumors, cancers, pathogenic microorganisms, inflammation, oxidation and aging, enhancing immunity, protecting liver, hypnotizing, removing stones, reducing blood sugar and the like. The triterpenes have effects in regulating immunity, resisting tumor, HBV, liver protection, leukemia, aging, inflammation, brain memory improvement, and preventing calculus.
At present, the sources of poria cocos are mostly traditional and mainly artificially cultivated, but the method is easily influenced by external environment and production conditions and has the defects of long production period, high labor demand, unstable product properties and the like. The existing research shows that the content of tuckahoe terpenoid compounds in tuckahoe peel and tuckahoe fermentation mycelium is obviously higher than that of tuckahoe blocks. Therefore, the poria cocos functional components are produced by adopting a liquid fermentation technology, and the advantages of shortening the growth period, being stable in active components, being capable of realizing large-scale automatic production and the like can be achieved. Since the 70 s in the 20 th century, Chinese and foreign scholars have extracted and separated various triterpenoids from poria peel, poria sclerotium or strain culture solution. The Gaozui et al researches the extraction process and the optimal culture conditions of the total triterpene produced by liquid fermentation of the tuckahoe, and the Zhang Xueli et al researches the optimal process of extracting the triterpenic acid compounds in the tuckahoe, and provides theoretical data for extraction research experiments. The accumulation dynamics of 3 main triterpenic acid components in tuckahoe fermentation mycelium such as Zhang Yang, but the chemical composition of tuckahoe triterpenoid is yet to be researched. By regulating the liquid fermentation condition of tuckahoe, pangolin and the like find that the growth of mycelium is closely related to the accumulation of metabolites of the mycelium.
The physiological activity of microbial metabolites is a research hotspot for extracting natural products at home and abroad at present, but related reports on the research on the physiological activity of the metabolites of the poria cocos mycelium are very limited, so that the research on related scientific researches has very good practical significance.
The fermented tea is a product which takes tea leaves as a substrate and leads the tea leaves to generate deep biochemical changes by natural inoculation or artificial inoculation of microorganisms and the growth and metabolism of the microorganisms, and the typical representative is black tea (Pu' er tea and Fuzhuan tea). In the process of pile fermentation of dark tea, complex substance conversion occurs under the action of extracellular enzyme produced by microorganisms and the action of respiration and heat production of the microorganisms, such as carbohydrate is decomposed into micromolecular sugar and soluble sugar, and sensory 'sweet' is shown; the protein contained in the leaves acts on a plurality of amino acids in the protease, so that the tea soup is fresh and fresh; reacting the fragrant substances with saccharides to generate terpene alcohol compounds which show a stale fragrance; tea polyphenol is converted into tea pigment through fermentation, and unique red-brown soup color is given to the tea pigment; meanwhile, the microorganisms generate amino acid, vitamin, mineral substance and other nutrient substances through self metabolism, and the nutrient substances enter the tea soup together to be drunk by people, so that the tea soup is beneficial to the health of human bodies.
The Weiweihui artificially inoculates Poria cocos mycelia with different concentrations into the coarse and old green tea for fermentation, and finds that the method can shorten the flowering period, improve the original coarse and astringent quality of the green tea and form unique color, aroma and taste quality. Zhao Yunlin and the like research that tuckahoe mycelia are inoculated on a green tea sample, and the temperature and the humidity are controlled, so that the fermented tea has the characteristics of the traditional green tea and the Fuzhuan tea, and the internal quality and the additional value of the green tea are improved. In addition, researchers combine the preparation process of the poria cocos mycelium with lotus leaf tea, Pu' er tea, Taiwan tea and fresh summer and autumn tea leaves, so that the quality of the tea leaves is improved, the additional value of the tea leaves is provided, and a new tea leaf processing process is developed. In recent years, fermented tea is widely loved by consumers due to unique quality characteristics and health care effects, wherein the Fuzhuan tea is a unique one of black tea, receives more and more attention of people and arouses research interests of a plurality of scholars, and the unique Poria cocos mycelium in the Fuzhuan tea is a hot spot of attention and research of people. The tuckahoe mycelium serving as a dominant strain beneficial to human health is yet to be further developed and utilized in the aspects of tea product processing and new tea product development.
In common words: the spring tea is fragrant, the summer tea is astringent, and the autumn tea is easy to drink without picking. The spring tea has the advantages of mellow and fresh taste, elegant and lasting fragrance, and suitability for processing famous tea, and the summer and autumn tea has higher content of polyphenols and lower content of amino acid, vitamin, aromatic substances and the like, so that the summer and autumn tea tastes bitter and astringent and lacks fragrance. However, according to the growth rule of Lithocarpus litseifolius leaves and the content change of active ingredients of the Lithocarpus litseifolius leaves in different growth stages, only tender leaves are developed and utilized, and the resource is greatly wasted, so that the full utilization of the crude and old fresh leaves of Lithocarpus litseifolius is an urgent problem to be solved in the Lithocarpus litseifolius production base.
[ summary of the invention ]
The invention aims to research the poria cocos mycelium lithocarpus litseifolius leaf and lithocarpus litseifolius fermented tea by using the crude and old lithocarpus litseifolius leaves, analyze the change of chemical components and quality characteristics in the fermentation process of a JH-1 lithocarpus litseifolius leaf and lithocarpus litseifolius fermented tea product, obtain a feasible fermentation process, and finally obtain the poria cocos mycelium lithocarpus litseifolius leaf and lithocarpus litseifolius fermented tea product. Finally, on the basis of obtaining the pachyma cocos mycelium Lithocarpus litseifolius Lithocarpus Litseikovi C57BL6J mouse GDM model, the research of tea.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a preparation method of poria cocos mycelium Lithocarpus litseifolius nakai fermented tea comprises the following steps:
step one, preparation of a poria cocos mycelium agent;
selecting Poria cocos mycelium for storing strain, inoculating into PDB culture liquid medium, standing and culturing at 28 deg.C for 24 hr, shaking, mixing, diluting at multiple ratio of 10
5-10
8Coating the dilution on a PDA culture solid medium, and performing inverted culture at 28 ℃ for 3d until a typical single colony appears;
selecting typical single colony, inoculating into PDB culture liquid medium, culturing at 28 deg.C for 3d, collecting culture solution, centrifuging at 4 deg.C and 5000rpm for 5min, collecting thallus, washing with sterile physiological saline for 3 times, adding nutrient solution without salt ion, shaking, mixing, freezing at-20 deg.C overnight, freezing at-80 deg.C for 40min, freeze drying at 1 mg/bottle, bottling, and storing in-20 deg.C refrigerator to obtain Poria cocos mycelial preparation;
step two, fermenting poria cocos mycelium Litsea litsea leaves;
accurately weighing a certain amount of de-enzymed Lithocarpus litseifolius leaves and drinking water with a mass volume of 1:1, mixing uniformly, putting into a dry-wet separation fermentation container, sterilizing at 121 ℃ for 30min, taking out, cooling, inoculating Poria cocos mycelium agent according to 1mg/100g Lithocarpus litseifolius leaves, shaking uniformly, and culturing at 28 ℃ for 4 d;
step three, preparing fermented tea;
sterilizing the fully fermented Lithocarpus litseifolius leaves at 121 ℃ for 30min, forming and drying to obtain the Poria cocos mycelium Lithocarpus litseifolius leaf fermented tea.
Preferably, the fermentation container is a wide-mouth fermentation bottle with an interlayer filter screen.
Preferably, in the second step, 500.00g of lithocarpus litseifolius and 500mL of drinking water are accurately weighed, and 5-10 poria cocos mycelium agents are inoculated.
The invention also provides application of the poria cocos mycelium lithocarpus litseifolius fermented tea in relieving the GDM model of a high-fat-fed C57BL6J mouse, which comprises the following steps:
step one, preparing a fermented tea extract and freeze-drying;
weighing 500g of tea leaves, adding 60% ethanol solution with the mass volume fraction of 8 times, leaching for 4h at room temperature, crushing, performing ultrasonic-assisted leaching for 10min, then leaching for 50min at 60 ℃, filtering, repeating the above operation for 2 times, combining filtrates, performing rotary evaporation concentration on the extract to about 100mL, and performing freeze drying to obtain the fermented tea extractant;
II, feeding C57BL6J mouse GDM model with high fat;
grouping experimental animals and establishing a model;
providing 8 w-age C57BL/6J mice, including 40 female mice, 20 male mice and 18-22 g in weight, after all the mice are fed with feed adaptively for 1w, randomly marking two markers for the female mice, dividing the mice into a common feed feeding group and a high-fat feed feeding group according to numbers, continuously feeding all the mice for 1w, measuring fasting glucose tolerance and insulin tolerance every 2d, and eliminating the mice with diabetes symptoms at the early stage;
mouse GDM model mitigation;
after 1w of continuous feeding, closing the male and female mice according to a ratio of 1:2, detecting vaginal embolus, calculating as 1 st day when the vaginal embolus is detected, after the pregnancy is established, determining fasting glucose tolerance and insulin tolerance every 2 days for 1-12 th day of the pregnancy, determining fasting glucose tolerance and insulin tolerance every 1 st day for 12-18 th day of the pregnancy, terminating the experiment after 18 th day of the pregnancy at 4 pm, excluding the mice with diabetes symptoms in the ordinary feed feeding group, and perfusing the stomach of the high-fat feed feeding group with fermented tea extract at the beginning of 9 th day of the pregnancy until the experiment is finished, wherein the optimal dose is 90 mg/kg/d; mice were euthanized and blood, placenta, fetus and internal organs were collected for subsequent experiments, while the placental and fetal indices were determined.
Preferably, the leaching comprises water leaching and organic solvent leaching, and the leaching temperature comprises normal temperature and water bath leaching at 40-80 ℃.
Preferably, the administration mode of the fermented tea extract to the mice is oral administration, including free feeding, drinking and gastric lavage, and the administration dosage is 1-1000 mg/kg/d.
Preferably, the dose administered is 90 mg/kg/d.
The invention has the following beneficial effects:
1. after fermentation, the leaf color of Lithocarpus litseifolius is changed from yellow to reddish brown, obvious Poria cocos mycelium is attached to the surface, the soup color is orange red and bright, the aroma is changed from original slight green grass smell to mellow aroma, and the taste is more mellow;
2. the lithocarpus litseifolius fermented tea has the dry matter content of 96.21%, the water extract content of 49.54%, the crude fiber content of 19.42%, the total flavone content of 85.30mg/100g, the tea polyphenol content of 22.5%, the total free amino acid content of 0.75%, contains various amino acids, and the content of 7 essential amino acids of human bodies such as lysine, phenylalanine, methionine, valine, threonine, leucine and isoleucine is increased in different degrees;
3. from 12d of gestation, the GDM model 5d of the C57BL6J mice fed with high fat is drenched at the dose of 90mg/kg/d, the anxiety phenotype of the mice disappears, and the glucose tolerance level gradually returns to normal.
[ description of the drawings ]
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without inventive efforts, wherein:
FIG. 1 is an amino acid analysis diagram of Lithocarpus litseifolius leaves;
FIG. 2 is an amino acid analysis chart of a poria cocos mycelium Lithocarpus litseifolius leaf fermented tea;
FIG. 3 is a graph showing the cellulose content of Lithocarpus litseifolius leaves after fermentation with Poria cocos mycelia;
FIG. 4 is a graph showing the content of probiotic factors after fermentation with Poria cocos mycelium;
FIG. 5 is a graph of weight gain in pregnant mice after drenching with the lixiviant;
FIG. 6 is a glucose tolerance performance diagram of pregnant mice after drenching with the extractive agent;
FIG. 7 is a graph showing the comparison of the weight of the fetus with that of the model and control group after the pregnant mouse is drenched with the leaching agent;
FIG. 8 is a graph showing the comparison of the weight of placenta after the pregnant mouse is drenched with the leaching agent and the weight of the placenta of a model and a control group.
[ detailed description ] embodiments
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to specific embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a preparation method of poria cocos mycelium Litsea litsea Litsea Lit:
step one, preparation of a poria cocos mycelium agent;
selecting Poria cocos mycelium for preservation, inoculating into PDB culture liquidStanding at 28 deg.C for 24 hr, shaking, mixing, diluting by multiple times, and diluting with 10%
5-10
8Coating the dilution on a PDA culture solid medium, and performing inverted culture at 28 ℃ for 3d until a typical single colony appears;
selecting typical single colony, inoculating into PDB culture liquid medium, culturing at 28 deg.C for 3d, collecting culture solution, centrifuging at 4 deg.C and 5000rpm for 5min, collecting thallus, washing with sterile physiological saline for 3 times, adding nutrient solution without salt ion, shaking, mixing, freezing at-20 deg.C overnight, freezing at-80 deg.C for 40min, freeze drying at 1 mg/bottle, bottling, and storing in-20 deg.C refrigerator to obtain Poria cocos mycelial preparation;
step two, fermenting poria cocos mycelium Litsea litsea leaves;
accurately weighing a certain amount of de-enzymed Lithocarpus litseifolius leaves and drinking water with a mass volume of 1:1, mixing uniformly, putting into a dry-wet separation fermentation container, sterilizing at 121 ℃ for 30min, taking out, cooling, inoculating Poria cocos mycelium agent according to 1mg/100g Lithocarpus litseifolius leaves, shaking uniformly, and culturing at 28 ℃ for 4 d;
specifically, 500.00g of lithocarpus litseifolius and 500mL of drinking water are accurately weighed, 5-10 poria cocos mycelium agents are inoculated, and the fermentation container is a wide-mouth fermentation bottle with an interlayer filter screen.
Step three, preparing fermented tea;
sterilizing the fully fermented Lithocarpus litseifolius leaves at 121 ℃ for 30min, forming and drying to obtain the Poria cocos mycelium Lithocarpus litseifolius leaf fermented tea.
As shown in fig. 3 and 4, it can be seen that, after the fermentation of the poria cocos mycelium, the content of the cellulose in lithocarpus litseifolius leaves is significantly reduced, and the content of the probiotic factors such as the hemicellulose in the lithocarpus litseifolius leaves is significantly increased.
The poria cocos mycelium Lithocarpus litseifolius leaf fermented tea prepared by the preparation method provided by the invention is evaluated:
1. sensory evaluation;
after the Lithocarpus litseifolius is fermented, the leaf color is changed from yellow to reddish brown, obvious Poria cocos mycelium is attached to the surface, the liquor color is orange red and bright, the aroma is changed from original slight green grass smell to mellow aroma, and the taste is more mellow. Therefore, through fermentation, the sensory quality of the lithocarpus litseifolius leaves is changed to a certain extent, the taste is good, and the lithocarpus litseifolius is suitable for drinking.
2. The content of the substances in the poria cocos mycelium Lithocarpus litseifolius leaf fermented dried tea;
the dry matter content of the poria cocos mycelium lithocarpus litseifolius leaf fermented tea is slightly increased to 96.21% compared with that before fermentation, which is probably caused by the increase of fermentation metabolites.
3. The content of the water extract of the tuckahoe mycelium Lithocarpus litseifolius leaf fermented tea;
the water extract content of the poria cocos mycelium Lithocarpus litseifolius leaf fermented tea is slightly increased to 48.54% compared with that before fermentation, which is probably caused by the fermentation effect of microorganisms and the generation of a large amount of soluble fermentation metabolites.
4. The crude fiber content of the poria cocos mycelium Lithocarpus litseifolius leaf fermented tea;
the crude fiber content of the pachyma cocos mycelium Lithocarpus litseifolius leaf fermented tea is greatly reduced from 81.2% to 19.42% compared with that before fermentation, which is probably because the pachyma cocos mycelium secretes a large amount of extracellular enzymes in the metabolic process, and the extracellular enzymes have the capacity of degrading cellulose, so that the crude fiber content is greatly reduced; in addition, the crude fiber can also be used as an energy source substance required by the growth and metabolism of microorganisms.
5. The content of the total flavonoids in the poria cocos mycelium Lithocarpus litseifolius leaf fermented tea;
the total flavone content of the pachyma cocos mycelium lithocarpus litseifolius leaf fermented tea is slightly increased from 9.6% to 15.30% compared with that before fermentation, which is probably because the metabolism of microorganisms, the change of temperature, pH, oxygen and other external conditions are favorable for the stability of the total flavone, and new flavonoid compounds are generated in the metabolite of the poria cocos, so that the total flavone content is increased.
6. The content of tea polyphenol is fermented by the tuckahoe mycelium Lithocarpus litseifolius leaves;
the tea polyphenol content of the pachyma cocos mycelium Lithocarpus litseifolius leaf fermented tea is slightly reduced from 24.87% to 22.5% compared with that before fermentation, which is probably because in the fermentation process, the tea polyphenol is gradually converted into tea pigments such as theaflavin, thearubigins and the like due to oxidation, so that the tea color is yellow and orange, the bitter taste is eliminated, and the tea pigment is mellow and sweet.
7. The total amount of free amino acids in the poria cocos mycelium Lithocarpus litseifolius leaf fermented tea;
the total content of free amino acids in the poria cocos mycelium lithocarpus litseifolius leaf fermented tea is remarkably increased from 0.32% to 0.75% compared with the total content of the free amino acids in the tea before fermentation, which is probably due to the fact that the protease secreted by the poria cocos mycelium decomposes protein into the free amino acids, so that the content of the free amino acids is remarkably increased.
The amino acid components of Lithocarpus litseifolius leaf and Poria mycelium Lithocarpus litseifolius leaf fermented tea are analyzed and determined, and the determination results are shown in figure 1 and figure 2.
As can be seen from FIG. 1, 16 amino acids were detected in Lithocarpus polystachyus leaves, among which 7 essential amino acids: lysine, phenylalanine, methionine, valine, threonine, leucine, and isoleucine.
As can be seen by comparing fig. 1 and fig. 2, the amino acid content of the lithocarpus litseifolius leaves after fermentation through the poria cocos mycelium is increased to different degrees, wherein the methionine is increased to the maximum extent, and the content after fermentation is increased by about 7 times compared with that before fermentation, and the lithocarpus litseifolius leaves can be used as an effective source for acquiring methionine.
In conclusion, after fermentation, the leaf color of lithocarpus litseifolius is changed from yellow to reddish brown, obvious poria cocos mycelium is attached to the surface, the liquor color is orange red and bright, the aroma is changed from original slight green grass smell to mellow aroma, and the taste is more mellow. Therefore, through fermentation, the sensory quality of the lithocarpus litseifolius leaves is changed to a certain extent, the taste is good, and the lithocarpus litseifolius is suitable for drinking.
The lithocarpus litseifolius fermented tea has the dry matter content of 96.21%, the water extract content of 49.54%, the crude fiber content of 19.42%, the total flavone content of 85.30mg/100g, the tea polyphenol content of 22.5%, the total free amino acid content of 0.75%, contains various amino acids, and the content of 7 essential amino acids of human bodies such as lysine, phenylalanine, methionine, valine, threonine, leucine and isoleucine is increased in different degrees.
The invention also provides application of the pachyma cocos mycelium Lithocarpus litseifolius Lithocarpus Lit;
the application of the poria cocos mycelium lithocarpus litseifolius fermented tea in relieving the GDM (GDM) model of a high-fat-fed C57BL6J mouse is characterized by comprising the following steps of:
step one, preparing a fermented tea extract and freeze-drying;
weighing 500g of tea leaves, adding 60% ethanol solution with the mass volume fraction of 8 times, leaching for 4h at room temperature, crushing, performing ultrasonic-assisted leaching for 10min, then leaching for 50min at 60 ℃, filtering, repeating the above operation for 2 times, combining filtrates, performing rotary evaporation concentration on the extract to about 100mL, and performing freeze drying to obtain the fermented tea extractant;
the extraction comprises water extraction and organic solvent extraction, the extraction temperature comprises normal temperature and 40-80 ℃ water bath extraction, the administration mode of the fermented tea extract to mice is oral administration, comprises free feeding, drinking water and gastric lavage, and the administration dosage is 1-1000mg/kg/d, preferably 90 mg/kg/d.
II, feeding C57BL6J mouse GDM model with high fat;
grouping experimental animals and establishing a model;
providing 8 w-age C57BL/6J mice, including 40 female mice, 20 male mice and 18-22 g in weight, after all the mice are fed with 1w of feed adaptively, randomly marking two markers by the female mice, dividing the mice into a common feed feeding group (Normal is 12) and a high-fat feed feeding group (2 groups, namely GDM models is 14; PF-treat is 14) according to numbers, continuously feeding all the mice with 1w, measuring fasting glucose tolerance and insulin tolerance at intervals of 2d, and excluding the mice with the symptom of diabetes at the early stage;
mouse GDM model mitigation;
after 1w of continuous feeding, closing the male and female mice according to a ratio of 1:2, detecting vaginal embolus, calculating as 1 st day when the vaginal embolus is detected, after the pregnancy is established, determining fasting glucose tolerance and insulin tolerance every 2 days for 1-12 th day of the pregnancy, determining fasting glucose tolerance and insulin tolerance every 1 st day for 12-18 th day of the pregnancy, terminating the experiment after 18 th day of the pregnancy at 4 pm, excluding the mice with diabetes symptoms in the ordinary feed feeding group, and perfusing the stomach of the high-fat feed feeding group with fermented tea extract at the beginning of 9 th day of the pregnancy until the experiment is finished, wherein the optimal dose is 90 mg/kg/d; mice were euthanized and blood, placenta, fetus and internal organs were collected for subsequent experiments, while the placental and fetal indices were determined.
It is further noted that 18d pregnant mice were euthanized, internal organs collected, and the WesternBlot assay of liver tissue showed significant increases in IRS and AKT phosphorylation and significant upregulation of GLUT4 expression levels compared to the model control group. From 9d of gestation, the GDM model 5d of the C57BL6J mice fed with high fat is drenched at the dose of 90mg/kg/d, the anxiety phenotype of the mice disappears, and the glucose tolerance level is gradually restored to normal.
Referring to figure 5, drenching with the extract slowed the rate of weight gain in pregnant mice; referring to fig. 6, infusion of the extract partially restored the glucose tolerance capacity of pregnant mice; referring to fig. 7, after the pregnant mouse is infused with the leaching agent, the weight of the fetus is significantly reduced compared to the model, and the weight of the fetus is close to that of the control group, so that the excessive growth of the fetus is relieved, and the generation of giant fetus is avoided; referring to fig. 8, after the pregnant mice were infused with the extract, the weight of the placenta was significantly reduced compared to the model, and the weight was close to the control group, which relieved the excessive increase of placenta index in the pregnant mice.
According to the preparation method and the application of the poria cocos mycelium lithocarpus litseifolius koehne fermented tea, poria cocos fermentation is beneficial to release of lithocarpus litseifolius koehne active ingredients, and the two active ingredients are complementary and enhanced in medicinal and edible substances; after fermentation, the reduction of tea polyphenol provides certain guarantee for the use safety in the gestation period; according to a complex theoretical system, the flavone and the triterpenoid in the leavening agent are presumed to be beneficial to improving insulin sensitivity and increasing glycogen synthesis, so that the blood sugar pressure is relieved, and the glycolipid balance capacity of the organism is increased by up-regulating GLUT. Therefore, the Lithocarpus litseifolius-Poria cocos mycelium starter can relieve the phenotype of a GDM model mouse, generates a protective effect, and is a very promising medicinal and edible homolog.
While embodiments of the invention have been disclosed above, it is not limited to the applications set forth in the description and embodiments, which are fully applicable to various fields of endeavor for which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described without departing from the generic concept as defined by the claims and the equivalents thereof.
Claims (7)
1. A preparation method of poria cocos mycelium Lithocarpus litseifolius nakai fermented tea is characterized by comprising the following steps:
step one, preparation of a poria cocos mycelium agent;
selecting Poria cocos mycelium for storing strain, inoculating into PDB culture liquid medium, standing and culturing at 28 deg.C for 24 hr, shaking, mixing, diluting at multiple ratio of 10
5-10
8Coating the dilution on a PDA culture solid medium, and performing inverted culture at 28 ℃ for 3d until a typical single colony appears;
selecting typical single colony, inoculating into PDB culture liquid medium, culturing at 28 deg.C for 3d, collecting culture solution, centrifuging at 4 deg.C and 5000rpm for 5min, collecting thallus, washing with sterile physiological saline for 3 times, adding nutrient solution without salt ion, shaking, mixing, freezing at-20 deg.C overnight, freezing at-80 deg.C for 40min, freeze drying at 1 mg/bottle, bottling, and storing in-20 deg.C refrigerator to obtain Poria cocos mycelial preparation;
step two, fermenting poria cocos mycelium Litsea litsea leaves;
accurately weighing a certain amount of de-enzymed Lithocarpus litseifolius leaves and drinking water with a mass volume of 1:1, mixing uniformly, putting into a dry-wet separation fermentation container, sterilizing at 121 ℃ for 30min, taking out, cooling, inoculating Poria cocos mycelium agent according to 1mg/100g Lithocarpus litseifolius leaves, shaking uniformly, and culturing at 28 ℃ for 4 d;
step three, preparing fermented tea;
sterilizing the fully fermented Lithocarpus litseifolius leaves at 121 ℃ for 30min, forming and drying to obtain the Poria cocos mycelium Lithocarpus litseifolius leaf fermented tea.
2. The method for preparing the pachyma cocos mycelium Lithocarpus litseifolius Koehne fermented tea according to claim 1, wherein the fermentation container is a wide-mouth fermentation bottle with an interlayer filter screen.
3. The preparation method of the pachyma cocos mycelium Lithocarpus litseifolius (rehd) Lithocarpus (.
4. The application of the poria cocos mycelium lithocarpus litseifolius fermented tea in relieving the GDM (GDM) model of a high-fat-fed C57BL6J mouse is characterized by comprising the following steps of:
step one, preparing a fermented tea extract and freeze-drying;
weighing 500g of tea leaves, adding 60% ethanol solution with the mass volume fraction of 8 times, leaching for 4h at room temperature, crushing, performing ultrasonic-assisted leaching for 10min, then leaching for 50min at 60 ℃, filtering, repeating the above operation for 2 times, combining filtrates, performing rotary evaporation concentration on the extract to about 100mL, and performing freeze drying to obtain the fermented tea extractant;
II, feeding C57BL6J mouse GDM model with high fat;
grouping experimental animals and establishing a model;
providing 8 w-age C57BL/6J mice, including 40 female mice, 20 male mice and 18-22 g in weight, after all the mice are fed with feed adaptively for 1w, randomly marking two markers for the female mice, dividing the mice into a common feed feeding group and a high-fat feed feeding group according to numbers, continuously feeding all the mice for 1w, measuring fasting glucose tolerance and insulin tolerance every 2d, and eliminating the mice with diabetes symptoms at the early stage;
mouse GDM model mitigation;
after 1w of continuous feeding, closing the male and female mice according to a ratio of 1:2, detecting vaginal embolus, calculating as 1 st day when the vaginal embolus is detected, after the pregnancy is established, determining fasting glucose tolerance and insulin tolerance every 2 days for 1-12 th day of the pregnancy, determining fasting glucose tolerance and insulin tolerance every 1 st day for 12-18 th day of the pregnancy, terminating the experiment after 18 th day of the pregnancy at 4 pm, excluding the mice with diabetes symptoms in the ordinary feed feeding group, and perfusing the stomach of the high-fat feed feeding group with fermented tea extract at the beginning of 9 th day of the pregnancy until the experiment is finished, wherein the optimal dose is 90 mg/kg/d; mice were euthanized and blood, placenta, fetus and internal organs were collected for subsequent experiments, while the placental and fetal indices were determined.
5. The application of the pachyma cocos mycelium Lithocarpus litseifolius (rehd.) nakai fermented tea to alleviating effect of high-fat fed C57BL6J mouse GDM model as claimed in claim 4, wherein the leaching comprises water leaching and organic solvent leaching, and the leaching temperature comprises normal temperature and 40-80 ℃ water bath leaching.
6. The use of the pachyma cocos mycelium lithocarpus litseifolius fermented tea for alleviating the alleviating effect of high-fat fed C57BL6J mice in a GDM model is characterized in that the fermented tea extract is orally administered to the mice in a dosage of 1-1000mg/kg/d, wherein the dosage comprises free feeding, drinking water and gastric lavage.
7. The use of the pachyma cocos mycelium lithocarpus litseifolius fermented tea for alleviating the GDM (lipid-fed C57BL 6J) mouse model as claimed in claim 6, wherein the administration dose is 90 mg/kg/d.
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| CN115868615A (en) * | 2023-01-10 | 2023-03-31 | 烟台新时代健康产业有限公司 | Process for improving antioxidant activity of pine pollen by utilizing poria cocos fermentation |
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