Application of a strain of Phellinus igniarius in the preparation of solid fermentation bacterial matrix

Abstract

The invention provides an application of a strain of Morigensis igniarius in preparing solid fermentation mycelium, belonging to the technical field of microbial fermentation. Mulberry leaves, white mulberry bark and tricholoma scaly SH-18 (preservation number GDMCC NO: 62276) are synergistically fermented to obtain solid fermentation mycelium with higher content of polyphenols, polysaccharides and triterpenoid active ingredients, providing a new idea for preparing functional products synthesized by participation of polyphenols, polysaccharides or triterpenoid active ingredients, improving the utilization rate of mulberry leaves and white mulberry bark, and avoiding waste of resources.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to the application of a strain of Phellinus igniarius in preparing solid fermentation bacterial mass. Background Art

[0002] Phellinus igniarius is a wood-rotting fungus with great medicinal potential. It is often found on the trunks of mulberry trees. Its fruiting bodies vary in color, usually yellow-brown. It is also known as mulberry, mulberry ear, etc. Phellinus igniarius is a type of hard polyporous fungus. Its taxonomic classification is still unclear. It is usually a general term for a variety of medicinal fungi belonging to the Hymenochaetaceae family.

[0003] Phellinus contains rich bioactive ingredients such as polysaccharides, flavonoids and polyphenols. For example, the Chinese invention patent with the patent publication number "CN114989986A" (application number 202210478125.X) discloses a wild mulberry fungus-Inonotus hispidus rich in multiple active ingredients, with a preservation number of GDMCC NO: 62276. The mulberry fungus is rich in multiple active ingredients, and the polysaccharides, polyphenols, flavonoids, triterpenes and other active ingredients contained in it are at a high level. It is a variety with great medicinal potential and great development potential.

[0004] Mulberry leaf is a plant that can be used as both medicine and food, and its medicinal value has a long history in my country. Studies have found that mulberry leaves are not only rich in nutrients such as lipids, proteins, carbohydrates, vitamins, organic acids and minerals, but also contain biologically active ingredients such as flavonoids, polysaccharides, alkaloids, phytosterols and volatile oils. Modern pharmacological studies have shown that mulberry leaves can be used to improve diabetes, and the main active ingredients that play a hypoglycemic role are polysaccharides and alkaloids. my country's mulberry leaf resources are very rich, but the overall use is relatively single. Most mulberry leaves are used as animal feed. Every year, a large amount of mulberry leaves are discarded, resulting in a serious waste of mulberry leaf resources.

[0005] Mulberry bark is the dried root bark of the mulberry tree. It is in the shape of a twisted roll, with a smooth inner surface that is white or light yellow, and a rough, scaly outer surface that is yellow or brown. Mulberry bark has been passed down as a traditional Chinese medicine until now. Nowadays, traditional Chinese medicine practitioners often use it to treat lung heat cough, edema, asthma, bacterial infection, obstructive pulmonary disease, etc.

[0006] Developing new uses for mulberry leaves and mulberry bark and improving the utilization structure of traditional mulberry resources are of great significance to improving economic and social benefits and expanding the sericulture industry. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides an application of a strain of Phellinus igniarius in preparing solid fermentation mycelium, and mulberry leaves, mulberry bark and Poria cocos (GDMCC NO: 62276) are synergistically fermented to obtain solid fermentation mycelium with higher content of polyphenols, polysaccharides and triterpenoid active ingredients.

[0008] The technical solution of the present invention is as follows:

[0009] The invention discloses an application of a strain of Phellinus igniarius in preparing solid fermentation bacterial matrix. The application method comprises the following steps: inoculating the Phellinus igniarius into a fermentation medium for fermentation to obtain solid fermentation bacterial matrix; the fermentation medium comprises mulberry leaves and mulberry bark; the Phellinus igniarius is Inonotus hispidus SH-18, and its preservation number is GDMCC NO: 62276.

[0010] Preferably, the fermentation conditions are: fermentation in the dark for 7 to 14 days at a temperature of 22 to 30° C. and a humidity of 70 to 90%.

[0011] More preferably, the inoculation amount of the Phellinus igniarius is 8-9% (m / m) of the fermentation medium.

[0012] Further preferably, the mass ratio between the mulberry leaves and the white mulberry bark is (2-3):1.

[0013] Preferably, the method for preparing the fermentation medium is:

[0014] (1) Wash, dry and wither fresh mulberry leaves;

[0015] (2) Grinding and sieving the mulberry bark to obtain mulberry bark powder;

[0016] (3) removing the petioles of the dried and withered mulberry leaves obtained in step (1), cutting the leaves into thin strips, and then mixing the strips with the mulberry bark powder obtained in step (2);

[0017] (4) Add nutrients and sterilize.

[0018] Preferably, the temperature during the drying and withering in step (1) is 20-30° C. and the humidity is 55-65%.

[0019] Preferably, the mesh size of the sieve during the sieving in step (2) is 60 to 200 meshes.

[0020] Preferably, the sterilization conditions in step (3) are: 121° C., sterilization for 20 min.

[0021] Preferably, the nutrient elements in step (4) include the following components: 10-30 g / L glucose, 0.1-1 g / L potassium dihydrogen phosphate, and 0.1-1 g / L magnesium sulfate.

[0022] The solid fermentation bacteria are used in the preparation of functional products, wherein the ingredients of the functional products include one or more active ingredients of polyphenols, polysaccharides or triterpenes.

[0023] Beneficial effects:

[0024] (1) The present invention provides an application of a strain of Phellinus igniarius in preparing solid fermentation biomass, wherein mulberry leaves, white mulberry bark and Porites pubescens SH-18 (GDMCC NO: 62276) are synergistically fermented to obtain a solid fermentation biomass having a higher content of polyphenols, polysaccharides and triterpenoid active ingredients.

[0025] (2) The preparation method provided by the present invention is simple and efficient, and provides a new idea for preparing functional products in which polyphenols, polysaccharides or triterpenoid active ingredients participate in the synthesis, and improves the utilization rate of mulberry leaves and mulberry bark, thus avoiding waste of resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the picture of mulberry bark powder;

[0027] Figure 2 This is a picture of the fermentation medium;

[0028] Figure 3 This is a picture of solid fermentation mycelium covered with Phellinus igniarius hyphae. DETAILED DESCRIPTION

[0029] The following is explained in conjunction with specific embodiments:

[0030] Source of experimental materials:

[0031] Inonotus hispidus SH-18: The deposit number is GDMCC NO:62276.

[0032] Phellinus linteus S1: from the strain bank of Qilu University of Technology.

[0033] Phellinus gilvus S2: from the strain bank of Qilu University of Technology.

[0034] Embodiment 1:

[0035] 1. Preparation of fermentation medium:

[0036] (1) Pick fresh mulberry leaves of uniform size, such as one bud and one leaf or one bud and two leaves, or two leaves of equal tenderness, remove diseased and insect-infested leaves and yellowed and withered leaves, and then wash them; dry and wither the washed mulberry leaves at a temperature of 20-30°C and a humidity of 55-65% for 12 hours until the moisture content of the mulberry leaves drops to about 60%.

[0037] (2) Grind the mulberry bark into powder and pass it through a 60-mesh sieve to obtain mulberry bark powder, as follows: Figure 1 shown.

[0038] (3) removing the petioles of the dried and withered mulberry leaves from step (1), cutting them into thin strips, and then mixing them with the mulberry bark powder from step (2); wherein the mass ratio between the mulberry leaves and the mulberry bark is 2:1.

[0039] (4) Add 10 g / L glucose, 0.1 g / L potassium dihydrogen phosphate, and 0.1 g / L magnesium sulfate, and sterilize at 121° C. for 20 min to obtain a fermentation medium. Figure 2 shown.

[0040] 2. Preparation of solid fermentation bacteria:

[0041] (1) Inoculating the SH-18 fungus on a PDA solid culture medium, culturing it at 30°C for 7 days to obtain activated mycelium; inoculating the activated mycelium into a liquid culture medium, culturing it at 30°C and 200 rpm for 7 days to obtain seed mycelium;

[0042] The components of the liquid culture medium are: glucose 30g / L, yeast powder 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 1g / L, vitamin B 1 0.1g / L.

[0043] (2) inoculating the seed mycelium of step (1) into the above fermentation medium to prepare for fermentation;

[0044] Wherein, the inoculation amount of the seed mycelium is 8% (m / m) of the fermentation medium.

[0045] (3) Fermenting in the dark for 10 days at a temperature of 30° C. and a humidity of 80%; after the fermentation is completed, a solid fermentation mycelium covered with Phellinus igniarius hyphae is obtained, as follows: Figure 3 shown.

[0046] Comparative Example 1:

[0047] Different from Example 1, when preparing the fermentation medium, the mulberry bark is replaced with mulberry leaves of equal weight, all the mulberry leaves are dried and withered, and then nutrient elements are added for sterilization, and the remaining steps are the same as Example 1; solid fermentation bacterial mass is obtained.

[0048] Comparative Example 2:

[0049] Different from Example 1, when preparing the fermentation medium, the mulberry leaves are replaced with an equal weight of mulberry bark, all the mulberry bark is ground into powder, and nutrient elements are added to sterilize, and the remaining steps are the same as Example 1; solid fermentation bacterial mass is obtained.

[0050] Comparative Example 3:

[0051] Preparation of solid fermentation bacteria:

[0052] The difference from Example 1 is that the F. schizodactylifera S1 is used as the fermentation strain to be inoculated into the fermentation medium, and the remaining steps are the same as those in Example 1; and solid fermentation bacterial mass is obtained.

[0053] Comparative Example 4:

[0054] Preparation of solid fermentation bacteria:

[0055] The difference from Example 1 is that the yellowish woody pore fungus S2 is used as the fermentation strain to be inoculated into the fermentation medium, and the remaining steps are the same as those in Example 1; solid fermentation bacterial mass is obtained.

[0056] Experimental Example 1:

[0057] Determination of polyphenol content:

[0058] The polyphenol content in the solid fermentation bacterial mass prepared in Example 1 and Comparative Examples 1 to 4 was determined, and the determination steps were as follows:

[0059] (1) Extraction of polyphenols: The solid fermentation bacteria were dried and ground into powder. The powder was accurately weighed and 25 times the volume of 70% ethanol solution was added. Ultrasonic extraction was performed at 55°C for 1 h. The mixture was centrifuged at 6000 rpm for 10 min and the supernatant was collected. The precipitate after centrifugation was extracted twice with 70% ethanol solution according to the above method. The supernatants of the three extractions were combined and used for the determination of polyphenol content.

[0060] (2) The polyphenol content was determined by the Folin-phenol colorimetric method. The determination principle of this method is: Folin reagent (2,4-dinitrophenylhydrazine) reacts with phenolic substances to form a light blue to green complex, which has an absorption peak at a wavelength of 760nm; the absorbance of the reaction solution at a wavelength of 760nm is measured by a spectrophotometer, and the polyphenol content in the solid fermentation microbial mass is calculated by drawing a standard curve.

[0061] The measurement results are shown in Table 1.

[0062] Table 1. Determination results of polyphenol content

[0063]

[0064] As shown in Table 1, compared with Comparative Examples 1 to 2, Example 1 uses a fermentation medium containing both mulberry leaves and white mulberry bark to culture Phellinus igniarius, and the solid fermentation mycelium obtained by using mulberry leaves or white mulberry bark as the fermentation medium has a higher polyphenol content than that obtained by using mulberry leaves or white mulberry bark as the fermentation medium alone;

[0065] Compared with Comparative Examples 3 to 4, in Example 1, the solid fermentation mycelium obtained by using the pubescens SH-18 as the fermentation strain and using a fermentation medium to which mulberry leaves and mulberry bark were added simultaneously had a higher polyphenol content than that obtained by using the phylloscopus S1 or the phylloscopus S2 as the fermentation strain. After the fermentation was completed, the polyphenol content in the solid fermentation mycelium was approximately 363.53 times that of the polyphenol content of the pubescens SH-18 itself.

[0066] Experimental Example 2:

[0067] Determination of polysaccharide content:

[0068] The polysaccharide content in the solid fermentation bacterial mass prepared in Example 1 and Comparative Examples 1 to 4 was determined, and the determination steps were as follows:

[0069] Extraction of polysaccharides: Dry the solid fermentation bacteria and grind it into powder. Accurately weigh the powder, add 200 times the volume of distilled water, boil it in a boiling water bath for 3 hours, filter it through a suction filter, and collect the filtrate for the determination of polysaccharide content.

[0070] The polysaccharide content was determined by the phenol-sulfuric acid method. The determination principle of this method is: sugar substances are dehydrated under the action of concentrated sulfuric acid to generate furfural, and furfural can condense with phenol to form an orange-red compound with an absorption peak at a wavelength of 490nm; the absorbance of the reaction solution at a wavelength of 490nm is determined by a spectrophotometer, and the polysaccharide content in the solid fermentation microbial mass is calculated by drawing a standard curve.

[0071] The measurement results are shown in Table 2.

[0072] Table 2. Determination results of polysaccharide content

[0073]

[0074]

[0075] As shown in Table 2, compared with Comparative Examples 1 to 2, Example 1 uses a fermentation medium containing both mulberry leaves and white mulberry bark to culture Phellinus igniarius, and the solid fermentation mycelium obtained by using mulberry leaves or white mulberry bark as the fermentation medium has a higher polysaccharide content;

[0076] Compared with Comparative Examples 3 to 4, in Example 1, the solid fermentation mycelium was prepared by using the fermentation medium to which mulberry leaves and mulberry bark were added at the same time. Compared with the solid fermentation mycelium obtained by using the Folium S1 or the Folium S2 as the fermentation strain, the polysaccharide content in the solid fermentation mycelium was higher. After the fermentation was completed, the polysaccharide content in the solid fermentation mycelium was about 43.65 times the polysaccharide content of the Folium SH-18 itself.

[0077] Experimental Example 3:

[0078] Determination of triterpene content:

[0079] The triterpenoid content in the solid fermentation bacterial mass prepared in Example 1 and Comparative Examples 1 to 4 was determined, and the determination steps were as follows:

[0080] Extraction of triterpenes: dry the solid fermentation bacteria and grind it into powder, accurately weigh the powder, add 25 times the volume of 70% ethanol solution, extract at 55℃ for 1h, centrifuge at 6000rpm for 10min, collect the supernatant; extract the precipitate after centrifugation again with 70% ethanol solution twice according to the above method; combine the supernatants of three times for the determination of triterpene content.

[0081] The triterpene content was determined by the vanillin-perchloric acid colorimetric method. The determination principle of this method is: triterpenoids react with vanillin under the oxidation of perchloric acid to generate colored substances, and the colored substances have an absorption peak at a wavelength of 548nm; the absorbance of the reaction solution at a wavelength of 548nm is determined by a spectrophotometer, and the triterpene content in the solid fermentation bacteria is calculated by drawing a standard curve.

[0082] The measurement results are shown in Table 3.

[0083] Table 3. Determination results of triterpenoid content

[0084]

[0085] As shown in Table 3, compared with Comparative Examples 1 to 2, Example 1 uses a fermentation medium containing both mulberry leaves and white mulberry bark to culture Phellinus igniarius, and the solid fermentation mycelium obtained by using mulberry leaves or white mulberry bark as the fermentation medium has a higher triterpene content than that obtained by using mulberry leaves or white mulberry bark as the fermentation medium alone;

[0086] Compared with Comparative Examples 3 to 4, in Example 1, the solid fermentation mycelium obtained by using the pubescens SH-18 as the fermentation strain and using a fermentation medium to which mulberry leaves and mulberry bark were added simultaneously had a higher triterpene content than that obtained by using the phylloscopus S1 or the phylloscopus S2 as the fermentation strain. After the fermentation was completed, the triterpene content in the solid fermentation mycelium was approximately 282.2 times the triterpene content of the pubescens SH-18 itself.

[0087] Experimental Example 4:

[0088] Determination of flavonoid content:

[0089] The flavonoid content in the solid fermentation bacteria prepared in Example 1 and Comparative Examples 1 to 4 was determined, and the determination steps were as follows:

[0090] Extraction of flavonoids: dry the solid fermentation bacteria and grind it into powder. Accurately weigh the powder, add 25 times the volume of 70% ethanol solution, extract at 55℃ for 1h, centrifuge at 6000rpm for 10min, and collect the supernatant; extract the precipitate after centrifugation again with 70% ethanol solution twice according to the above method; combine the supernatants of three times for the determination of flavonoid content.

[0091] The flavonoid content was determined by aluminum nitrate colorimetric method. Under neutral or weak alkaline conditions and the presence of sodium nitrite, flavonoid compounds and aluminum salts formed chelates, which had an absorption peak at a wavelength of 500nm. The absorbance of the reaction solution at a wavelength of 500nm was determined by a spectrophotometer, and the flavonoid content in the solid fermentation microbial mass was calculated by drawing a standard curve.

[0092] The measurement results are shown in Table 4.

[0093] Table 4. Determination results of flavonoid content

[0094]

[0095] As shown in Table 4, compared with Comparative Examples 1 to 2, Example 1 uses a fermentation medium containing both mulberry leaves and white mulberry bark to culture Phellinus igniarius, and the solid fermentation mycelium obtained by using mulberry leaves or white mulberry bark as the fermentation medium has a small difference in flavonoid content;

[0096] Compared with Comparative Examples 3 to 4, in Example 1, the solid fermentation mycelium was prepared by using the fermentation medium to which mulberry leaves and mulberry bark were added at the same time. Compared with the solid fermentation mycelium obtained by using the Feloporium S1 or the Feloporium S2 as the fermentation strain, the content of flavonoids was not higher, which indicates that the combination of mulberry leaves and mulberry bark as the fermentation medium cannot significantly improve the metabolic activity of Feloporium SH-18 for flavonoids.

[0097] In summary, the synergistic fermentation of Pleurotus eryngii SH-18 with mulberry leaves and mulberry bark can produce higher levels of polyphenols, polysaccharides and triterpenoid active ingredients than other mulberry fungi. This may be because the enzymes in Pleurotus eryngii SH-18 can catalyze the transformation of the chemical components in mulberry leaves and mulberry bark. At the same time, certain components in mulberry leaves and mulberry bark can regulate the metabolism of Pleurotus eryngii, thereby enhancing the metabolic activity of Pleurotus eryngii and allowing it to metabolize and produce more polyphenols, polysaccharides and triterpenoid active ingredients.

Claims

1. Use of a strain of Phellinus igniarius in preparing solid fermentation bacterial matrix, characterized in that: The application method is to inoculate the mulberry fungus into a fermentation medium for fermentation to obtain solid fermentation bacteria; the fermentation medium includes mulberry leaves and mulberry bark; the mulberry fungus is a thick hair fibrous fungus ( Inonotus hispidus ) SH-18, deposit number is GDMCC NO:62276; Among them, the inoculation amount of the Phellinus igniarius is 8-9% (m / m) of the fermentation medium; the mass ratio between the mulberry leaves and the white mulberry bark is (2-3):

1.

2. The use according to claim 1, characterized in that The fermentation conditions are as follows: fermentation in the dark for 7 to 14 days at a temperature of 22 to 30° C. and a humidity of 70 to 90%.

3. The use according to claim 1, characterized in that The preparation method of the fermentation medium is: (1) Wash, dry and wither fresh mulberry leaves; (2) Grinding and sieving the mulberry bark to obtain mulberry bark powder; (3) removing the petioles of the dried and withered mulberry leaves from step (1), cutting them into small strips, and then mixing them with the mulberry bark powder from step (2); (4) Add nutrients and sterilize.

4. The use according to claim 3, characterized in that The temperature during the drying and withering in step (1) is 20-30° C. and the humidity is 55-65%.

5. The use according to claim 3, characterized in that The mesh size of the sieve during the sieving in step (2) is 60 to 200 meshes.

6. The use according to claim 3, characterized in that The sterilization conditions in step (4) are: 121° C., sterilization for 20 min.

7. The use according to claim 3, characterized in that The nutrient elements in step (4) include the following components: 10-30 g / L glucose, 0.1-1 g / L potassium dihydrogen phosphate, and 0.1-1 g / L magnesium sulfate.

8. Use of the solid fermentation microbial substance according to claim 1 in the preparation of a functional product, wherein the ingredients of the functional product include one or more of polyphenols, polysaccharides or triterpenoid active ingredients.

Images