CN108901594B

CN108901594B - Method for improving polysaccharide content in fruiting bodies or hyphae of lentinus edodes by utilizing vetiver

Info

Publication number: CN108901594B
Application number: CN201810823349.3A
Authority: CN
Inventors: 高玉千; 邱立友; 王晓婷; 李涛; 张朝辉; 田芳; 丁亚通
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2018-07-25
Filing date: 2018-07-25
Publication date: 2021-03-23
Anticipated expiration: 2038-07-25
Also published as: CN108901594A

Abstract

The invention relates to a method for improving polysaccharide content in mushroom fruiting bodies or hyphae by using vetiver grass, which comprises the steps of adding vetiver grass scraps into a mushroom cultivation culture material, and then planting by adopting a conventional method in the field to obtain mushroom fruiting bodies; or adding vetiver grass powder into PDA solid or liquid culture medium, and preparing to obtain Lentinus Edodes mycelium by conventional method in the art. The method is simple to operate, can obviously improve the polysaccharide content in the fruiting bodies or hyphae of the lentinus edodes, and is suitable for large-scale production.

Description

Method for improving polysaccharide content in fruiting bodies or hyphae of lentinus edodes by utilizing vetiver

Technical Field

The invention belongs to the technical field of mushroom planting, and particularly relates to a method for improving polysaccharide content in fruiting bodies or hyphae of mushrooms by utilizing vetiver grass.

Background

The Lentinus edodes is rich in nutrition and bioactive components, and lentinan has multiple immunity promoting effects. At present, the mushroom is the second major edible mushroom in the world after the agaricus bisporus. The existing mushroom production has two problems, which seriously affect the economic and social benefits of mushroom production, one is that the polysaccharide content in mushroom hypha and fruiting body is low, which not only increases the cost of mushroom polysaccharide medicine, but also reduces the edible and medicinal value of mushroom; the other is that the main raw material for cultivating the mushroom is wood chips, and the contradiction of the mushroom forest is more and more prominent.

At present, the existing mushroom cultivation materials mainly comprise broadleaf tree sawdust, willow, fir and other part of needle-leaved tree sawdust, cotton seed hulls, straws, corn stalks, wheat straws and the like, and the yield of lentinan is about 3 percent more. With the increasing expansion of the cultivation scale of the lentinus edodes, a large amount of wood chip resources with slow growth need to be consumed, which is in necessary contradiction with the construction of modern ecological forestry and ecological civilization. Therefore, the method actively searches for a proper substitute cultivation material, reduces the excessive dependence on the traditional wood chip resources, and promotes the green sustainable development of the mushroom industry, thereby improving the economic benefit, the social benefit and the ecological benefit of the mushroom industry, and is the main development direction of the industry.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for improving the polysaccharide content in the fruiting bodies or the mycelia of the lentinus edodes by utilizing vetiver, which is simple to operate, can obviously improve the polysaccharide content in the fruiting bodies or the mycelia of the lentinus edodes and is suitable for large-scale production.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for increasing polysaccharide content in fruiting body or mycelium of Lentinus Edodes by using vetiver comprises adding vetiver grass bits into culture medium for Lentinus Edodes cultivation, and planting by conventional method in the field to obtain fruiting body of Lentinus Edodes; or adding vetiver grass powder into PDA solid or liquid culture medium, and preparing to obtain Lentinus Edodes mycelium by conventional method in the art.

Specifically, the sum of the mass of the shiitake cultivation compost and the vetiver grass cuttings is 100%, and the addition amount of the vetiver grass cuttings is 30-50%.

Further, the mushroom cultivation compost comprises the following raw materials in percentage by mass: 8-12% of bran, 1-2% of lime and the balance of broad-leaved tree sawdust.

The method for improving the polysaccharide content in the fruiting bodies or the hyphae of the lentinus edodes by utilizing the vetiver comprises the following steps:

1) preparing materials: weighing the raw materials in proportion, adding water for stirring, and controlling the ratio of the materials to the water to be 1: 1.10-1.15; then bagging and sterilizing;

2) inoculation: inoculating Lentinus Edodes strain by acupoint pressing method, with 6-8 acupoints per bag; transferring the inoculated strain to a strain room for spawn running and fungus culturing, enabling the hyphae to grow full of bags and start to form a tumor-shaped object, and then turning color in summer after putting on a shelf;

3) cultivating flower mushrooms: is obtained by conventional bud forcing, bud breeding and squating, flower forcing and flower protecting.

The method for improving the polysaccharide content in the fruiting bodies or the mycelia of the shiitake mushrooms by using the vetiver grass comprises the following steps of adding 2-10 g of vetiver grass powder into 100ml of PDA solid culture medium; adding 1-4 g of vetiver grass powder into 100ml of PDA liquid culture medium.

The method for increasing the polysaccharide content in the fruiting bodies or hyphae of the lentinus edodes by utilizing the vetiver is preferably as follows: pulverizing cleaned and dried vetiver into powder, adding into PDA solid culture medium in proportion, sterilizing, pouring into flat plate, inoculating Lentinus Edodes strain, and culturing at 25-28 deg.C for 8-15 days to obtain Lentinus Edodes mycelium.

The method for increasing the polysaccharide content in the fruiting bodies or hyphae of the lentinus edodes by utilizing the vetiver is preferably as follows: pulverizing cleaned and dried herba Cymbopogonis Citrari into powder, adding into PDA liquid culture medium in proportion, sterilizing, inoculating Lentinus Edodes strain, and shake-culturing at 25-28 deg.C and 180 + -20 r/min for 8-15 days.

Vetiver, also known as vetiver, is a perennial herb of gramineae, has the characteristics of strong adaptability, fast growth and reproduction, developed root system, cold resistance, barren resistance and the like, is called as a 'herb plant with the longest root system in the world', and is commonly used as a water and soil conservation plant. Vetiver is rich in lignocellulose and is a good material for replacing wood with grass. In addition, the vetiver also contains various plant secondary metabolites, and some components can promote the improvement of the yield of the shiitake mushrooms. The invention is found by experiments that: adding vetiver grass into mushroom cultivation compost (mainly containing sawdust) and PDA solid or liquid culture medium, and culturing mushroom by bag cultivation or flat plate or shake flask, the polysaccharide content in fruiting body and mycelium of mushroom can be remarkably increased. The method has simple operation steps and is suitable for large-scale production.

Drawings

FIG. 1 shows the growth of hyphae of Lentinus edodes on solid plates of example 2 with different addition amounts of vetiver grass;

FIG. 2 is a graph showing the measurement of polysaccharides in the mycelia of Lentinus edodes on a solid plate at different addition amounts of vetiver grass in example 2;

FIG. 3 is a graph showing the results of measurement of polysaccharides in PDA broth obtained from liquid culture medium with different addition amounts of vetiver.

Detailed Description

The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.

Example 1 vetiver grass bag-cultured shiitake mushrooms were added.

A method for improving the content of polysaccharide in a mushroom fruiting body by using vetiver grass specifically comprises the following steps:

preparing materials: selecting dry, moldy and pollution-free vetiver stem and leaf, and pulverizing into grass scraps with a pulverizer, wherein the particles with the diameter of 5-8 mm account for more than 60%;

the sum of the mass of the mushroom cultivation compost and the vetiver grass cuttings is 100%, and the addition amount of the vetiver grass cuttings is 30-50%. In this example, the added amount of vetiver grass dust is 30% and 50% respectively, and the culture material for culturing pure shiitake mushroom is used as a control. The mushroom cultivation compost comprises the following raw materials in percentage by mass: 10% of bran, 1% of lime and 89% of broad-leaved tree sawdust;

and weighing the raw materials according to the formula proportion and the bag making amount. Pre-wetting vetiver grass chips, broad-leaved tree wood chips and bran in advance, adding water according to the required water content, wet-mixing, and holding the mixed materials by hands, wherein water seeps from finger joints but does not drip. The material-water ratio is 1: 1.1-1.15, and then adjusting the pH value to 7-7.5.

Bagging: adopts a double-layer bag, and the inner bag is a cutting-free bag. The inner bag is made of plastic bags with the specification of 55 multiplied by 17 multiplied by 0.002cm, is ultrathin (the bag is not cut during fruiting, time and labor are saved), and meanwhile, the bags are tightly contacted with the material surface, so that the disease incidence rate is reduced. The outer bag is a plastic bag with the specification of 60 multiplied by 18 multiplied by 0.004 cm. 3) And (3) sterilization: sterilizing at normal pressure by adopting a circulating steam bag, raising the temperature to 100 ℃ within 4-6 hours, and maintaining for 24 hours; during autoclaving, the pressure is maintained at 1.2-1.5 kg/cm²And maintaining for 1.5-2h, and discharging the bag when the pressure is reduced to 0.

Inoculation: the inoculation chamber must be clean, dry and well sealed. Before inoculation, sulfur, formaldehyde, potassium permanganate aerosol disinfection box or acetic acid is used for thorough sterilization, and an ozone lamp is used for sterilization. Under aseptic condition, removing outer bag, inoculating Lentinus Edodes L26 strain in a acupoint mode with 6-8 acupoints per bag, sealing with sterile adhesive tape, covering with outer bag, and transferring into bacteria culture room for spawn running.

Spawn running and fungus culturing: the shed type layer is adopted to pile up bacteria and culture bacteria, 6-8 layers are piled up, and a sunshade net is hung outside the shed while a straw mat, corn stalks and the like are used for shading. The relative humidity of air in the culture room is kept at 60-70%, the air is ventilated, darkly lighted and clean, and the temperature is kept at 22-27 ℃. The duty refining management is carried out in the middle, formaldehyde, lime, carbendazim and the like are regularly selected for disinfection, pile turning, hole puncturing and impurity detection, and the holes are not punctured at high temperature, so that bacteria burning caused by vigorous breathing and heating of hypha is prevented. About 70 pricks are completed two months after spawn running. The mycelium grows full of the bag and starts to form a tumor-shaped object, and then the color turns over summer after the mycelium is put on the shelf.

Cultivating flower mushrooms: adopts a conventional layered rack type cement shed frame with 7-8 layers. And (4) building a mushroom shed before mushroom bags enter the shed and grow mushrooms, and reserving a proper footpath. Each shed is provided with 1000-3000 bags of bacteria bags.

a) The inducement of primordium is carried out in a centralized way indoors or in a fruiting shed. After full color change in spring cultivation in 8 months and autumn cultivation, vibrating mushroom stick with proper strength, and adopting conventional heating, humidifying, ventilation enhancing and other control promoting measures to promote day and night temperature difference to be above 10 ℃ (usually 8-22 ℃), controlling air relative humidity to be 85%, water content in material to be 50-55%, scattering light and oxygen to be sufficient, and promoting hypha to further kink to form mushroom buds.

b) The outer bag of the fungus bag is removed in time after the squat bud of the bud grows, and the mushroom bud with strong growth vigor can automatically break the bag because the inner bag is ultra-thin. And (5) performing conventional positioning and bud thinning after bag breaking. Squatting for 5-7 days when the bud is about 2cm, keeping the temperature at 8-12 deg.C, keeping the relative humidity of air at 85-90%, scattering light, and ventilating. When the diameter of the mushroom bud reaches 3-4cm and the mushroom bud is thick and strong 5-8 days after the bud squat, a preliminary crack pattern is formed.

c) The flower promoting method adopts large temperature difference (day and night temperature difference is more than 10 ℃), large dry-wet difference (more than 15%), ventilation, full illumination and other control methods to continuously promote the flower for 2-4 times, and promote the mushroom cap to form white flower cracks (namely white flower mushroom).

d) After the flower-keeping white flower mushrooms are formed, the relative humidity of air in the greenhouse is kept below 70%, the temperature is 8-15 ℃, the air is fully illuminated and ventilated, and the commercial mushrooms can grow after 5 days.

The diameter of the cap of the harvested and processed flower mushroom is 4-5cm, and the flower mushroom can be harvested in stages and batches according to the requirement before opening the cap, dried without sulfur hazard, and packaged in stages. When the mushroom is picked, no mushroom feet are left, and water is properly supplemented when the water in the mushroom material of the second tide is deficient.

Managing the picked mushroom sticks of the mushrooms, cultivating the mushrooms for 7-10 days under the conditions of temperature of 22-26 ℃, humidity of 80% -85%, dark light and ventilation, and supplementing water in time or certain nutrient solution when water is deficient to prepare for producing the damp mushrooms.

Mature commodity lentinan is collected, and then the lentinan content in the pileus and the stipe is respectively detected according to NY/T1676-.

As can be seen from table 1: after the vetiver is added, the polysaccharide content in the mushroom pileus and the stipe is obviously improved, and particularly the polysaccharide content in the stipe is increased by 3.21-3.48 times. The polysaccharide in the pileus is gradually increased along with the increase of the addition amount of the vetiver. When 50% of vetiver is added, the polysaccharide in the pileus is increased by 2.36 times.

Example 2 addition of vetiver grass for plate culture of Lentinus edodes

A method for improving polysaccharide content in mushroom mycelia by using vetiver grass comprises the following specific steps:

cleaning and drying vetiver grass, cutting into thin strips of about 3cm, and then putting into a grinder to be ground for 30-60s to be ground into fine powder. Then adding the mixture into PDA solid culture medium according to six gradient ratios of 2%, 4%, 6%, 8% and 10% (namely adding 2g, 4g, 6g, 8g and 10g vetiver grass powder into each 100ml PDA solid culture medium), taking the PDA solid culture medium without adding vetiver grass as a control (Ck), sterilizing at 121 ℃ for 30min, then pouring the plate, and inoculating lentinus edodes L26 strain. Culturing at 25-28 deg.C for 10 days, observing hypha growth, scraping hypha, extracting and purifying hypha, and measuring lentinan content in hypha by phenol-sulfuric acid method. The results are shown in FIG. 1, FIG. 2 and Table 2.

As can be seen from the culture plate in FIG. 1: with the increase of the addition amount of vetiver grass, the growth rate of the mushroom hyphae is decreased gradually, but the growth vigor is increased gradually. As can be seen from fig. 2: after hyphae are scraped and added into DNS, the color is gradually deepened and changes in a gradient way with the increase of the addition amount of the vetiver grass. As can be seen from table 2: with the increase of the addition amount of the vetiver grass, the content of polysaccharide in the mushroom hypha is gradually increased.

From this embodiment, it can be seen that: when mushroom L26 strain is cultured on a plate, vetiver grass is added into the PDA solid culture medium, although the growth speed of hyphae cannot be increased, the growth of the hyphae is facilitated, and the hyphae are more white, dense and strong. Table 2 the results of the measurement of the content of lentinan in the mycelia show that: the addition of the vetiver can greatly improve the yield of the polysaccharide. When the addition amount of the vetiver grass is 10 percent (the maximum addition amount), the polysaccharide content is the highest and is 9 times of that of the blank control.

Example 3: liquid culture of Lentinus edodes by adding vetiver

cleaning and drying vetiver grass, cutting into thin strips of about 3cm, and then putting into a grinder to be ground for 30-60s to be ground into fine powder. Then, the mixture was added to PDA liquid medium at a ratio of 1%, 2% and 4% (i.e., 1g, 2g and 4g of vetiver grass powder was added to 100ml of PDA liquid medium), and PDA liquid medium without vetiver grass was used as a control (Ck). Inoculating Lentinus Edodes L26 strain, shake culturing at 25-28 deg.C and 180 r/min for 10 days. After the fermentation liquor in the shake flask is extracted and purified, the content of lentinan in the fermentation liquor is measured by adopting a phenol-sulfuric acid method. The results are shown in FIG. 3 and Table 3.

The invention utilizes a phenol-sulfuric acid method to determine lentinan solution, and the determination principle of the method is as follows: the sugar is hydrolyzed into monosaccharide molecules under the action of concentrated sulfuric acid, and is rapidly dehydrated to produce furfural derivatives, the substances are condensed with phenol to be orange yellow compounds, and the absorbance and the polysaccharide content are in direct proportion in a linear relation at 490nm and within a certain concentration range. Generally, the darker the color, indicating a higher level of hydrolytic condensate, the more monosaccharide molecules the polysaccharide is hydrolyzed to, and a higher level of polysaccharide, fig. 3 is a result that can be directly observed with the naked eye. As can be seen from the color of the polysaccharide solution in fig. 3: with the gradual increase of the addition amount of the vetiver grass, the content of polysaccharide also increases. The content of the specific polysaccharide is obtained by conversion of a standard curve and absorbance, and the result is shown in table 3. As can be seen from table 3: the addition of the vetiver can greatly improve the yield of the polysaccharide. When the addition amount of the vetiver grass is 4 percent (the maximum addition amount), the polysaccharide content is the highest and is 13 times of that of the blank control.

TABLE 3 content of polysaccharides in PDA broth of liquid culture medium with different addition of vetiver grass

In the above examples 2 and 3, before the content of lentinan is measured by the phenol-sulfuric acid method, the extraction and purification treatment of the mycelium or fermentation broth is as follows:

1) extracting lentinan: extracting the mycelium or fermentation liquid with 98 deg.C hot water for 3 hr, filtering, evaporating and concentrating 10 times, and standing overnight with 4 times of 95% ethanol. Centrifuging at 5000r/min for 15min, and discarding the supernatant; washing the precipitate with 85% ethanol; centrifuging at 5000r/min for 10min, and discarding the supernatant; washing the precipitate with 85% ethanol; centrifuging at 5000r/min for 10min, discarding supernatant, and dissolving precipitate with hot water to obtain crude polysaccharide solution;

2) and (3) purifying lentinan: adopting a Sevage deproteinization method: the crude polysaccharide solution was added with a mixture of chloroform and n-butanol (V: V =4: 1) of equal volume, shaken and shaken. Centrifuging at 5000r/min for 10min, and carefully sucking out supernatant with liquid transfer gun to obtain lentinan solution. The measurement of the polysaccharide content in the lentinan solution is directly carried out by a phenol-sulfuric acid method, which is a conventional technique in the field, and thus, the detailed description is omitted.

Claims

1. A method for improving polysaccharide content in mushroom hypha by using vetiver grass is characterized in that vetiver grass powder is added into a PDA solid or liquid culture medium, and then mushroom hypha is prepared by adopting a conventional method in the field;

adding 6g, 8g and 10g of vetiver grass powder into each 100ml of PDA solid culture medium respectively, and measuring the content of lentinan in the hyphae by adopting a phenol-sulfuric acid method, wherein the content of the polysaccharide in the lentinan is 1.2g/L, 1.3g/L and 1.8g/L respectively;

adding 1-4 g of vetiver grass powder into 100ml of PDA liquid culture medium.

2. The method for increasing the content of polysaccharide in the mushroom mycelia using vetiver grass as claimed in claim 1, wherein the cleaned and dried vetiver grass is pulverized into powder, proportionally added to PDA solid medium, sterilized and then poured out of a flat plate, inoculated with mushroom strain, and cultured at 25-28 ℃ for 8-15 days to obtain mushroom mycelia.

3. The method for increasing the content of polysaccharide in the mushroom mycelia by using vetiver grass as claimed in claim 1, wherein the cleaned and dried vetiver grass is ground into powder, the powder is proportionally added into PDA liquid culture medium, sterilized and inoculated with mushroom strain, and shake-flask culture is carried out at 25-28 ℃ and 180 +/-20 r/min for 8-15 days to obtain the mushroom mycelia.