Fungus fermentation extract and application thereof to reduction of cadmium residue in rice grains

Abstract

The invention provides a fungus fermentation extract and application of the fungus fermentation extract to reduction of rice grain cadmium residues, the fungus is an endophytic fungus derived from northern wild plants, the fungus is Coprinus micaceus, and the strain preservation number is CGMCC No.4063; the fermentation extract of the coprinus comatus (CBS-D) has the effect of reducing cadmium residues in rice grains, in the environment with the pH value being slightly acid, when the fermentation extract of the coprinus comatus (CBS-D) is applied to spraying rice, the cadmium residues in the rice grains growing under the condition that the soil cadmium (Cd + 2) concentration ranges from 40 mg / Kg to 200 mg / Kg can be well controlled, and the rice grain cadmium removal rate is greatly increased. The method is easy to operate, safe and economical, new pollution is not introduced to the soil environment, and the method has good application prospects for safe production of rice in cadmium-polluted areas in China and yield increase and remediation of cadmium-polluted soil at the same time.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of reducing cadmium-contaminated rice grains, and particularly relates to a fungal fermentation extract and application of the fungal fermentation extract in preventing cadmium from remaining in rice grains. BACKGROUND

[0002] Cadmium, as a heavy metal with high toxicity, has a wide range of pollution sources, including not only naturally occurring cadmium elements in the natural environment, but also from mining, industrial wastewater discharge, industrial dust deposition and other human activities. These factors have jointly caused cadmium pollution in a large area of farmland, especially in rice fields.

[0003] The currently limited application of cadmium pollution control technology in rice fields is to apply a ceramic particle with cadmium adsorption function, with an initial investment of about 5000 yuan per mu, which can be used for 10 years. Regardless of the application conditions and stability of this technology, the cost investment cannot cover the balance between rice planting and income, and cannot fundamentally change the threat of cadmium pollution in rice fields to food safety. In addition, it has been found that the mycelium of some fungi has the effect of adsorbing cadmium, but in the application scenario of cadmium-contaminated rice fields, it is difficult to play an actual application effect. SUMMARY

[0005] Therefore, the present application aims to provide a fungal fermentation extract and application of the fungal fermentation extract in preventing cadmium from remaining in rice grains, so as to solve the problems of cadmium-contaminated rice grains, cadmium residue exceeding the standard affecting food safety, and persistent soil cadmium pollution being difficult to repair.

[0006] In order to achieve the above-mentioned application purpose, the present application provides the following technical solutions:

[0007] The present application provides 1, a fungal fermentation extract, characterized in that the fungus is Crystalliferous Coprinus, and the fermentation extract of the Crystalliferous Coprinus is a whole cell mixture extracted by ethanol. The active substances detected by a mass spectrometer include cyclic dipeptides formed by leucine and isoleucine, indole derivatives, piperazine, etc.

[0008] As a preferred, the application of the fungal fermentation extract in reducing cadmium residue in rice grains.

[0009] As a preferred, the fungal fermentation extract is applied in preventing rice grains from absorbing and accumulating harmful metal cadmium, and in a low-concentration cadmium pollution situation, the fungal fermentation extract mainly prevents the movement and enrichment of cadmium in rice plants to rice grains, and in a high-concentration cadmium situation, the fungal fermentation extract prevents rice grains from absorbing and accumulating harmful metal cadmium by enhancing the enrichment of harmful metal cadmium in rice roots and stems.

[0010] As preferred, the fermented extract of the Coprinus giganus is diluted to 5-1000 ng / ml in water solution to spray the rice leaves, while the rice soil is kept in the environment with PH value of 6-6.5.

[0011] As preferred, the extraction method of the fermented extract of the fungus comprises the following steps:

[0012] Step 1: strain activation and seed liquid preparation: the Coprinus giganus is inoculated from the slant medium to the PDA liquid medium for culture to obtain the seed liquid;

[0013] Step 2: preparation of corn and wheat bran solid culture medium: corn kernels are soaked in water, drained, and then the water content is adjusted to 65%-70%, wheat bran is added, and the solid fermentation bottle is divided and sterilized by high-pressure steam, and then cooled to room temperature for standby;

[0014] Step 3: solid fermentation culture: inoculation is performed according to the mass ratio of 1:10 of the seed liquid to the solid culture medium, and the culture is placed in a constant temperature incubator with relative humidity of 60%-65% for static fermentation, until the culture medium is covered with white mycelium and no contamination with miscellaneous bacteria, and the fermentation period is 25-30 days;

[0015] Step 4: solid fermentation material extraction: the fermented solid material is dried, crushed, and then sieved through a 40-60 mesh sieve; ethanol solution is added according to the solid-liquid ratio of 1:15, and water bath constant temperature oscillation extraction is performed for 1-2 h, followed by ultrasonic extraction; after standing and precipitating, the supernatant is taken; the residue is extracted once more, and the two supernatants are combined;

[0016] Step 5: concentration test of the extract: the combined supernatant is weighed to determine the concentration of the extract; through the above steps, the endophyte extract mother liquor is prepared, and the mother liquor concentration is 1-5 mg / ml.

[0017] As preferred, the culture process in step 1 is carried out at 25°C, 180 r / min oscillation culture for 72 h to obtain the seed liquid;

[0018] In step 2, the corn kernels are soaked in water for 72 hours, and the added wheat bran is 10% of the dry weight of the corn kernels; high-pressure steam sterilization is performed at 121°C for 120 min;

[0019] In step 4, the drying temperature is 60°C; the concentration of the ethanol solution is 20-30%, the water bath temperature is 60°C, and the oscillation frequency is 150 r / min; the ultrasonic extraction time is 30-40 min;

[0020] In step 5, the extraction rate of the endophyte extract mother liquor is 18-20% of the dried material after fermentation, and the concentration used is 1 mg / ml.

[0021] Compared with the prior art, the present application has the following beneficial effects:

[0022] The present application takes common corn bran as the main culture material, fully utilizes the nutritional characteristics of corn bran to promote the growth of the fungus and the synthesis of active substances. The fermentation extract of the fungus (CBS-D) has the effect of reducing the cadmium residue in rice grains. In an acidic soil environment, the fermentation extract (1 mg / ml, 5000 times dilution) of the fungus (CBS-D) is sprayed on the rice. After the rice matures, the whole plant (roots, straw, and ears) of the rice treated in different ways is harvested. The roots and straw are crushed and numbered, and the ears are threshed and numbered randomly. Then, the cadmium content is determined by a testing agency. The results show that the cadmium residue in the rice grains grown in the soil with a cadmium (Cd +2 ) concentration of 40 mg / Kg, 100 mg / Kg, and 200 mg / Kg can be well controlled by using the fermentation extract of the fungus (CBS-D), especially when the cadmium (Cd +2 ) concentration is in the range of 90-130 mg / Kg. The rice produces a stress response, and the cadmium (Cd +2 ) in the rice grains is extremely low. The present application is simple, safe, and economical, does not introduce new pollution to the soil environment, and has a good application prospect for safe production of rice in cadmium-polluted areas, simultaneous yield increase, and remediation of cadmium-polluted soil in China. DETAILED DESCRIPTION

[0023] The present application provides an extraction method of a fungal fermentation extract, which comprises the following steps:

[0024] Step 1: strain activation and seed liquid preparation: the fungus is inoculated from a slant medium to a PDA liquid medium, and is cultured at 25℃ and 180 r / min for 72 h to obtain a seed liquid;

[0025] Step 2: preparation of corn and bran solid culture medium: corn kernels are soaked in water for 72 h, drained, and then the water content is adjusted to 65%-70%. Bran is added at 10% of the dry weight of the corn kernels, and the mixture is divided into solid fermentation bottles, sterilized at 121℃ for 120 min, and cooled to room temperature for standby;

[0026] Step 3: solid fermentation culture: inoculation is performed at a seed liquid to solid culture medium ratio of 1:10, and the mixture is placed in a constant temperature incubator with a relative humidity of 60%-65% for static fermentation until the culture medium surface is covered with white mycelium and no contamination is observed. The fermentation period is 25-30 days;

[0027] Step 4: solid fermentation extraction: the fermented solid material is dried at 60°C, crushed and sieved through a 40-60 mesh sieve; 20-30% ethanol solution is added at a material-to-liquid ratio of 1:15, and the mixture is extracted in a 60°C water bath for 1-2 hours with constant temperature shaking at a frequency of 150 r / min, followed by ultrasonic extraction for 30-40 minutes; after standing and sedimentation, the supernatant is taken; the residue is extracted once more, and the supernatants from the two extractions are combined;

[0028] Step 5: concentration test of the extract: the combined supernatant is tested for concentration using the oven-drying method, with the unit being mg / ml; through the above steps, the endophyte extract mother liquor for application is prepared; the extraction rate is usually 18-20% of the dried material after fermentation, and the concentration used is 1 mg / ml.

[0029] Alternatively, the PDA liquid medium is prepared as follows:

[0030] (1) Take 200g of fresh peeled potatoes and cut them into 1cm 3 pieces;

[0031] (2) Add 1L of distilled water, boil for 30 minutes, and then filter through double-layered gauze;

[0032] (3) Add 20g of glucose to the filtrate, which is then dissolved by adding distilled water to make up to 1L (pH 6.5-7.0).

[0033] Specifically, ordinary corn bran is used as the main solid culture material to fully utilize the nutritional characteristics of corn bran to promote the growth of the fungus and the synthesis of active substances.

[0034] In this embodiment, the fungus is Coprinus acus, and the fermentation extract of the fungus is a whole-cell extract of ethanol. The dry matter concentration of this ethanol extract is very low, generally less than 10 mg / ml; the active substances include cyclic dipeptides formed by leucine and isoleucine, indole derivatives, piperazine, etc.; all components are detected by high-performance liquid chromatography, and the active components are collected and prepared by medium-pressure preparative liquid chromatography.

[0035] Step 1: medium-pressure liquid chromatography preparative chromatography purification and collection: the fermentation extract mother liquor is injected into a C18 reverse-phase preparative column (250mm x 20mm, 5μm), with 0.1% formic acid aqueous solution (A phase) and acetonitrile (B phase) as the mobile phase, and the gradient program is as follows: 0-10min, 5% B; 10-30min, from 5% to 30% B; 30-40min, from 30% to 50% B; 40-50min, from 50% to 95% B; 50-60min, maintain 95% B; control the flow rate at 8-10mL / min, the detection wavelength at 254-260nm, and the column temperature at 25-30°C, and monitor the elution curve in real time.

[0036] Step 2: elute under the above conditions, collect the elution peak components with retention time of 18-22 min (leucine-isoleucine cyclic dipeptide), 28-32 min (indole derivative), and 38-42 min (piperazine) respectively, and then concentrate the target components to dryness by a rotary evaporator at 45°C under reduced pressure to obtain the high-purity fungal fermentation extract.

[0037] The leucine-isoleucine cyclic dipeptide, indole derivative, piperazine and other components are the mass spectrometry detection results; the components collected from the high-purity fungal fermentation extract are detected for biological activity in the later stage, and neither the single nor the mixed test reaches the activity of the original solution; it is indicated that the product obtained by medium-pressure liquid phase separation is insufficient to represent the biological activity of the ethanol extraction original solution; since the amount of other components is too small, the further research on the effective components cannot be carried out at present.

[0038] In the present embodiment, the used crystal grain Coprinus micaceus is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Hua-yang District, Beijing, with the preservation number of CGMCC No. 4063.

[0039] In the present embodiment, the fungal fermentation extract prevents the application of cadmium to the cadmium residue in rice grains.

[0040] In the present embodiment, the fungal fermentation extract is applied to prevent the rice grains from absorbing and accumulating the harmful metal cadmium by enhancing the enrichment of the harmful metal cadmium in the rice roots and stems, but can prevent the rice grains from absorbing and accumulating the harmful metal cadmium.

[0041] In the present embodiment, the application of the fungal fermentation extract dilutes the fermentation extract of the crystal grain Coprinus micaceus to 5-1000 ng / mL aqueous solution to spray the rice, while keeping the rice soil in the environment with PH of 6-6.5.

[0042] In other embodiments, the fermentation extract of the crystal grain Coprinus micaceus is diluted to 10, 100, 200, 400, 600, 800 ng / mL aqueous solution to spray the rice leaf surface, and other conditions remain unchanged, which can achieve the purpose of the present application.

[0043] The technical solutions provided by the present application will be described in detail in combination with the embodiments below, but they should not be understood as limitations on the scope of protection of the present application.

[0044] Embodiment

[0045] The fermentation extract of the crystal grain Coprinus micaceus (CBS-D) is adjusted to a preparation with a concentration of 1 mg / mL, and after being diluted 5000 times with water, the rice leaf surface is sprayed at the rice transplanting stage, the tillering late stage, and the flowering stage; the pot soil is weighed in advance, and the soil cadmium (Cd 2+) concentration was adjusted to 40mg / kg (moderate pollution level), 100mg / kg (high pollution level), 200mg / kg (extremely high pollution level, crop growth limited) three levels; after irrigation, the pot soil environment was adjusted to pH 6, 6.5 and 7 using sulfuric acid; the pH value was monitored regularly during the test, and the pH value was corrected to the preset value, and the standard fertilizer amount was applied.

[0046] The control group was not applied with the fermented extract of Coprinus cinereus (CBS-D), and the cadmium (Cd) concentration in the soil was 40mg / kg. 2+ ) concentration was adjusted to 40mg / kg (moderate pollution level), 100mg / kg (high pollution level), 200mg / kg (extremely high pollution level, crop growth limited) three levels; after irrigation, the pot soil environment was adjusted to pH 6, 6.5 and 7 using sulfuric acid.

[0047] All tests were 10 pots for each treatment, normal fertilization and watering management; at harvest, the relatively normal plants were taken, and the rice roots, stems and grains were sampled and dried for storage for cadmium content detection; the cadmium concentration in the soil was 200mg / kg, which was close to the tolerance limit of rice, the plant growth was weak, and the seed setting amount was greatly reduced, and the control was more severely damaged. The test samples were entrusted to the Ministry of Agriculture and Rural Affairs of Tianjin Environmental Protection Testing Center (national level qualification certification) for testing, and the test data are shown in Table 1 below.

[0048] Table 1 Test data

[0049]

[0050]

[0051] The data in Table 1 shows that

[0052] 1. Without using CBS-D bacterial agent, when the cadmium pollution content in the soil reaches 40mg / Kg concentration, the cadmium content in the rice grain basically exceeds the national food safety standard, i.e. 1mg / Kg; far exceeding the foreign rice standard of 0.15mg / Kg.

[0053] 2. Under the condition of each concentration of cadmium pollution level, using CBS-D bacterial agent treatment, except that the cadmium content in the rice grain under the condition of 200mg / Kg ultra-high concentration of cadmium and pH 7.0 exceeds the national safety standard 1mg / Kg of rice, the cadmium in the rice grain under other pH conditions is far below the national safety standard or even the more stringent standard abroad.

[0054] 3. In soil with an ultra-high cadmium concentration of 200 mg / Kg, rice roots exhibited abnormal cadmium accumulation compared to medium and high cadmium pollution levels. In soils with cadmium concentrations of 40 mg / Kg and 100 mg / Kg, the cadmium accumulation in rice roots treated with CBS-D microbial agent exceeded that in untreated rice roots. In the cadmium treatment at a relatively severe pollution level of 100 mg / Kg, the cadmium accumulation level in rice roots treated with CBS-D microbial agent increased nearly 10 times compared to untreated rice roots. Removing approximately 500 kg of cadmium-rich rice roots per acre can remove approximately 35 grams of pure cadmium, providing a new approach for soil remediation and cadmium resource recovery.

[0055] 4. Especially soil cadmium (Cd) +2 When the concentration of cadmium (Cd) is in the range of 90-130 mg / kg, rice exhibits a stress response, and the concentration of cadmium (Cd) in the rice grains increases. +2 The cadmium content is extremely low, while it is high in rice straw and roots.

[0056] 5. From the perspective of cadmium-contaminated farmland remediation, the use of CBS-D microbial agent can almost solve the existing cadmium pollution problem in paddy fields in my country.

[0057] This invention uses ordinary corn and wheat bran as the main culture material, making full use of the nutritional characteristics of corn and wheat bran to promote the growth of *Coprinus granulosus* and the synthesis of active substances.

[0058] The fermentation extract of *Coprinus granulosus* (CBS-D) of this invention promotes rice growth and yield. In a slightly acidic environment, spraying rice with the fermentation extract of *Coprinus granulosus* (CBS-D) reduces soil cadmium (Cd). +2 This invention effectively controls cadmium residues in rice grains grown under conditions with concentrations ranging from 40 to 200 mg / kg. It is simple to operate, safe, economical, and does not introduce new pollution into the soil environment. It has great application prospects for safe rice production in cadmium-polluted areas of my country, as well as for increasing yields and remediating cadmium-polluted soils.

Claims

1. A fungal fermentation extract, characterized in that, The fungus is Coprinus micaceus, and the fermentation extract of Coprinus micaceus is a whole cell mixture capable of being extracted by ethanol, and the main active substances are cyclo-dipeptide formed by leucine and isoleucine, indole derivatives and piperazine.

2. The fungal fermentation extract of claim 1, characterized in that, The Coprinus micaceus is Coprinus micaceus, which is preserved in China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 4063.

3. Use of the fermentation extract of the fungus according to claim 1 for reducing cadmium residue in rice grains.

4. Use according to claim 3, characterized in that, The use of the fermentation extract of the fungus for preventing the absorption and accumulation of harmful metal cadmium in rice grains is achieved by enhancing the enrichment of harmful metal cadmium in the root system and stem of rice, thereby preventing the absorption and accumulation of harmful metal cadmium in rice grains.

5. The use of a fungal fermentation extract according to claim 3, characterized in that, The fermentation extract of the Coprinus micaceus is diluted to 5-1000 ng / ml aqueous solution to spray the leaves of the rice, and the soil of the rice is kept in an environment with a pH of 6-6.

5.

6. The extraction method of the fungal fermentation extract as set forth in claim 1, characterized by, The extraction method comprises the following steps: Step 1: strain activation and seed liquid preparation: the Coprinus micaceus is inoculated from the slope medium to the PDA liquid medium for culture to obtain the seed liquid; Step 2: preparation of corn and wheat bran solid culture medium: corn kernels are soaked in water, drained, and then the water content is adjusted to 65%-70%, wheat bran is added, and the solid fermentation bottle is sterilized by high-pressure steam, and then cooled to room temperature for standby; Step 3: solid fermentation culture: inoculation is performed according to the mass ratio of seed liquid to solid culture medium of 1:10, and the culture is placed in a constant temperature incubator with a relative humidity of 60%-65% for static fermentation, until the culture medium is covered with white mycelium and there is no contamination of miscellaneous bacteria, and the fermentation period is 25-30 days; Step 4: solid fermentation extract: the fermented fixed material is dried, crushed, and then sieved through a 40-60 mesh sieve; ethanol solution is added according to the solid-liquid ratio of 1:15, and water bath constant temperature oscillation extraction is performed for 1-2 h, followed by ultrasonic extraction; after standing and precipitating, the supernatant is taken; the residue is extracted once more, and the supernatants of the two times are combined; Step 5: concentration test of the extract: the concentration of the extract is determined by the weighing method; through the above steps, the endophyte extract mother liquor for application is prepared, and the mother liquor concentration is 1-5 mg / ml.

7. The extraction method of the fermentation extract of the fungus according to claim 6, wherein the culture process in step 1 is carried out at 25°C with 180 r / min oscillation for 72 h to obtain the seed liquid; in step 2, the corn kernels are soaked in water for 72 hours, and the added wheat bran is 10% of the dry weight of the corn kernels; high-pressure steam sterilization is performed at 121°C for 120 min; in step 4, the drying temperature is 60°C; the concentration of the ethanol solution is 20-30%, the water bath temperature is 60°C, and the oscillation frequency is 150 r / min; the subsequent ultrasonic extraction time is 30-40 min; in step 5, the extraction rate of the endophyte extract mother liquor is 18-20% of the dried material after fermentation, and the concentration used is 1 mg / ml.

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