CN110117636B - Method for rapidly detecting whether Schizophyllum commune liquid strain reaches standard and verification method thereof - Google Patents
Method for rapidly detecting whether Schizophyllum commune liquid strain reaches standard and verification method thereof Download PDFInfo
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- CN110117636B CN110117636B CN201910383731.1A CN201910383731A CN110117636B CN 110117636 B CN110117636 B CN 110117636B CN 201910383731 A CN201910383731 A CN 201910383731A CN 110117636 B CN110117636 B CN 110117636B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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Abstract
A method for rapidly detecting whether a Schizophyllum commune liquid strain reaches the standard and a verification method thereof are provided, wherein a liquid culture medium is prepared, and then Schizophyllum commune liquid strain seed liquid is put into a fermentation tank for culture according to 2-4% of the mass of the culture medium in the tank to obtain fermentation liquid; taking 20-30ml of liquid in the tank by using a sterile bottle, shaking the bottle, pouring out, taking 50ml again for later use, measuring after using a pH meter for calibration, keeping the pH value unchanged for 5min, reading, and when the pH value reaches 5.4 +/-0.1, enabling the schizophyllum commune liquid strain to reach the use standard. The verification method comprises the steps of measuring the dry weight of hyphae, measuring the vitality of the hyphae and the diameter of the mycelium pellet, and determining that the end-point pH value of the fermentation liquor is maintained between 5.4 +/-0.1 when the dry weight is the maximum and the vitality of the mycelium pellet is the maximum. The invention realizes convenient monitoring of whether the liquid strain is qualified or not by using the end point pH value through a specific culture medium and a strict liquid strain culture method.
Description
Technical Field
The invention belongs to the technical field of edible fungus liquid strain detection methods, and particularly relates to a detection method capable of quickly detecting whether a Schizophyllum commune liquid strain meets use standards.
Background
Schizophyllum rare edible fungi are popular among many people, especially in Yunnan, and are health-care food materials for treating gynecological diseases, children night sweat and the like. Moreover, the Schizophyllum commune polysaccharide contained in the composition is a preferable emerging component for skin care and health care, and is also one of the material sources of the anti-tumor drugs. However, the cultivation is started late, the cultivation technology is immature, the cultivation scale is small, and the yield is difficult to meet the market demand. In recent years, with the continuous innovation of cultivation technology, the industrial cultivation of schizophyllum commune is also effective, and based on the results, the research on the liquid strain of schizophyllum commune is urgent, and the detection quality is crucial and must be strictly controlled due to the large risk of the liquid strain.
The research on the liquid strain of the schizophyllum commune in the prior art mainly focuses on the extraction of polysaccharide, the application research on the cultivation of the liquid strain of the schizophyllum commune is less, and the quality (whether the quality reaches the use standard) of the liquid strain of the schizophyllum commune has very important influence on the cultivation effect. At present, no standard can be referred to for detection of liquid strains of edible fungi, and the edible fungi are qualified as long as the hypha quantity, the hypha activity and the hypha ball size of the liquid strains are ensured to meet the production requirement. However, the process of detecting the hypha quantity, the hypha activity and the hypha ball size of the liquid strain is complicated, long and troublesome in time consumption, low in detection efficiency, high in professional quality requirement on detection personnel and not beneficial to large-scale production.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art and provide a quick detection method for a Schizophyllum commune liquid strain, so as to ensure that the liquid strain required by industrial cultivation of the Schizophyllum commune is effective and qualified, and provide a simple and easy-to-observe method for a non-professional person to better master and judge the Schizophyllum commune liquid strain.
The technical scheme adopted by the invention is as follows:
the method for rapidly detecting whether the Schizophyllum commune liquid strain reaches the standard comprises the following steps:
(1) preparing a liquid culture medium: the culture medium comprises the following components: bran 2%, glucose 1.5%, peptone 0.2%, KH2PO40.2％、MgSO40.15％；
(2) Performing liquid strain amplification culture, namely putting the seed liquid of the schizophyllum commune liquid strain into a fermentation tank containing a culture medium, wherein the putting amount is 2-4% of the mass of the culture medium in the tank, the pressure in the tank is maintained at 0.04-0.05Mpa, the temperature is kept at 24 ℃, and the culture is performed for 2-4d to obtain a fermentation liquid;
(3) detecting the pH value of the fermentation liquor: taking 20-30ml of liquid in the tank by using a sterile bottle, shaking the bottle, pouring out, taking 50ml again for later use, measuring after using a pH meter for calibration, keeping the pH value unchanged for 5min, reading, and when the pH value reaches 5.4 +/-0.1, enabling the schizophyllum commune liquid strain to reach the use standard.
The method for verifying whether the liquid strain of the schizophyllum commune reaches the standard or not quickly comprises the following steps:
(1) measurement of hyphal dry weight: filtering the rest fermentation liquid in the fermentation tank with two layers of gauze, cleaning with clear water for 2-3 times, placing the filtered mycelium into a culture dish with known weight after no water dripping, drying at 90 deg.C to constant weight, and weighing;
(2) and (3) measuring hypha activity: absorbing 1ml of strain by using a pipette under the aseptic condition, adding the strain into 9ml of sterile water, uniformly mixing, absorbing 1ml of strain on a flat plate, culturing for 24 hours in a constant-temperature incubator at 26 ℃, and then counting the colony germination number;
(3) measuring the diameter of the bacterial ball: picking representative 15 bacteria balls on filter paper by using toothpicks, measuring by using a vernier caliper after the water absorption is finished and the bacteria balls present the shape, and calculating the average diameter of the bacteria balls;
(4) determining the relationship among the fermentation end point pH value, the mycelium dry weight and the mycelium pellet activity, wherein the relationship is as follows: with the increase of pH value, the dry weight of hyphae shows a trend of increasing first and then decreasing, the initial pH value is between 5.5 and 6.0, and the final pH value of the fermentation liquid is maintained between 5.4 +/-0.1 when the dry weight is the maximum and the mycelium pellet activity is the most vigorous.
Compared with the prior art, the invention has the following advantages:
(1) the detection method is rapid and only needs 5 min;
(2) the detection method is reliable, and after repeated test and verification, the pH value is determined to be maintained at 5.4 +/-0.1;
(3) the technical requirement for detection is not high, only one pH meter is needed, and the non-professional person can operate the pH meter.
(4) The method adopts liquid strains, and compared with the method using solid strains, the method shortens the culture period by at least 10 days, and has good consistency of the age of the strains, fast spawn running and vigorous growth potential of hyphae;
(5) the invention utilizes the optimized liquid strain culture technology, shortens the culture period, can be used only for 2-4d, has good strain quality and is an important basis for ensuring production;
(6) the liquid strain of the schizophyllum commune cultured by the method can be directly applied to culture, the culture time is short, the fungus age is consistent, the hyphae germinate fast and the activity is good, the current situation that the liquid strain of the schizophyllum commune is only used for the research of polysaccharide extraction is changed, and a good foundation is laid for the industrial culture of the schizophyllum commune.
The invention realizes that whether the liquid strain is qualified or not is conveniently monitored by using the end point pH value through a specific culture medium and a strict liquid strain culture method, and the repeated verification proves that the tank-placing standard is reached when the pH value is between 5.4 +/-0.1.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. The materials, reagents and the like used are commercially available unless otherwise specified.
The method for rapidly detecting whether the Schizophyllum commune liquid strain reaches the standard comprises the following steps:
(1) preparing a liquid culture medium: the culture medium comprises the following components: bran 2%, glucose 1.5%, peptone 0.2%, KH2PO40.2％、MgSO40.15 percent, and the balance of water; accurately weighing, boiling bran into juice, filtering to remove residues, and fixing the volume. The liquid medium defined in the present invention is a prerequisite for achieving the object of the present invention,
(2) liquid strain amplification culture: preparing a culture medium according to the amount of the fermentation tank, putting the schizophyllum commune liquid strain seed liquid into the fermentation tank containing the culture medium, sterilizing at 121 ℃ and 0.1Mpa for 40min, cooling to room temperature after sterilization, and detecting the sterilization condition of the liquid strain culture medium. The Schizophyllum commune liquid strain seed liquid can be purchased or prepared by self, is prepared 4d in advance, is subjected to aseptic detection, is free from bacteria, mould and other mixed bacteria, and is used after indexes to be detected are qualified, the adding amount of the Schizophyllum commune liquid strain is 2-4% of the mass of a culture medium in a tank, the pressure in the tank is maintained at 0.04-0.05Mpa, the temperature is kept at 24 ℃, and the Schizophyllum commune liquid strain is cultured for 2-4d to obtain a fermentation liquid;
(3) detecting the pH value of the fermentation liquor: taking 20-30ml of liquid in the tank by using a sterile bottle, shaking the bottle, pouring out, taking 50ml again for later use, measuring after using a pH meter for calibration, keeping the pH value unchanged for 5min, reading, and when the pH value reaches 5.4 +/-0.1, enabling the Schizophyllum commune liquid strain to reach the use standard (the tank placing standard).
In order to verify whether the method for rapidly detecting whether the Schizophyllum commune liquid strain reaches the standard is correct, the method adopts the following steps:
(1) measurement of hyphal dry weight: filtering the rest fermentation liquid in the fermentation tank with two layers of gauze, cleaning with clear water for 2-3 times, placing the filtered mycelium into a culture dish with known weight after no water dripping, drying at 90 deg.C to constant weight, and weighing;
(2) and (3) measuring hypha activity: absorbing 1ml of strain by using a pipette under the aseptic condition, adding the strain into 9ml of sterile water, uniformly mixing, absorbing 1ml of strain on a flat plate, culturing for 24 hours in a constant-temperature incubator at 26 ℃, and then counting the colony germination number;
(3) measuring the diameter of the bacterial ball: picking representative 15 bacteria balls on filter paper by using toothpicks, measuring by using a vernier caliper after the water absorption is finished and the bacteria balls present the shape, and calculating the average diameter of the bacteria balls;
(4) and determining the relation between the pH value of the fermentation end point and the dry weight and the vitality of the mycelium pellet. Repeated tests show that the dry weight and activity of the hyphae are in certain relation with the pH value of the fermentation liquor, and the relation is as follows: with the increase of the pH value, the dry weight of the hyphae shows a trend of increasing first and then decreasing, when the initial pH value is between 5.5 and 6.0, the final pH value of the fermentation liquid is maintained between 5.4 plus or minus 0.1 when the dry weight is the maximum and the vitality of the hyphae is the maximum, namely when the pH value is maintained at 5.4 plus or minus 0.1, the hyphae quantity and the vitality of the hyphae are optimal.
Therefore, on the premise of ensuring that the liquid strain is free from other mixed bacteria pollution, according to the culture medium and the culture method, whether the liquid strain reaches the use standard can be balanced only by detecting the pH value.
Claims (2)
1. The method for rapidly detecting whether the Schizophyllum commune liquid strain reaches the standard is characterized by comprising the following steps of:
(1) preparing a liquid culture medium: the culture medium comprises the following components: bran 2%, glucose 1.5%, peptone 0.2%, KH2PO40.2％、MgSO40.15％；
(2) Performing liquid strain amplification culture, namely putting the schizophyllum commune liquid strain seed liquid into a fermentation tank containing a culture medium, wherein the putting amount is 2-4% of the mass of the culture medium in the tank, the pressure in the tank is maintained at 0.04-0.05Mpa, the temperature is kept at 24 ℃, and the liquid strain is cultured for 2-4d to obtain a fermentation liquid;
(3) detecting the pH value of the fermentation liquor: taking 20-30ml of liquid in the tank by using a sterile bottle, shaking the bottle, pouring out, taking 50ml again for later use, measuring after using a pH meter for calibration, keeping the pH value unchanged for 5min, reading, and when the pH value reaches 5.4 +/-0.1, enabling the schizophyllum commune liquid strain to reach the use standard.
2. A method of validating the method of claim 1, the method comprising:
(1) measurement of hyphal dry weight: filtering the rest fermentation liquid in the fermentation tank with two layers of gauze, cleaning with clear water for 2-3 times, placing the filtered mycelium into a culture dish with known weight after no water dripping, drying at 90 deg.C to constant weight, and weighing;
(2) and (3) measuring hypha activity: absorbing 1ml of strain by using a pipette under the aseptic condition, adding the strain into 9ml of sterile water, uniformly mixing, absorbing 1ml of strain on a flat plate, culturing for 24 hours in a constant-temperature incubator at 26 ℃, and then counting the colony germination number;
(3) measuring the diameter of the bacterial ball: picking representative 15 bacteria balls on filter paper by using toothpicks, measuring by using a vernier caliper after the water absorption is finished and the bacteria balls present the shape, and calculating the average diameter of the bacteria balls;
(4) determining the relationship among the fermentation end point pH value, the mycelium dry weight and the mycelium pellet activity, wherein the relationship is as follows: with the increase of pH value, the dry weight of hyphae shows a trend of increasing first and then decreasing, the initial pH value is between 5.5 and 6.0, and the final pH value of the fermentation liquid is maintained between 5.4 +/-0.1 when the dry weight is the maximum and the mycelium pellet activity is the most vigorous.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1306084A (en) * | 2000-01-18 | 2001-08-01 | 罗星野 | Artificially cultured schizophyllum sporophores and its culture process |
| CN101643759A (en) * | 2009-07-31 | 2010-02-10 | 中国农业科学院农产品加工研究所 | Method for preparing Schizophyllum commune Fr polysaccharides and dedicated culture medium thereof |
| CN102805335A (en) * | 2012-07-20 | 2012-12-05 | 黄晓青 | Method for preparing edible fungus health-care product |
| CN109355203A (en) * | 2018-11-27 | 2019-02-19 | 云南菌视界生物科技有限公司 | A kind of cultural method of schizophyllum commune liquid spawn |
-
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- 2019-05-09 CN CN201910383731.1A patent/CN110117636B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1306084A (en) * | 2000-01-18 | 2001-08-01 | 罗星野 | Artificially cultured schizophyllum sporophores and its culture process |
| CN101643759A (en) * | 2009-07-31 | 2010-02-10 | 中国农业科学院农产品加工研究所 | Method for preparing Schizophyllum commune Fr polysaccharides and dedicated culture medium thereof |
| CN102805335A (en) * | 2012-07-20 | 2012-12-05 | 黄晓青 | Method for preparing edible fungus health-care product |
| CN109355203A (en) * | 2018-11-27 | 2019-02-19 | 云南菌视界生物科技有限公司 | A kind of cultural method of schizophyllum commune liquid spawn |
Non-Patent Citations (2)
| Title |
|---|
| 裂褶菌液体发酵条件及发酵产物的药理活性研究;李雪;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20091115(第11期);第E057-14页 * |
| 裂褶菌的栽培研究;马布平 等;《内蒙古农业大学学报（自然科学版）》;20190131;第40卷(第1期);第11-21页 * |
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