CN106680266A - Method for testing mushroom dregs of industrialized pleurotus eryngii production - Google Patents
Method for testing mushroom dregs of industrialized pleurotus eryngii production Download PDFInfo
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Abstract
The invention relates to a method for testing the content of lignocelluloses in mushroom dregs of industrialized pleurotus eryngii production. The method comprises the following steps: (1) performing boiling treatment on the mushroom dregs in the industrialized pleurotus eryngii production by using a neutral detergent and anhydrous sodium sulfite, centrifuging and collecting residues, that is, neutral washed fiber, and testing total sugar of supernate by using a phenol sulfuric acid method; (2) treating the neutral washed mushroom dregs by using an acid detergent and anhydrous sodium sulfite, centrifuging and collecting residues, that is, acid washed fiber, and testing total sugar of supernate by using the phenol sulfuric acid method; and (3) dissolving the acid washed mushroom dregs by using a sulfuric acid solution, centrifuging and collecting residues, performing drying and incineration so as to obtain the content of lignin, centrifuging supernate, and testing total sugar by using the phenol sulfuric acid method. By adopting the method, the content of lignocelluloses can be accurately quantified, errors can be greatly reduced, and slight variation of the content of lignocelluloses in culture materials within a short term in the growth process of edible mushrooms can be obtained.
Description
Technical field
The invention belongs to Pleurotus eryngii industrial production field, the survey of bacteria residue in more particularly to a kind of Pleurotus eryngii industrial production
Determine method.
Background technology
Edible fungus industrial bottle is planted business and is primarily referred to as being cultivated throughout the year in air conditioner facility with plastic bottles, by cultivation
The mechanization and standardization production throughout the year stable in factory to realize edible fungi.Because mushroom house environment does not receive season in factory
Affect, realize the year-round supply of edible fungi, meet demand of the consumer to fresh food bacterium product.In view of edible fungi bottle is planted
The advantage of technology, at present Hypsizygus marmoreuss, Grifola frondosa, Pleurotus nebrodensis, Flammulina velutiper (Fr.) Sing, Pleurotus eryngii, HUAZIGU, agrocyb eaegerita, Hericium erinaceus (Bull. Ex Fr.) Pers. etc.
Bottle plant production be industrially widely used.
Pleurotus eryngii (Pleurotus eryngii Quel), also known as perverse celery picks up the ears.It is under the jurisdiction of Eumycota, Basidiomycetess, umbrella
Zoopagales, Pleurotaceae, pleurotus.Pleurotus eryngii bacterial context is plump, and quality is tender and crisp, particularly stem dense structure, solid, milky white, can be complete
Portion eats, and stem cunning more crisp than cap, tasty and refreshing, is referred to as " Pleurotus ostreatus king ", " dry scallop mushroom ", with happy almond flavor and such as
The mouthfeel of Carnis Haliotidiss, is adapted to fresh-keeping, processing, firmly gets liking for people.The sporophore list life of Pleurotus eryngii or all living creatures, cap width 2-
12cm, just in arch-shaped, after it is gradually open and flat, to infundibulate, there is silk-like sheen on surface to central scrobicula when ripe, smooths, is dried, thin
Threadiness, lid edge is involute when young, is in wavy or drastic crack after maturation；Bacterial context white, with almond, without galactopoiesiss；Lamella
Prolong life, intensive, slightly wide, milky, edge and both sides are flat, there is lamellula；Stem 2-8cm to 0.5-3cm, bias is raw or side is given birth to.
Pleurotus eryngii belongs to middle fruiting performances at low temperature mushroom, and fruit body development preference temperature encloses 10-15 DEG C.
Can be used in the assay method of bacteria residue at present generally using traditional Fan Shi washing methods, the drying transfer of sample sucking filtration
Process causes very big error to result, it is impossible to obtain during Growth of Pleurotus eryngii wood fibre cellulose content in compost in a short time
Minor variations, limit for during Growth of Pleurotus eryngii to lignocellulose using the research of process, therefore can not go deep into
Carry out Pleurotus eryngii industrial production recipe determination research, hinder the industrialization development of Pleurotus eryngii.
The content of the invention
The technical problem to be solved is to provide a kind of assay method of bacteria residue in Pleurotus eryngii industrial production, should
Method is capable of the content of accurate quantitative analysis lignocellulose, greatly reduces error, can obtain during edible fungi growth in a short time
The minor variations of wood fibre cellulose content in compost.
The assay method of bacteria residue in a kind of Pleurotus eryngii industrial production of the present invention, including：
(1) the bacteria residue Jing neutral detergents and anhydrous sodium sulfite during Pleurotus eryngii industrial is produced is (wooden for removing
Element) process is boiled, it is centrifuged, residue is collected, obtain neutral detergent fiber；Supernatant determines total sugar using Phenol sulfuric acid procedure；
(2) bacteria residue Jing acid detergents and anhydrous sodium sulfite after neutral detergent is processed, and residue is collected in centrifugation, is obtained
Acid detergent fiber；Supernatant determines total sugar using Phenol sulfuric acid procedure；
(3) Jing after sulfuric acid solution digestion, centrifugation is dried the bacteria residue after acidic cleaning, and ashing obtains content of lignin；From
Supernatant after the heart determines total sugar using Phenol sulfuric acid procedure.
Neutral detergent in the step (1) is consisted of：EDTA 0.0186g/ml, sodium borate 0.0068g/ml, ten
Sodium dialkyl sulfate 0.03g/ml, disodium hydrogen phosphate,anhydrous 0.00456g/ml, ethylene glycol 1%, pH value is 6.9~7.1.
The composition of the neutral detergent fiber in the step (1) includes hemicellulose, cellulose, lignin and silicate.
Acid detergent in the step (2) is consisted of：1mol/L contains the sulphuric acid of hexadecane trimethyl ammonium bromide
Solution.
The composition of the acid detergent fiber in the step (2) includes cellulose, lignin and silicate.
The anhydrous sodium sulfite is 1g with the mass volume ratio of neutral detergent or acid detergent:200ml.
Sulfuric acid solution percentage by volume in the step (3) is 72%.
Digestion temperature in the step (3) is 30 DEG C, and digestion time is 3h.
Ashing temperature in the step (3) is 550 DEG C, and ashing time is 3h.
Phenol sulfuric acid procedure in the step (1), (2) and (3) determines total sugar and is specially：Polysaccharide is first in the presence of sulphuric acid
Monosaccharide is hydrolyzed into, and is dehydrated generation alditol derivant, then generate orange-yellow compound with phenol, then with colorimetric method for determining.
Beneficial effect
(1) present invention is quantitative determined using Phenol sulfuric acid procedure to the polysaccharide in Digestive system after digestion, is then convert into
The content of lignocellulose, reaches accurate quantitative analysis, greatly reduces error, can obtain being cultivated in a short time during edible fungi growth
The minor variations of wood fibre cellulose content in material；
(2) present invention adopts ultracentrifugal method, and disengaging time shortens, efficiency is greatly improved；Therefore for albumen contains
Measure the bacteria residue of higher edible fungi advantageously, and error is little, and the minor variations of wood fibre cellulose content in sample can be carried out
Tracking, has a good application prospect.
Description of the drawings
Fig. 1 is the standard curve that Phenol sulfuric acid procedure determines total sugar in embodiment 1.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.In addition, it is to be understood that after the content for having read instruction of the present invention, people in the art
Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Scope.
Embodiment 1
First, solution is prepared
(1) neutral detergent solution
Accurately weigh 18.6g disodiumedetate (EDTA, C10H14O8Na2·2H2O, analyzes pure) and 6.8g boric acid
Sodium (Na2B4O7·10H2O, analyzes pure) beaker is put into, a small amount of distilled water is added, after heating for dissolving, add 30g dodecanes
Base sodium sulfate (C12H25NaO4S, analyzes pure) and 10ml ethylene glycol (C4H10O2, analyze pure), 4.56g disodium hydrogen phosphate,anhydrous
(Na2HPO4, analyze pure), it is diluted to 1000ml in volumetric flask, wherein pH value is about 6.9~7.1 (pH value does not typically need to adjust
It is whole).
(2) acidic cleaning liquid
Measure about 27.87ml concentrated sulphuric acids (to analyze pure, proportion 1.84,98%), add slowly and have been loaded with 500ml distilled water
In beaker, 1000ml volumetric flask constant volumes are injected after cooling, demarcated；Weigh 20g hexadecane trimethyl ammonium bromides (CTAB is analyzed pure)
1000ml 1N sulphuric acid is dissolved in, is filtered if necessary.
(3) 72% sulphuric acid are prepared
433.53g deionized waters are weighed in 1L glass beakers, is placed on magnetic stirring apparatuss, be stirred continuously, along Glass rod
It is adherent slowly to pour the concentrated sulphuric acids of 1199.68g 98% into.
(4) 5% phenol are prepared
80 grams of phenol (the pure redistillation reagent of analysis) plus 20 grams of water are allowed to dissolve, and can put lucifuge long term storage in refrigerator.Face
5% phenol is prepared with 80% phenol with front.(determine be both needed to now match somebody with somebody every time).
2nd, method
(1) factory culture Pleurotus eryngii flow process
Dispensing, bottling, sterilizing, cooling, inoculation, preculture, culture, mycelium stimulation, mycelia recovery, flower bud sporophore, develop, adopt
Receive, cut mushroom, packaging, dig bottle, respectively after sterilization, after mycelium stimulation, three bottles of samples are respectively taken after fruiting, claim weight in wet base, drying to claim dry weight,
Record water content, three bottles mix sampling, beat powder, sieve, and determine wood fibre cellulose content.
(2) prepared by standard curve
Glucose (105 DEG C, drying constant weight processed) 0.0100g (AR) is taken, with distillation water dissolution 100mL is settled to, taken clean
Dry test-tube 1-7, with liquid-transfering gun plus glucose solution 0mL, 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL, difference
Plus then distilled water adds 5% phenol 0.5mL to 1mL, adds 2.5mL98% concentrated sulphuric acids.Concussion is mixed, boiling water bath 15min,
Flowing water cooling, detects absorbance at 490nm, draw standard curve.(drawing standard curve when determining every time).
(3) neutral detergent solution washing
1.0g samples, add 100mL neutral detergent solutions, 0.5g anhydrous sodium sulfite in being placed in 100 DEG C of water-baths, to keep
Micro-boiling 60min, takes 2mL cleaning mixture 12000rpm centrifugation 5min, and supernatant Phenol sulfuric acid procedure determines total sugar, remainder
10000rpm is centrifuged 10min, abandons supernatant and collects residue.Composition includes hemicellulose, cellulose, lignin and silicate.
(4) acidic cleaning liquid washing
By neutral detergent residue as in 100ml volumetric flasks, 100mL acidic cleaning liquids are added, 0.5g anhydrous sodium sulfite,
In being placed in 100 DEG C of water-baths, micro-boiling 60min is kept, take 2mL cleaning mixture 12000rpm centrifugation 5min, supernatant Phenol sulfuric acid procedure
Total sugar is determined, remainder 10000rpm centrifugation 10min abandon supernatant and collect residue.Composition includes cellulose, lignin and silicic acid
Salt.
(5) 72% sulphuric acid digest
By acidic cleaning residue as in 100ml volumetric flasks, 10mL72% sulphuric acid is added, 3h is digested at 30 DEG C, dilution is fixed
Hold 100L, take 2mL Digestive systems 12000rpm centrifugation 5min, supernatant Phenol sulfuric acid procedure determines total sugar, remainder
10000rpm is centrifuged 10min, abandons supernatant, and clear water washs repeated centrifugation once (residual sulphuric acid makes residue be carbonized when preventing drying),
Collect residue.
(6) lignin is determined
Residue after the digestion of 72% sulphuric acid, is dried after constant weight and weighs, insoluble for pickling lignin and acid insoluble ash, Jing
Content is drawn after 550 DEG C of 3h ashing.
(7) computing formula
M (polysaccharide)=n (neutral detergent) * 180*0.9；
M (hemicellulose)=n (acidic cleaning) * 150*0.9；
M (cellulose)=n (digestion of 72% sulphuric acid) * 180*0.9；
M (lignin)=m (digestion of 72% sulphuric acid)-m (ash).
(8) interpretation of result
1. batch production Growth of Pleurotus eryngii situation
Batch production Growth of Pleurotus eryngii situation
2. ligocellulose degradation's situation：
1. the drafting of standard curve
Standard substance absorbance
| Standard substance (mg/ml) | 0 | 0.01 | 0.02 | 0.03 | 0.04 | 0.05 | 0.06 |
| Absorbance 1 | 0.047 | 0.138 | 0.232 | 0.315 | 0.397 | 0.468 | 0.566 |
| Absorbance 2 | 0.045 | 0.136 | 0.230 | 0.312 | 0.400 | 0.479 | 0.568 |
| Absorbance 3 | 0.045 | 0.139 | 0.232 | 0.316 | 0.401 | 0.485 | 0.572 |
| Meansigma methodss | 0.046 | 0.138 | 0.231 | 0.314 | 0.399 | 0.477 | 0.569 |
According to seven standard substance and correspondence absorbance values standard curve is made as shown in figure 1, showing that normal equation is：
Y=8.625x+0.0518, R2=0.9994.
2. Pleurotus eryngii bacteria residue determination of polysaccharide
After neutral detergent solution washing, total sugar content in cleaning mixture, initial data such as following table are determined using Phenol sulfuric acid procedure:
The meansigma methodss of three absorbances are substituted into into normal equation, total sugar concentration X=(Y-0.0518)/8.625 is drawn；
The total sugar mole that neutral detergent solution is obtained is：N (neutral detergent)=total sugar/180* volumes * extension rate=X*
V*a/180；
Show that polysaccharide quality is：M (polysaccharide)=n (neutral detergent) * 180*0.9；
Because sampling quality is 1g, show that polyoses content is：Polyoses content=polysaccharide quality * 100%=m*
100%；
Final sample polyoses content such as following table is obtained after three parallel sample results averageds：
3. Pleurotus eryngii bacteria residue hemicellulose level is determined
After acidic cleaning liquid washing, total sugar content in cleaning mixture, initial data such as following table are determined using Phenol sulfuric acid procedure：
The meansigma methodss of three absorbances are substituted into into normal equation, total sugar concentration X=(Y-0.0518)/8.625 is drawn；
The total sugar mole that acidic cleaning liquid is obtained is：N (acidic cleaning)=total sugar/180* volumes * extension rate=X*
V*a/180；
Show that hemicellulose quality is：M (hemicellulose)=n (acidic cleaning) * 150*0.9；
Because sampling quality is 1g, show that hemicellulose level is：Hemicellulose level=hemicellulose quality *
100%=m*100%；
Final sample hemicellulose level such as following table is obtained after three parallel sample results averageds：
4. Pleurotus eryngii bacteria residue content of cellulose is determined
After the digestion of 72% sulphuric acid, total sugar content in cleaning mixture, initial data such as following table are determined using Phenol sulfuric acid procedure:
The meansigma methodss of three absorbances are substituted into into normal equation, total sugar concentration X=(Y-0.0518)/8.625 is drawn；
72% sulphuric acid digests the total sugar mole for obtaining：N (digestion of 72% sulphuric acid)=total sugar/180* volumes * dilution times
Number=X*V*a/180；
Show that cellulose quality is：M (cellulose)=n (digestion of 72% sulphuric acid) * 180*0.9；
Because sampling quality is 1g, show that content of cellulose is：Content of cellulose=cellulose quality * 100%=
M*100%；
Final sample content of cellulose such as following table is obtained after three parallel sample results averageds：
5. Pleurotus eryngii bacteria residue content of lignin is determined
Quality is after sulphuric acid digestion：M (digestion of 72% sulphuric acid)=m (constant weight after drying)-m (crucible constant weight)；
The quality of ash deducts crucible constant weight for quality after ashing：M (ash)=m (after ashing)-m (crucible constant weight)；
Lignin quality then deducts ash quality i.e. for quality after the digestion of 72% sulphuric acid：M (lignin)=m (72% sulphuric acid
Digestion)-m (ash)=m (constant weight after drying)-m (crucible constant weight)-(m (after ashing)-m (crucible constant weight))=m is (permanent after drying
Weight)-m (after ashing), as a result such as following table：
6. result collects
Industrial pleurotus eryngii culture cycle lignocellulose changes of contents
Claims (10)
1. the lignocellulose content assaying method of bacteria residue during a kind of Pleurotus eryngii industrial is produced, including：
(1) the bacteria residue Jing neutral detergents and anhydrous sodium sulfite during Pleurotus eryngii industrial is produced boils process, centrifugation, collects
Residue, obtains neutral detergent fiber；Supernatant determines total sugar using Phenol sulfuric acid procedure；
(2) bacteria residue Jing acid detergents and anhydrous sodium sulfite after neutral detergent is processed, and residue is collected in centrifugation, obtains acidity
Washing fiber；Supernatant determines total sugar using Phenol sulfuric acid procedure；
(3) Jing after sulfuric acid solution digestion, centrifugation is dried the bacteria residue after acidic cleaning, and ashing obtains content of lignin；After centrifugation
Supernatant using Phenol sulfuric acid procedure determine total sugar.
2. the assay method of bacteria residue during a kind of Pleurotus eryngii industrial according to claim 1 is produced, it is characterised in that：It is described
Neutral detergent in step (1) is consisted of：EDTA 0.0186g/ml, sodium borate 0.0068g/ml, sodium lauryl sulphate
0.03g/ml, disodium hydrogen phosphate,anhydrous 0.00456g/ml, ethylene glycol 1%, pH value is 6.9~7.1.
3. the assay method of bacteria residue during a kind of Pleurotus eryngii industrial according to claim 1 is produced, it is characterised in that：It is described
The composition of the neutral detergent fiber in step (1) includes hemicellulose, cellulose, lignin and silicate.
4. the assay method of bacteria residue during a kind of Pleurotus eryngii industrial according to claim 1 is produced, it is characterised in that：It is described
Acid detergent in step (2) is consisted of：1mol/L contains the sulfuric acid solution of hexadecane trimethyl ammonium bromide.
5. the assay method of bacteria residue during a kind of Pleurotus eryngii industrial according to claim 1 is produced, it is characterised in that：It is described
The composition of the acid detergent fiber in step (2) includes cellulose, lignin and silicate.
6. the assay method of bacteria residue during a kind of Pleurotus eryngii industrial according to claim 1 is produced, it is characterised in that：It is described
Anhydrous sodium sulfite is 1g with the mass volume ratio of neutral detergent or acid detergent:200ml.
7. the assay method of bacteria residue during a kind of Pleurotus eryngii industrial according to claim 1 is produced, it is characterised in that：It is described
Sulfuric acid solution percentage by volume in step (3) is 72%.
8. the assay method of bacteria residue during a kind of Pleurotus eryngii industrial according to claim 1 is produced, it is characterised in that：It is described
Digestion temperature in step (3) is 30 DEG C, and digestion time is 3h.
9. the assay method of bacteria residue during a kind of Pleurotus eryngii industrial according to claim 1 is produced, it is characterised in that：It is described
Ashing temperature in step (3) is 550 DEG C, and ashing time is 3h.
10. the assay method of bacteria residue during a kind of Pleurotus eryngii industrial according to claim 1 is produced, it is characterised in that：Institute
State the measure total sugar of the Phenol sulfuric acid procedure in step (1), (2) and (3) to be specially：Polysaccharide is first hydrolyzed into list in the presence of sulphuric acid
Sugar, and generation alditol derivant is dehydrated, then orange-yellow compound is generated with phenol, then with colorimetric method for determining.
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