CN104914025B - A kind of method of quick detection lucidum spore powder miospore value - Google Patents
A kind of method of quick detection lucidum spore powder miospore value Download PDFInfo
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- CN104914025B CN104914025B CN201510295671.XA CN201510295671A CN104914025B CN 104914025 B CN104914025 B CN 104914025B CN 201510295671 A CN201510295671 A CN 201510295671A CN 104914025 B CN104914025 B CN 104914025B
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Abstract
The present invention relates to the assay method of the spore value of the assay method of fungal spore broken wall situation, especially Reishi sporule.The present invention is a kind of method for the spore value for determining Reishi sporule, it, which is characterized by, takes conidia powder B to be measured that appropriate suspension is made, after constant volume, it is well mixed, in the micro- Microscopic observation statistical unit visual field intact spore number and exosporium-broken spore number of tool C6 eyepiece micrometers, the percent value that spore value i.e. intact spore number accounts for intact spore number and exosporium-broken spore number summation is calculated.The advantage of the invention is that being counted using C6 micrometers to the area of finely dispersed conidia powder intact spore and exosporium-broken spore, purpose simple to operate, cheap, that testing result is representative is reached.
Description
Technical field
, especially can quick detection breaking trachytectum of glossy ganoderma the present invention relates to a kind of fungal spore powder miospore value detection method
Afterwards and without with the spore value in the non-ganoderma spove powder of batch, belong to the field of Chinese medicines.
Background technology
Reishi sporule is that ganoderma lucidum is thin in growth and maturity phase, avette reproduction ejected from glossy ganoderma lamella and its small
Born of the same parents, and the seed of ganoderma lucidum.It has condensed the elite of ganoderma lucidum, has the whole inhereditary materials and health-care effect of ganoderma lucidum.Lucidum spore powder
Shell include two layers of sporoderm being made up of very hard chitin and glucan；Sporoderm quality is hard, has acid and alkali-resistance, resists
The properties such as oxidation, pressure-resistant heat resistanceheat resistant, the anti-digestion of resistance to enzyme, limit human body and nutritional ingredient in Reishi sporule are absorbed.Sporoderm
After broken or elimination, the active ingredient wrapped tightly by sporoderm farthest could directly be absorbed by human body stomach.There is data
Show, the lucidum spore powder of broken wall is advantageous to the dissolution of the compositions such as triterpene, Thick many candies, crude fat, wherein Thick many candies content ratio not
Ganoderma spove powder is higher by 70%.If directly eat non-broken wall lucidum spore powder, human body be only capable of using 12% or so it is effective
Composition, and the lucidum spore powder after broken wall is taken, utilization rate of active components is up to more than 95%.Therefore, ganoderma spove powder
Can the size of sporoderm-broken rate be exactly that utilize the key point of effective component of ganoderma lucidum spore powder to the full extent.It is numerous in the market
Lucidum spore powder, sporoderm-broken rate is often by as the important indicator for weighing this product quality.
At present, the quality standard of ganoderma spove powder is mainly sporoderm-broken rate, the measure Primary Reference Ministry of Agriculture of sporoderm-broken rate
Standard《The measure of NY/T1677-2008 ganoderma spove powder sporoderm-broken rates》And national standard《GB/T29344-2012 ganoderma lucidums
Conidia powder pick and process technical specification》Appendix A " assay method of ganoderma spove powder sporoderm-broken rate ".In the two standards,
Sporoderm-broken rate measuring principle is all that the spore of non-broken wall is counted by blood counting chamber, is then calculated contained by Unit Weight
The quantity of intact spore, utilizes formula
Sporoderm-broken rate of spore（%）=（Complete spore before complete spore/broken wall after 1- broken walls）×100%
Calculate " sporoderm-broken rate " of spore.The standard curve that need to be established between lucidum spore powder quality and spore number, profit
The spore number with non-spore powder with crushed sporoderm in batch certain mass spore powder with crushed sporoderm to be measured is determined with this standard curve, obtains broken wall
The sporoderm-broken rate of conidia powder.But such a method need to be with non-spore powder with crushed sporoderm in batch spore powder with crushed sporoderm to be measured, and in real life
Consumer's purchase is ganoderma spove powder, can not obtain the non-ganoderma spove powder of same batch, and then can not be broken to it
Wall rate is detected, thus the present invention proposes to detect its spore value.
In addition to blood counting chamber method, the assay method of the sporoderm-broken rate of open report also has water load combination microtechnic at present
Method, suspension method combination physics technology detecting method and chemical fingerprint detection method.But the inspection of chemical composition analysis method
It is complicated to survey step, instrument is expensive, and testing cost is higher.
The content of the invention
It is an object of the invention to overcome above-mentioned weak point of the prior art, there is provided one kind makes the averagely scattered journey of spore
Degree is higher, the method for more accurately determining ganoderma spove powder spore value.Spore value is in ganoderma spove powder
Intact spore number accounts for the percent value of intact spore number and exosporium-broken spore number summation.Because exosporium-broken spore number can not count, use
C6 type eyepiece micrometers, are counted to its area, and small lattice internal substance is designated as 1 small lattice more than 1/2, and discontented 1/2 is designated as 0
Small lattice, lucky 1/2 is designated as 0.5 small lattice.To non-wall-broken ganoderma spore and, wall-broken ganoderma spore counts respectively, non-broken wall spirit
Ganoderma lucidum spore number accounts for non-wall-broken ganoderma spore number and the mean percent ratio of exosporium-broken spore number summation, as spore value.
The present invention is a kind of method of the spore value of quick detection Reishi sporule, it is characterised in that：
（1）Take the lucidum spore powder B of spore value to be measured, be dried to constant weight, and using a certain amount of chloraldurate test solution with
Repeatedly softly ground well on glass mortar, ultrasound mix, after be settled to same volume, be well mixed, obtain corresponding suspension；
（2）Take above-mentioned lucidum spore powder suspension point to drop on slide, under the microscope, use C6 type eyepiece micrometers
Chi, small lattice number shared by intact spore and broken spore is read respectively；Small lattice include intact spore or broken spore is designated as more than 1/2
1 small lattice, discontented 1/2 is designated as 0 small lattice, and lucky 1/2 is designated as 0.5 small lattice；Utilize formula
Spore value（%）=[intact spore number ÷（Intact spore number+exosporium-broken spore number）]×100%
Calculate spore value；Wherein, spore value refers to that the intact spore number in ganoderma spove powder accounts for intact spore number
With the percent value of exosporium-broken spore number summation, intact spore number refers to the grid shared by intact spore, and exosporium-broken spore number refers to brokenly
Grid shared by broken spore.And using conidia powder A spore value as judgment criteria.
The present invention is chloraldurate from suspension, and after conidia powder is uniformly dispersed, quantitative point sample is in slide, through experiment
Compare, it is good compared with zinc sulfate test solution and other effects from the dispersion effect of chloraldurate test solution conidia powder, because its density and viscosity are favourable
In physical stability of the spore in suspension, but this is not the most important reason of the present invention.
For accurate measurement, used spore powder with crushed sporoderm need to be the ganoderma spove powder for being not added with any auxiliary material, and
Impurity should be removed, 60 DEG C are dried to constant weight.
Reassembled to increase dispersiveness and reduction spore of the spore in chloraldurate, machinery is carried out after constant volume and is stirred
Mix, such as ultrasound mixes.
After breaking trachytectum, the spore count of contained complete non-broken wall is less in the single visual field, for accurate measurement, institute
The amount of the spore powder with crushed sporoderm taken should suitably increase, if conidia powder amount is too many, spore powder with crushed sporoderm is assembled on slide, can not under the visual field
Form individual layer conidia powder.
The advantage of the invention is that immobilized using same lucidium spore powder wall breaking and intact spore number, in conidia powder suspension
In dispersed, simple to operate cheap, testing result representative purpose accurate so as to reach technology.
Brief description of the drawings
Spore distribution (note under Fig. 1 microscopes in zinc sulfate ethanol solution：A. non-wall-broken ganoderma spore; B.
Wall-broken ganoderma spore).
Non- exosporium-broken spore distribution (note under Fig. 2 microscopes after the processing of this patent method：A. non-wall-breaking lucidum spore
Son;B. wall-broken ganoderma spore).
Exosporium-broken spore distribution (note under Fig. 3 microscopes after the processing of this patent method：A. non-wall-breaking lucidum spore
Son;B. wall-broken ganoderma spore).
C6 eyepiece micrometers miospore distribution (note under Fig. 4 microscopes：A. non-wall-broken ganoderma spore;B. broken wall
Reishi sporule).
The spore value analysis result of sample in Fig. 5 embodiments 1.
The spore value analysis result of sample in Fig. 6 embodiments 2.
The spore value analysis result of sample in Fig. 7 embodiments 3.
The spore value analysis result of sample in Fig. 8 embodiments 4.
Embodiment
Specific steps and condition of the invention further explained below：
Embodiment 1：
1st, spore suspension is prepared
（1）Take 4 parts（The sample of different manufacturers or same producer's different batches is respectively derived from, is shown in Table 1, sample number into spectrum is
P01-P04）The lucidum spore powder A of appropriate known sporoderm-broken rate（Broken wall Shuai≤98%）With commercially available unknown sporoderm-broken rate conidia powder B（Spore
Sub- powder B uses conventional method, i.e. national standard《GB/T29344-2012 lucidum spore powder pick and process technical specifications》Appendix A
" assay method of ganoderma spove powder sporoderm-broken rate ", measures its sporoderm-broken rate>98%；1 is shown in Table, sample number into spectrum P05), in baking oven
Dried 5 hours at 60 DEG C.
（2）Accurate quantification weighs the conidia powder A and B of drying（W=0.04g）.
（3）Above-mentioned conidia powder is put into diameter 5mm glass mortar, repeatedly spore is gently ground well on a small quantity with chloraldurate test solution
Sub- powder disperses its agglomerate as far as possible, and with chloraldurate constant volume into 5ml volumetric flasks, then ultrasonic 20min, make spore in suspension
In it is fully dispersed.
2nd, observation and counting
（1）The above-mentioned detection spore suspensions of 10 μ l are drawn with liquid-transfering gun, the edge of cover glass is placed in, liquid is slowly oozed
Enter, unnecessary liquid is absorbed with blotting paper, and after waiting a moment, spore, as shown in Figure 2,3, spore are observed under 400 power microscopes
It is average scattered.
（2）Under the microscope of 10 × 40 multiples, 5 × 5mm on C6 micrometers is read respectively2In big lattice intact spore number and
Exosporium-broken spore number, moving stage scale, 25 visuals field are made to be evenly distributed in whole slide, the average value in 25 visuals field is spore
Value.Parallel 6 slides of every part of prepare liquid, calculate their average, NAAnd NB。
3rd, data calculate
（1）Spore value（%）=[intact spore number ÷（Intact spore number+exosporium-broken spore number）]×100%
Intact spore number is small lattice number shared by intact spore, and exosporium-broken spore number is small lattice number shared by exosporium-broken spore, tool
Body result is see Fig. 5
The spore value analysis result of Fig. 5 samples（See Figure of description）
Note：Sample source is as follows：P01（Sichuan Fu Zheng medicine companies Co., Ltd, lot number：140801）；P02（Chengdu
Kang Hua pharmaceutcal corporation, Ltd, lot number：L140701）；P03（Chengdu Kanghua Pharmaceutical Co., Ltd., lot number：L131101）；P04
（Chengdu Kanghua Pharmaceutical Co., Ltd., lot number：L140101）；P05（Lotus pond Chinese Medicinal Materials Markets）.
From the data in Fig. 5, using detection method provided by the invention can quick detection go out the spore value of sample,
And result is accurate, its corresponding sporoderm-broken rate of broken wall situation of Reishi sporule is reacted in error range, it is simple to operate low
It is honest and clean, there is promotional value.
Embodiment 2：
1st, spore suspension is prepared
（1）Take the lucidum spore powder C of appropriate known sporoderm-broken rate（Non- broken wall）, dried 5 hours at 60 DEG C of baking oven.
（2）Accurate quantification weighs the conidia powder C of drying（W=0.04g）.
（3）Above-mentioned conidia powder is put into diameter 5mm glass mortar, repeatedly spore is gently ground well on a small quantity with chloraldurate test solution
Sub- powder disperses its agglomerate as far as possible, and with chloraldurate constant volume into 5ml volumetric flasks, then ultrasonic 20min, make spore in suspension
In it is fully dispersed.
2nd, observation and counting
（1）The above-mentioned detection spore suspensions of 10 μ l are drawn with liquid-transfering gun, the edge of cover glass is placed in, liquid is slowly oozed
Enter, unnecessary liquid is absorbed with blotting paper, and after waiting a moment, spore, as shown in Figure 2,3, spore are observed under 400 power microscopes
It is average scattered.
（3）Under the microscope of 10 × 40 multiples, 5 × 5mm on C6 micrometers is read respectively2In big lattice intact spore number and
Exosporium-broken spore number, moving stage scale, 25 visuals field are made to be evenly distributed in whole slide, the average value in 25 visuals field is spore
Value.Parallel 6 slides of every part of prepare liquid, calculate their average, NC。
4th, data calculate
（1）Spore value（%）=[intact spore number ÷（Intact spore number+exosporium-broken spore number）]×100%
Intact spore number is small lattice number shared by intact spore, and exosporium-broken spore number is small lattice number shared by exosporium-broken spore, tool
Body result is see Fig. 6
The spore value analysis result of Fig. 6 samples（See Figure of description）
Embodiment 3：
1st, spore suspension is prepared
（1）Take the lucidum spore powder D of appropriate known sporoderm-broken rate（Sporoderm-broken rate 50%）, dried 5 hours at 60 DEG C of baking oven.
（2）Accurate quantification weighs the conidia powder D of drying（W=0.04g）.
（3）Above-mentioned conidia powder is put into diameter 5mm glass mortar, repeatedly spore is gently ground well on a small quantity with chloraldurate test solution
Sub- powder disperses its agglomerate as far as possible, and with chloraldurate constant volume into 5ml volumetric flasks, then ultrasonic 20min, make spore in suspension
In it is fully dispersed.
2nd, observation and counting
（1）The above-mentioned detection spore suspensions of 10 μ l are drawn with liquid-transfering gun, the edge of cover glass is placed in, liquid is slowly oozed
Enter, unnecessary liquid is absorbed with blotting paper, and after waiting a moment, spore, as shown in Figure 2,3, spore are observed under 400 power microscopes
It is average scattered.
（4）Under the microscope of 10 × 40 multiples, 5 × 5mm on C6 micrometers is read respectively2In big lattice intact spore number and
Exosporium-broken spore number, moving stage scale, 25 visuals field are made to be evenly distributed in whole slide, the average value in 25 visuals field is spore
Value.Parallel 6 slides of every part of prepare liquid, calculate their average ND。
5th, data calculate
（1）Spore value（%）=[intact spore number ÷（Intact spore number+exosporium-broken spore number）]×100%
Intact spore number is small lattice number shared by intact spore, and exosporium-broken spore number is small lattice number shared by exosporium-broken spore, tool
Body result is see Fig. 7
The spore value analysis result of Fig. 7 samples（See Figure of description）
Embodiment 4：
1st, spore suspension is prepared
（1）Take the lucidum spore powder E of appropriate known sporoderm-broken rate（Sporoderm-broken rate 75%）, dried 5 hours at 60 DEG C of baking oven.
（2）Accurate quantification weighs the conidia powder E of drying（W=0.04g）.
（3）Above-mentioned conidia powder is put into diameter 5mm glass mortar, repeatedly spore is gently ground well on a small quantity with chloraldurate test solution
Sub- powder disperses its agglomerate as far as possible, and with chloraldurate constant volume into 5ml volumetric flasks, then ultrasonic 20min, make spore in suspension
In it is fully dispersed.
2nd, observation and counting
（1）The above-mentioned detection spore suspensions of 10 μ l are drawn with liquid-transfering gun, the edge of cover glass is placed in, liquid is slowly oozed
Enter, unnecessary liquid is absorbed with blotting paper, and after waiting a moment, spore, as shown in Figure 2,3, spore are observed under 400 power microscopes
It is average scattered.
（5）Under the microscope of 10 × 40 multiples, 5 × 5mm on C6 micrometers is read respectively2In big lattice intact spore number and
Exosporium-broken spore number, moving stage scale, 25 visuals field are made to be evenly distributed in whole slide, the average value in 25 visuals field is spore
Value.Parallel 6 slides of every part of prepare liquid, calculate their average NE。
6th, data calculate
（1）Spore value（%）=[intact spore number ÷（Intact spore number+exosporium-broken spore number）]×100%
Intact spore number is small lattice number shared by intact spore, and exosporium-broken spore number is small lattice number shared by exosporium-broken spore, tool
Body result is see Fig. 8
The spore value analysis result of Fig. 8 samples（See Figure of description）.
Claims (1)
- A kind of 1. method of quick detection lucidum spore powder miospore value, it is characterised in that：Lucidum spore powder B to be measured is taken, is dried to constant weight, and with a certain amount of chloraldurate test solution in repeatedly light on glass mortar It is soft to grind well, ultrasound mix, after be settled to certain volume, be well mixed, corresponding lucidum spore powder suspension is obtained, wherein using Density and the larger chloraldurate test solution of viscosity mix conidia powder, it is not lumpd, and are easy to observation to count；Take above-mentioned lucidum spore powder suspension point to drop on slide, under the microscope, using C6 type eyepiece micrometers, read respectively Go out small lattice number shared by intact spore and broken spore；Wherein, small lattice include intact spore or broken spore is designated as 1 more than 1/2 Small lattice, discontented 1/2 is designated as 0 small lattice, and lucky 1/2 is designated as 0.5 small lattice；Utilize formulaSpore value（%）=×100%Calculate spore value；Wherein, spore value refers to that the intact spore number in ganoderma spove powder accounts for intact spore number with breaking The percent value of wall spore count summation, intact spore number refer to the grid shared by intact spore, and exosporium-broken spore number refers to broken spore Grid shared by son；Described well mixed ultrasound mixing after referring to constant volume.
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|---|---|---|---|---|
| US6440420B1 (en) * | 2001-03-19 | 2002-08-27 | Xin Liu | Method for extracting oleaginous substances from germination-activated Ganoderma lucidum spores |
| CN101446581A (en) * | 2008-10-06 | 2009-06-03 | 福建仙芝楼生物科技有限公司 | Method for measuring wall breaking rate of ganoderma lucidum spores |
| CN104404123A (en) * | 2014-10-30 | 2015-03-11 | 广州市祈和电器有限公司 | Method for detecting cell wall breaking rate of wall-broken dry fruit, vegetable and coarse cereal raw materials |
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| CN103445024A (en) * | 2013-09-06 | 2013-12-18 | 山西宏福农牧科技有限公司 | Mixed ration feed for dairy cattle |
| CN104345024A (en) * | 2014-10-30 | 2015-02-11 | 广州市祈和电器有限公司 | Method for detecting cell wall breaking ratio in wall breaking fresh fruit/vegetable raw material |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6440420B1 (en) * | 2001-03-19 | 2002-08-27 | Xin Liu | Method for extracting oleaginous substances from germination-activated Ganoderma lucidum spores |
| CN101446581A (en) * | 2008-10-06 | 2009-06-03 | 福建仙芝楼生物科技有限公司 | Method for measuring wall breaking rate of ganoderma lucidum spores |
| CN104404123A (en) * | 2014-10-30 | 2015-03-11 | 广州市祈和电器有限公司 | Method for detecting cell wall breaking rate of wall-broken dry fruit, vegetable and coarse cereal raw materials |
Non-Patent Citations (1)
| Title |
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| "木香细胞破壁率的测定";蔡萍等;《湖南中医杂志》;20101130;第26卷(第6期);第1-2部分 * |
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