Cell wall breaking rate is often regarded as one of the important indexes to evaluate the method of pine pollen cell wall breaking and the quality of product, but there is no unified standard detection method so far. This article discusses the different detection methods of pine pollen cell wall breaking rate, analyzes the advantages and disadvantages of various methods, and puts forward the existing shortcomings and development trend for your reference.
Pine pollen powder is not only the life source of pine trees, but also known as “micro nutritional treasure house”. It contains above 10% protein, more than 20 kinds of free amino acids easily absorbed by human body, more than 10 kinds of vitamins, trace elements, natural active enzymes and flavonoids. It has high nutritional value and medical and health care function. The outer wall of pine pollen is hard, which is mainly composed of dense substances such as sporopollenin, cellulose, cutin and pollen. Its chemical properties are very stable and has the characteristics of acid and alkali resistance. As a result, the leaching of effective substances in pollen wall is greatly hindered, so that its effective substances can not be fully utilized by human body. In order to improve the utilization value of pine pollen powder, people have done a lot of exploration on the outer wall of pine pollen, among which “pollen cell wall breaking technology” is one of the main research directions. The results show that the utilization rate of pollen can be improved by breaking the cell wall of pollen. At present, pollen cell wall breaking methods mainly include compound cell wall breaking method, physical cell wall breaking method, biochemical cell wall breaking method and so on. However, no matter which cell wall breaking method and process conditions are adopted, there should be a wall breaking rate detection method to test the wall breaking degree of pollen.
Microscopic determination
1.1, Slide direct counting method& Pap hemocytometer method
The determination method of slide direct counting method is to use a micro syringe (or hemoglobin pipette) to suck 10μL of wall broken pollen slurry and non wall broken pollen slurry prepared with equal concentration on ordinary glass slide, add 20mm*20mm cover glass, put it under the microscope, move the propeller, and count the field of view one by one with a high-power mirror at the center of the cover glass, from left to right and from top to bottom. The formula to calculate the pine pollen cell wall breaking rate (η) is: cell wall breaking rate (%) = (number of pollen cells in the control sample – number of intact pollen cells retained in the test sample) / pollen cells in the control sample Count * 100%.
The Pap hemocytometer method is more reasonable than the slide direct reading method. Because the counting room has a depth of 0.1 mm, the pollen cells are not squeezed, the four grids are evenly distributed and have good reproducibility. If there is no micro syringe, you can also take a small drop of sample in the counting room with an eyedropper, but the filling should be appropriate. The operation method is made of a thick glass slide, and the whole counting room is divided into nine grids. The area of each large grid is mm2 and its depth is 0.1mm. Each grid in the four corners is divided into 16 small grids, draw 10μL of cell wall broken pine pollen slurry and non cell wall broken pollen slurry prepared into equal concentration aqueous solution and fill into the counting chamber along the edge of the cover glass respectively. Wait for 3 – 5min, count under a high-power microscope, and divide the total number of four large cells in the four corners by 4 to obtain the number of pollen cells per large cell. The pine pollen cell wall breaking rate was calculated by comparing the number of cells per mm3 and the formula (η) is as: cell wall breaking rate (%) = 100% – {100% *( A/4 *10) / ( B/4* 10) }(A refers to the total number of four cells of the tested pollen, and B refers to the total number of four cells of the control pollen.).
The cell wall breaking rate of more than 50 batches of pine pollen was measured and examined by the Pap hemocytometer method. Compared with the slide direct counting method, it has the advantages of rapidity, simplicity, good repeatability and reliable results.
1.2, Staining Smear Method
This method is mainly based on the slide direct counting method, and uses the principle that the protoplast of pine pollen is light red and the wall is purplish red after staining, resulting in a large contrast between the two, and the pollen is easy to count to determine the wall breaking rate of pine pollen. Dye the cell wall broken and unbroken pine pollen with quantitative alkaline fuchsin staining solution for 1 to 1.5 hours respectively, then dilute the dyed pollen solution by 5 times, mix it evenly, take the quantitative pollen solution with a pipette, put it on the carrier, spread it in a certain area, dry it in a 40 ℃ oven, transparent with xylene for 2 to 3 minutes, and seal it with Canadian gum after taking it out, Then make observation and statistics. Under the light microscope, equipped with an eyepiece micrometer, use the 5-point sampling method to calculate the number of broken and unbroken pollen within 0.5 cm2 of each point, and calculate the wall breaking rate of pollen according to the following formula: Wall breaking rate of pine pollen (%) =100%*(number of complete pollen before wall breaking – number of complete pollen after wall breaking) / number of complete pollen before wall breaking.
It can be seen from the above that this method is more reasonable than the slide direct counting method. The contrast in color makes the pollen easy to count, which further provides the reliability of microscopic observation results. However, there are also some disadvantages of uneven distribution, overlap or omission of pollen in the slide direct counting method.
1.3, Gravimetric method combined with microscopic quantitative method
The method is based on the above slide direct counting method, referring to the micro quantitative method of traditional Chinese medicine, taking complete pine pollen grains as the reference material, and using gravimetric method combined with micro quantitative method to determine the wall breaking rate of pollen. The formula of pollen wall breaking rate is as follows: Pollen wall breaking rate (%) = {1 – (number of sample pollen / number of control pollen) * (weight of control pollen / number of sample pollen)} * 100%. The number of pollen in the sample is the total average of 9 visual fields observed in the secondary sample of the sample pollen; the number of control pollen is the total average of 9 visual fields observed in the secondary sample of control pollen; the weight of control pollen is the actual weight of control pollen when preparing suspension (mg); the sample pollen weight is the actual weight of the sample when preparing the suspension (mg). This method is simple and easy. Chloral hydrate test solution is used for loading, and the average value of pollen grains in 9 fields is observed in the same sample twice suspension, and the relative deviation is less than 5%. At the same time, the principle of gravimetric analysis is added to improve the accuracy and reliability of the measurement results. However, the raw material must be a single pollen and shall not be mixed with other pollen and impurities, otherwise it is easy to affect the measurement results.
1.4, Standard Curve Method
The principle of standard curve method is a quantitative analysis method to explore the relationship between independent variables and dependent variables by using points of different concentrations. The first step of the experiment is to weigh 0g, 1.00G, 5.00g, 10.00g, 15.00g and 20.00g of unbroken pine pollen respectively, dilute them to 20.00g with 100% broken pine pollen and mix them evenly to prepare standard samples with wall breaking rates of 100%, 95%, 75%, 50%, 25% and 0% respectively. Weigh 0.50g of each standard sample, dissolve it with water and fix the volume to 25.00ml to make a suspension of 20.00mg/ml, then take 0.25ml of the shaken suspension on the glass slide, expand it into an oval sheet with an area of about 5cm2, observe under the microscope (eyepiece * 15, objective lens * 10), observe 5 fields, and record the number of unbroken pollen cells in each field, Each standard sample shall be operated in parallel for 3 times to obtain the arithmetic average. The standard curve was drawn with the broken wall rate as the ordinate and the arithmetic average of the number of unbroken pollen cells detected in each visual field as the abscissa. According to the number of non broken pollen cells detected in the sample, calculate the pollen wall breaking rate according to the following formula: Y = a + bX.
In the formula: Y is the wall breaking rate of pine pollen (%); X is the arithmetic mean of unbroken pine pollen (PCs.); a. b are constant. This method is suitable for the determination of wall breaking rate of pine pollen, and is also suitable for the determination of wall breaking rate of many kinds of pollen. This method has the characteristics of high precision, high accuracy, simplicity and wide linear range.
Laser Particle Size Analysis
In recent years, with the development of the research on the wall breaking rate of traditional Chinese medicine, more and more scientific researchers are no longer limited to the traditional microscopic method to study the wall breaking rate, but are constantly looking for new methods to determine the wall breaking rate. Among them, laser particle size analysis method is a more forward-looking analysis method, which has great development advantages. It can not only overcome the characteristics of large subjective influencing factors of microscopic determination method, but also measure the cell wall breaking rate of traditional Chinese medicine with no obvious microscopic characteristics. In addition, it has the advantages of simple operation, reliable results and high accuracy. Its principle is to use
According to Mie scattering theory, the energy distribution of scattered light is directly related to the distribution of particle diameter. The particle size distribution characteristics can be obtained by receiving and measuring the energy distribution of scattered light. According to the characteristics of the tested medicinal materials, wet method and dry method can be selected. Wet measurement is to mix the sample with a liquid to prepare a suspension, and disperse it with the help of stirring, ultrasonic and other physical conditions to disperse the aggregated particles into original particles, and keep the original particles in a good dispersion state in the dispersion; The dry method is used to determine the solid sample with water solubility or without suitable dispersion medium. The agglomerated particles are impact dispersed through the high-speed air flow provided by the air compressor, and then collected by the connected vacuum cleaner after being measured in a closed air tank. Considering the relationship between the average diameter of traditional Chinese medicine cells, particle size after ultra-fine comminution and cell wall breaking rate, the ideal calculation method of crushing model is obtained, and the formula of wall breaking rate(η) is as follows:
η1=［1- ( 1-1 /n) 3］*100% ( n ＞ 1) ;
η2=100% ( n≤1) ;
η= η1 + η2;
In the formula, “n” is the ratio of particle size to cell diameter.
Chemical Composition Method
Relevant literature studies show that there is a certain relationship between wall breaking rate and chemical composition. On this basis, the relationship between different wall breaking rate and proline content in pollen with different wall breaking rate is discussed, and a new method for determining wall breaking rate of black pine pollen is proposed. After the absorbance of proline content at 520nm in samples with different wall breaking rates is measured by spectrophotometry, the wall breaking rate of pine pollen is calculated according to the following formula:
Y ( % ) = ( a + bX)* 100%
In the formula: Y represents the wall breaking rate of pollen (%);
X represents the absorbance of proline content at 520nm;
a. b: constant.
The results showed that the relationship between pollen wall breaking rate and its corresponding proline absorbance was y = 11 886x- 0. 0637， Ｒ2 = 0.9897. It can be seen that the linear relationship is good. As long as the proline absorbance of a pollen wall broken is measured and substituted into the equation, the pollen wall broken rate can be obtained, which is simple and accurate, and can avoid the cumbersome operation process caused by microscopic measurement of wall broken rate. However, there are some defects. The content of proline available in different black pine pollen is different, and the measured wall breaking rate is different. Therefore, it affects the reliability of the results.
At present, although each method has its own advantages, there are still some shortcomings. The slide direct counting method is relatively simple and easy, but because the cover glass has the effect of extrusion, the distribution of pollen cells is not easy to be uniform, easy to overlap or omit, poor reproducibility and so on. Compared with the fragment direct counting method, the improved PAP blood cell counting method is faster, simpler, reliable and reproducible, but the filling of the counting room should be appropriate, otherwise it is easy to cause the accuracy of pollen cell reading. The staining smear method is more reasonable than the slide direct counting method. The contrast in color makes the pollen easy to count, which further provides the reliability of microscopic observation results. However, the pollen distribution will be uneven, with overlap or omission. The gravimetric analysis method combined with the microscopic quantitative method is simple and easy. The analysis principle of the gravimetric method is added to make the data more objective and further improve the measurement accuracy. The disadvantage is that it can only be a single pollen and shall not be mixed with other pollen and impurities, otherwise it is easy to affect the measurement results. The standard curve method has the advantages of high precision, high accuracy, simplicity and wide linear range, but it is subjective compared with laser particle size method and chemical composition. The laser particle size analysis method is simple, fast and objective, but it is easily affected by dispersants, the properties of medicinal materials, the water content of medicinal materials. In addition, the correlation between particle size and wall breaking rate needs to be further studied. The chemical composition method is simple and accurate. The correlation between pollen wall breaking rate and composition is further established and discussed. However, different pollen contents are different and there are many influencing factors, such as collection time, variety and batch. Therefore, the reliability of the measurement results is not very strong and there are some disadvantages.
At present, it is generally believed that the effective components of pine pollen can not be fully released without shell breaking treatment. Therefore, the rate of pollen wall breaking has become one of the important indexes to evaluate the method of pollen wall breaking and the quality of pollen wall breaking powder. According to the current literature research, the detection methods of pollen wall breaking rate mainly include microscopic determination, laser particle size determination and chemical composition determination. However, there is no unified conclusion on which detection method is the more reasonable detection method of wall breaking rate. It can be seen from the summary of the advantages and disadvantages of the above detection methods, Each detection method has certain advantages and disadvantages. Therefore, which detection method is reasonable needs to be further studied in the future. However, as far as the author is concerned, the laser particle size analysis method may be a hot spot in the study of pollen wall breaking rate in the future, and is expected to become a reasonable and standard detection method In addition, when analyzing or studying which pollen wall breaking rate detection method is more reasonable, we should first clarify whether the detection method itself is scientific, whether the inspection process is reasonable, and whether the factors causing error sources can be reduced to the most, so as to make the experimental results more reliable. Therefore, when discussing the detection methods of pine pollen wall breaking rate, we should compare and verify various detection methods according to the specificity of each pollen, so as to formulate a reasonable standard method of wall breaking rate.
Therefore, the future research should strengthen the research on the uncertainty of the detection method of wall breaking rate, so as to reduce the source of error and improve the reliability of the experimental results; Strengthen the correlation study between particle size and pollen wall breaking rate and the discussion of influencing factors, so as to realize the determination of pollen wall breaking by laser particle size analysis, make the detection more simple and easy to operate, and overcome the shortcomings of traditional microscopy; To some extent, the wall breaking rate is directly proportional to the chemical components in pollen, but their correlation is still lack of in-depth research; When measuring the wall breaking rate of a certain pollen, systematic research or joint analysis of multiple detection methods should be carried out to make the experimental results more reasonable and reliable.
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