CN112063529A

CN112063529A - Red card skeleton powder and preparation method thereof

Info

Publication number: CN112063529A
Application number: CN202010813481.3A
Authority: CN
Inventors: 李玉琢; 魏玲娟
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-08-13
Filing date: 2020-08-13
Publication date: 2020-12-11

Abstract

The invention relates to a red card skeleton powder and a preparation method thereof, belonging to the technical field of cellulose. The preparation method comprises the following steps: step one, inoculating activated Nocardia in a liquid culture medium, and culturing to obtain a bacterial liquid; step two, taking bacterial liquid, shaking up, centrifuging, discarding supernatant, adding distilled water for dilution, and mixing uniformly to obtain bacterial suspension; and thirdly, breaking the walls of the bacterial suspension by alkali liquor or ultrasonic waves, extracting cell wall frameworks, drying and grinding to obtain the red cardskeleton powder. The powder is white or quasi-white powder, and has no odor and odor; insoluble in water, dilute acids, dilute bases and most organic solvents; high purity, no impurity and high affinity. The gel additive can be added as an auxiliary material to obviously enhance the strength and toughness of the gel, improve the internal structural space of the gel, increase the drug carrying amount of the gel per unit mass, and achieve the effect of slow release.

Description

Red card skeleton powder and preparation method thereof

Technical Field

The invention belongs to the technical field of cellulose, and particularly relates to a red card skeleton powder and a preparation method thereof.

Background

Plant cellulose (cellulose) is a macromolecular polysaccharide composed of glucose and is the main component of plant cell walls. Bacterial Cellulose (BC for short) has the characteristics of high purity, high water retention, high tensile strength, good biocompatibility and the like compared with plant Cellulose, and therefore, is widely applied to many fields of biomedicine, food, cosmetics, textile industry, membrane filters and the like.

The existing bacterial cellulose appears in a gel form, has limited functions and effects and is not convenient for long-term storage; meanwhile, the powder is large in size and inconvenient to transport and carry, and the application of the powder prepared from the powder can be widened. However, the bacterial cellulose has a hyperfine three-dimensional network structure, the intermolecular force is strong, and the bacterial cellulose is difficult to be prepared into powder, which brings difficulty to processing and production. At present, bacterial cellulose powder can be prepared by using various drying modes such as spray drying and the like, but the prepared powder has nonuniform granularity, a plurality of excellent characteristics are lost, the cost is too high, and the method is not suitable for industrial production.

Disclosure of Invention

In view of the above, in order to solve the technical problems in the prior art, the invention provides a red cardskeleton powder and a preparation method thereof.

The technical scheme adopted by the invention for solving the technical problems is as follows.

The invention firstly provides a preparation method of a red card framework powder, which comprises the following steps:

step one, inoculating activated Nocardia in a sterilized liquid culture medium, wherein the inoculation amount of 35-75mL of the liquid culture medium is 0.25-0.1mL, and performing shake culture at 24-30 ℃ for more than 24 hours to obtain a bacterial liquid;

the liquid culture medium contains 1 wt% of carbon source and 1 wt% of nitrogen source, the carbon source is glucose, sucrose, starch or mannitol, the nitrogen source is yeast extract, protein gland, urea or a sulfuric acid string, and the pH is 6-8;

step two, taking bacterial liquid, shaking up, centrifuging, discarding supernatant, adding distilled water for dilution, and mixing uniformly to obtain bacterial suspension with the mass concentration of 1.5 g/L;

step three, placing the bacterial suspension into 5-5.5% KOH solution, and carrying out water bath at 100 ℃ for 10-50 min;

or, performing ultrasonic wall breaking on the bacterial suspension, wherein the wall breaking conditions are ultrasonic time 1-15s, interval time 5-15s, cycle number 40-90 times and power 100-;

and step four, extracting the cell wall skeleton, drying and grinding to obtain the red cardskeleton powder.

Preferably, in the first step, the activation process of nocardia is as follows: inoculating Nocardia genus into solid culture medium by streaking, and culturing at 28 deg.C for two days;

the solid medium contains 1 wt% of glucose, 1 wt% of yeast extract and 2 wt% of agar, and has a pH of 7.2.

Preferably, in the first step, the sterilization temperature is 115-118 ℃, and the sterilization time is 20 min.

Preferably, in the first step, the rotation speed of the shaking table is 160-.

Preferably, in the first step, the carbon source of the liquid culture medium is glucose powder, and the nitrogen source is yeast powder; the pH of the liquid culture medium is 7-8; more preferably 7.2 to 7.4.

Preferably, in the first step, the temperature for culturing is 26 ℃.

Preferably, in the first step, the inoculation amount of the 50mL liquid culture medium is 0.75 mL.

Preferably, in the second step, the rotation speed of the centrifugation is 4000r/min, and the time is 5 min.

Preferably, in the third step, the bacterial suspension is placed in a 5% KOH solution, and is subjected to water bath at 100 ℃ for 40 min;

in the third step, the wall breaking conditions are ultrasonic time 15s, interval time 15s, cycle number 40 times and power 500W.

The invention also provides the red card skeleton powder prepared from the red card skeleton powder.

Compared with the prior art, the invention has the beneficial effects that:

the red card framework powder is white or white-like powder, and is odorless and tasteless; insoluble in water, dilute acids, dilute bases and most organic solvents; high purity, no impurity and high affinity. The gel can be added as an auxiliary material to obviously enhance the strength and toughness of the gel, improve the internal structural space of the gel, increase the drug carrying amount of the gel per unit mass, and achieve the effect of slow release, namely, the content of other drugs can be improved and the release degree of active ingredients is increased. And is expected to be applied to the fields of human body dressings, nursing and cleaning products, tissue engineering scaffolds and the like.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows the cell counts of bacterial suspensions at different concentrations in example 7 of the present invention;

FIG. 2 is a graph showing the effect of different total disruption times on the bacterial disruption rate in example 8 of the present invention;

FIG. 3 is a graph showing the effect of time per sonication on the degree of disruption in example 9 of the present invention;

FIG. 4 is a graph showing the effect of ultrasonic output power on the crushing ratio in example 10 of the present invention;

FIG. 5 is a graph showing the effect of KOH concentration on bacterial disruption rate in example 12 of the present invention;

FIG. 6 is a graph showing the effect of treatment time on bacterial disruption rate in example 13 of the present invention.

Detailed Description

For a further understanding of the invention, preferred embodiments of the invention are described below in conjunction with the detailed description, but it is to be understood that the description is intended to further illustrate the features and advantages of the invention and not to limit the claims to the invention.

The preparation method of the red card framework powder comprises the following steps:

In the above technical scheme, in the step one, the activation process of nocardia is the prior art, and usually comprises: inoculating Nocardia genus into solid culture medium by streaking, and culturing at 28 deg.C for two days; the solid medium contained 1 wt% glucose, 1 wt% yeast extract and 2 wt% agar, and had a pH of 7.2. The Nocardia used in the invention is the prior art and is provided by the center for storing strains of the institute of Life sciences of the university of Fujian Master.

In the above technical scheme, in the first step, the sterilization temperature is 115-118 ℃, and the sterilization time is 20 min.

In the above technical solution, in the step one, the rotation speed of the shaking table is 160-.

According to the technical scheme, in the first step, a carbon source of a liquid culture medium is glucose powder, and a nitrogen source is yeast powder; the pH of the liquid culture medium is 7-8; more preferably 7.2 to 7.4.

In the technical scheme, in the step one, the culture temperature is 26 ℃.

In the above technical scheme, in the first step, the inoculation amount of 50mL of liquid culture medium is 0.75 mL.

In the technical scheme, in the second step, the rotating speed of the centrifugation is 4000r/min, and the time is 5 min.

In the technical scheme, in the third step, when 5% KOH suspension bacteria liquid is used, a good wall-breaking effect can be obtained after 40min of water bath at 100 ℃, and the wall-breaking rate can basically reach more than 90%; therefore, the bacterial suspension is preferably placed in a 5% KOH solution and subjected to water bath at 100 ℃ for 40 min.

According to the technical scheme, in the third step, the cell is crushed by a short-time multiple crushing mode and the bacterial suspension with the mass concentration of 1.5 g/L. The ultrasonic treatment time is more suitable at 15s, and the upper limit of the wall breaking rate can reach 84.36 percent within a 95 percent confidence interval; 40 cycles are more suitable working cycles, and the upper limit of the wall breaking rate of the device can reach 75.67 percent within a 95 percent confidence interval; 500W is more suitable ultrasonic power, and the upper limit of the wall breaking rate of the ultrasonic wave breaking device can reach 70.81% within a 95% confidence interval. Therefore, the ultrasonic wave time 15s, the interval time 15s, the number of operation cycles 40, and the ultrasonic wave power 500W are preferable. The probe of the ultrasonic wave is immersed at a liquid level of about 15 mm.

The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention will be further described in detail with reference to the following embodiments and the accompanying drawings.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. Materials, reagents, devices, instruments, apparatuses and the like used in the following examples are commercially available unless otherwise specified.

Example 1

Step one, inoculating activated Nocardia (cultured for 2 days) into a sterilized liquid culture medium (sterilized at 115 ℃ for 20min), wherein the inoculation amount of 50mL of the liquid culture medium is 0.75mL, and culturing for 24h in a shaking table (240r/min) at 28 ℃ to obtain a bacterial liquid;

the liquid culture medium is: preparing liquid culture media according to the proportion that a carbon source and a nitrogen source are 0.5g (1 wt%), and combining four carbon sources of glucose, sucrose, starch and mannitol and four nitrogen sources of yeast extract, protein gland, urea and sulfuric acid string two by two to prepare 16 different liquid culture media; adjusting the pH to 7.2;

step two, taking bacterial liquid, shaking up, centrifuging for 5min at 4000r/min, discarding supernatant, adding distilled water for dilution, and uniformly mixing by using a magnetic stirrer to obtain bacterial suspension with the mass concentration of 1.5 g/L;

step three, carrying out ultrasonic wall breaking on the bacterial suspension, wherein the wall breaking conditions are ultrasonic time 15s, interval time 15s, cycle number 40 times and power 500W;

The wet weight of the bacterial cells of the bacterial liquid obtained in the first step of example 1 was weighed. The wet weight test method of the thallus comprises the following steps: centrifuging the bacterial liquid for 10min at 5000r/min by using a centrifugal machine, discarding supernatant, weighing to obtain the wet weight of the thallus, and generally taking an average value by adopting three times of measurement. The test results are shown in table 1.

TABLE 1 Wet weight of bacterial cells of the bacterial suspension obtained in the first step of example 1

As can be seen from Table 1, different media are suitable for the cultivation of the bacteria of the invention; among them, the combination of glucose/yeast extract gave the greatest bacterial weight, indicating that the combination of glucose/yeast extract is most suitable for the growth of the bacterial cells.

Example 2

the liquid culture medium is: glucose/yeast extract culture medium (glucose 1 wt%, yeast extract 1 wt%), pH 5, 6, 7, 7.2, 8;

step two to step four are the same as in example 1.

The wet weight of the bacterial cells of the bacterial liquid obtained in the first step of example 2 was weighed. The wet weight of the cells was measured in the same manner as in example 1. The test results are shown in table 2.

TABLE 2 wet weight of bacterial cells of the bacterial suspension obtained in the first step of example 2

As is clear from Table 2, the pH of 5 is due to the fact that the cells hardly grow and the medium should be peracid. The increase of the pH value causes the increase of the cell weight, the cell weight reaches the maximum when the pH value is 7, and the cell weight slightly decreases when the pH value is 8, which is the reason that the culture medium is alkaline. The pH value is between 7 and 8, which is most suitable for the growth of the thallus.

Example 3

Step one, inoculating activated Nocardia (cultured for 2 days) into a sterilized liquid culture medium (sterilized for 20min at 115 ℃), wherein the inoculation amount of 50mL of the liquid culture medium is 0.75mL, and culturing for 24h at 28 ℃ to obtain a bacterial liquid;

the liquid culture medium is: glucose/yeast extract medium (glucose 1 wt%, yeast extract 1 wt%), pH 7.2;

the cultivation respectively comprises 160r/min of a shaking table, 200r/min of the shaking table, 240r/min of the shaking table, 280r/min of the shaking table and 320r/min of the shaking table, complete standing, 6h of shaking table (240r/min) cultivation, 6h of standing cultivation (circulation 24h) in a constant temperature incubator, 4h of shaking table (240r/min) cultivation and 4h of standing cultivation (circulation 24h) in the constant temperature incubator;

step two to step four are the same as in example 1.

The wet weight of the bacterial cells of the bacterial liquid obtained in the first step of example 3 was weighed. The wet weight of the cells was measured in the same manner as in example 1. The test results are shown in table 3.

TABLE 3 wet weight of bacterial cells of the bacterial suspension obtained in the first step of example 3

As can be seen from Table 3, the growth effect of the cells was poor and the yield was very low during the static culture; in the case of the alternate culture, the yield of the cells was increased, and the effect of the culture at the 6-hour interval was inferior to that at the 4-hour interval, i.e., the shorter the interval, the better the culture effect. When the continuous shaking table culture is carried out, the yield of the thalli is greatly improved compared with that of the thalli which is kept still and is cultured at intervals, and along with the improvement of the rotating speed of the shaking table, the thalli are greatly increased, but the increase is not obvious. When the rotating speed of the shaking table is 320r/min, the bacterial weight reaches the maximum.

Example 4

Step one, inoculating activated Nocardia (cultured for 2 days) into a sterilized liquid culture medium (sterilized at 115 ℃ for 20min), wherein the inoculation amount of 50mL of the liquid culture medium is 0.75mL, and performing shake culture (240r/min) at 24 ℃, 26 ℃, 28 ℃ and 30 ℃ for 24h respectively to obtain a bacterial liquid;

the liquid culture medium is: glucose/yeast extract medium (glucose 1 wt%, yeast extract 1 wt%), pH 7.2.

Step two to step four are the same as in example 1.

The wet weight of the bacterial cells of the bacterial liquid obtained in the first step of example 4 was weighed. The wet weight of the cells was measured in the same manner as in example 1. The test results are shown in table 4.

TABLE 4 wet weight of bacterial cells of the bacterial suspension obtained in the first step of example 4

As can be seen from Table 4, the productivity of the cells reached the maximum at the culture temperature of 26 ℃ indicating that it is most suitable for the growth of the cells at this temperature; when the temperature is lower than 26 ℃, the yield of the bacteria is in direct proportion to the temperature and is increased along with the increase of the temperature; when the temperature is higher than 26 ℃, the yield of the bacteria is inversely proportional to the temperature, and decreases with the increase of the temperature.

Example 5

Step one, respectively inoculating 0.75mL of activated Nocardia (cultured for 2 days) into 30mL, 45mL, 50mL, 60mL and 75mL of liquid culture medium after sterilization (sterilized at 115 ℃ for 20min), and performing shake culture (240r/min) at 28 ℃ for 24h to obtain a bacterial liquid;

Step two to step four are the same as in example 1.

The wet weight of the bacterial cells of the bacterial liquid obtained in the first step of example 5 was weighed. The wet weight of the cells was measured in the same manner as in example 1. The test results are shown in table 5.

TABLE 5 wet weight of bacterial cells of the bacterial suspension obtained in the first step of example 5

As can be seen from Table 5, when the volume of the culture medium is between 30 and 45mL, the cell yield is slightly reduced with the decrease of the dissolved oxygen amount, and the cell growth effect is proportional to the dissolved oxygen amount. When the volume of the culture solution is between 45 and 75mL, the yield of the thalli is gradually increased along with the reduction of the dissolved oxygen, and the growth effect of the thalli is inversely proportional to the dissolved oxygen. The reason is probably that when the volume of the culture solution is between 45 and 75mL, the factors of the nutrients in the culture medium are dominant, and the influence of the nutrients on the thalli is greater than that of the dissolved oxygen, so that the thalli can obtain more nutrients along with the increase of the volume of the culture solution, and the growth effect is better. On the other hand, when the volume of the culture medium is 45mL or less, the amount of dissolved oxygen in the cells is larger than that of the nutrients, and the cell yield decreases as the amount of dissolved oxygen decreases.

Example 6

Step one, respectively inoculating 0.25mL (0.5%), 0.5mL (1%), 0.75mL (1.5%) and 0.1mL (2%) of activated Nocardia (cultured for 2 days) into 75mL of liquid culture medium after sterilization (sterilized at 115 ℃ for 20min), and performing shaking culture at 28 ℃ for 24h (240r/min) to obtain a bacterial liquid;

step two to step four are the same as in example 1.

The wet weight of the bacterial cells of the bacterial liquid obtained in the first step of example 6 was weighed. The wet weight of the cells was measured in the same manner as in example 1. The test results are shown in table 6.

TABLE 6 wet cell weight of the bacterial suspension obtained in the first step of example 6

As can be seen from Table 6, the maximum yield of the obtained bacteria was obtained when the inoculum size was 0.50mL (1%); when the inoculation amount is less than 0.50mL, the yield of the bacteria and the inoculation amount are in positive correlation and increase along with the increase of the inoculation amount; when the inoculation amount is more than 0.50mL, the yield of the bacteria is inversely proportional to the inoculation amount, and the growth condition of the bacteria is reduced along with the increase of the inoculation amount. The reason is probably that the culture solution has a certain volume, the nutrient in the culture medium is limited, the nutrient is insufficient due to the overlarge inoculation amount, and the growth of thalli is inhibited, so that the yield of the thalli is reduced. When the volume of the culture solution is constant, the inoculum size has a maximum value, about 0.5mL, and when the inoculum size is less than 0.5mL, the obtained bacterial weight increases with the increase of the inoculum size, and when the inoculum size is more than 0.5mL, the growth condition of the thallus is reduced, which is supposed to be the reason of insufficient nutrients.

Example 7

Step one, inoculating activated Nocardia (cultured for 2 days) into a sterilized liquid culture medium (sterilized at 115 ℃ for 20min), wherein the inoculation amount of 50mL of the liquid culture medium is 0.75mL, and culturing for 24h, 48h, 72h, 96h, 168h and 216h in a shaking table (240r/min) at 28 ℃ to obtain a bacterial liquid;

step two, taking bacterial liquid, shaking up, centrifugally separating for 5min (the rotating speed is 4000r/min), discarding supernate, adding distilled water for diluting, and uniformly mixing by using a magnetic stirrer to obtain bacterial suspension with the mass concentration of 1.5 g/L;

and step three, performing ultrasonic wall breaking on the bacterial suspension, wherein the wall breaking conditions are ultrasonic time 15s, interval time 15s, cycle number 40 times and power 500W.

The cell number of the bacterial suspension after the cell wall breaking in the third step of example 7 is detected, and the cell number in the bacterial suspension after the cell wall breaking is obtained by counting with a blood counting chamber (the cell number is the average value of the cell number of the bacterial suspension in the cells in the blood counting chamber, and part of data is obtained by converting the cell number in each cell by the dilution factor). The results are shown in FIG. 1.

As can be seen from FIG. 1, as the number of cells increased, cell breakage increased first and then decreased. At high bacterial suspension concentrations, this results in increased viscosity which is detrimental to vacuole formation, expansion and explosion, while at low bacterial concentrations it reduces the chance of contact with cells during vacuole expansion and explosion.

Example 8

step three, performing ultrasonic wall breaking on the bacterial suspension, wherein the wall breaking conditions are as follows: the working power of the ultrasonic crusher is 300W, the interval is 15s after crushing for 12s every time, 10, 20, 30, 40, 50 and 60 cycles are respectively set, and crushing is carried out for three times, namely the whole crushing time is respectively 6min, 12min, 18min, 24min, 30min and 36 min.

The bacteria were counted on a hemacytometer at 87 × 10 n/ml (n ═ 6) before disruption. The impact of the whole crushing time on the crushing effect was examined and the results are shown in fig. 2. As can be seen from FIG. 2, the bacterial disruption rate is obviously increased along with the extension of the whole disruption time, which shows that the extension of the whole disruption time can increase the radiation of ultrasonic waves to the bacteria, thereby being beneficial to the disruption of the bacteria. However, since the ultrasonic disruption method is a relatively vigorous cell disruption method, the structure and properties of the protein are easily destroyed after a long time. So the whole crushing time does not exceed 30 min.

Example 9

The first to fourth steps are the same as example 8, and the wall breaking conditions are as follows: the working power of the ultrasonic crusher is 300W, the crushing time is respectively set to 1s, 2s, 3s, 4s, 5s, 10s, 12s and 15s, the interval is 15s, 90 cycles are set, and the crushing is carried out for 1 time.

The bacteria were counted on a hemacytometer at 81 × 10 n/ml (n ═ 6) before disruption. The effect of each ultrasonication time on the crushing effect was examined, and the results are shown in FIG. 3. As can be seen from FIG. 3, the cell disruption rate increased and then decreased with the increase of the time for each sonication at intervals of 2s, and the disruption rate decreased significantly with each sonication less than 2s or more than 2 s. The process that the ultrasonic wave breaks the cells through the cavitation effect is actually the process that cavitation bubbles are formed, vibrated, expanded, compressed and collapsed to be closed, the process needs a very short time to be completed, and the short-time and multiple working modes can ensure that the cavitation bubbles generated by the ultrasonic wave have enough time and more chances to complete the processes of expansion and explosion, thereby being beneficial to cell breaking.

Example 10

The first to fourth steps are the same as example 8, and the wall breaking conditions are as follows: the working power of the ultrasonic crusher is respectively 100W, 200W, 300W, 400W, 500W and 600W, the time is respectively set to be 5s after 2s, 60 cycles are set, and crushing is carried out for 1 time.

The bacteria were counted on a hemocytometer at 102X 10 n/ml before disruption. The influence of the output power on the crushing rate was examined. The structure is shown in fig. 4, and it can be seen from the figure that the output power of the ultrasonic wave has a great influence on the bacterial breakage rate, and the bacterial breakage rate increases with the increase of the output power of the ultrasonic wave, because the increase of the output power causes more cavities to be formed in the liquid, and further more cavitation bubbles are generated, so that the breakage effect is enhanced. The degree of increase in the crushing rate is not significant after the power is increased to 500W, and the output power of 500-600W is considered to be suitable in view of the requirements of the ultrasonic crusher per se.

Example 11

The first to fourth steps were the same as in example 8, the wall breaking conditions are shown in table 7, and the test results are shown in table 8.

Table 7 wall breaking conditions of example 11

TABLE 8 test results of example 11

As can be seen from table 8: the ultrasonic treatment time is more suitable at 15s, and the upper limit of the wall breaking rate can reach 84.36 percent within a 95 percent confidence interval; 40 cycles are more suitable working cycles, and the upper limit of the wall breaking rate of the device can reach 75.67 percent within a 95 percent confidence interval; 500W is more suitable ultrasonic power, and the upper limit of the wall breaking rate of the ultrasonic wave breaking device can reach 70.81% within a 95% confidence interval. Therefore, the ultrasonic wave time is preferably 15s, the time interval is preferably 15s, the cycle number is preferably 40 times, and the power is preferably 300W.

Example 12

Step one, step two and step four are the same as embodiment 8, and step three is: sucking 1ml of the bacterial suspension into a test tube, respectively adding 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% KOH1mL by mass percent, and carrying out water bath treatment at 100 ℃ for 30 min.

The alkali can dissolve the soluble polysaccharide layer in the cell wall and part of lipid, so that the permeability of the cell wall is increased, and the intracellular substances are easy to separate out. The bacteria of example 12 were tested for breakage rate, which is the average of the number of cells in each cell of the hemacytometer, and the results are shown in FIG. 5. As can be seen from FIG. 5, on the basis of the treatment time of 30min at the water bath temperature of 100 ℃, the wall-breaking rate of the cells is obviously increased along with the increase of the mass fraction of KOH, when the mass fraction of KOH reaches about 5%, the wall-breaking rate reaches the maximum, and when the mass fraction of KOH exceeds 90% and is continuously increased along with the mass fraction of KOH, the wall-breaking rate value fluctuates up and down at 90%, and is basically maintained at about 90%, and the wall-breaking rate tends to be stable. The possible reason is that the alkaline hydrolysis reaction gradually reaches equilibrium as the alkali concentration increases, and the cell wall-breaking rate gradually reaches equilibrium as the alkali concentration increases after the reaction reaches equilibrium. Therefore, in actual operation, better wall breaking effect can be obtained by selecting KOH with the mass fraction of 5% to carry out cell wall breaking.

Example 13

Step one, step two and step four are the same as embodiment 8, and step three is: sucking the bacterial suspension ImL into a test tube, adding 5.0% KOH solution ImL, treating in 100 deg.C water bath for 10min, 20min, 30min, 40min, 50min, and performing cell disruption.

The results of examining the breakage of the bacteria of example 13 and the effect of the treatment time on the alkali wall-breaking effect are shown in FIG. 6. As can be seen from fig. 6, on the basis that the mass fraction of KOH is 5% and the water bath temperature is 100 ℃, the wall-breaking rate is significantly improved along with the extension of the treatment time, and when the treatment time reaches 40min, the wall-breaking rate can reach more than 90%, the treatment time is continuously improved, and the increase of the wall-breaking rate is small. Therefore, 40min is preferably used as the treatment time for alkali wall breaking.

Example 14

step three, placing the bacterial suspension in a 5% KOH solution, and carrying out water bath at 100 ℃ for 40 min;

or, performing ultrasonic wall breaking on the bacterial suspension, wherein the wall breaking conditions are ultrasonic time 15s, interval time 15s, cycle number 40 times and power 500W.

The wet weight of the bacterial cells of the bacterial suspension obtained in the first step of example 14 was weighed. The wet weight of the cells was measured in the same manner as in example 1. The test results are shown in table 9.

Comparative example 1

To the liquid medium of example 14, 0.025g of MgSO was added respectively₄、KH₂PO₄、NaCl、CaCl₂Otherwise, the same as example 14.

Weighing wet bacteria of the bacteria liquid obtained in the step one of the comparative example 1. The wet weight of the cells was measured in the same manner as in example 1. The test results are shown in table 9.

TABLE 9 wet cell weights of the bacterial suspension obtained in example 14 and the first step of comparative example 1

As is clear from Table 9, the weight of the cells was less than that of example 14 when 0.025g of the inorganic salt was added to the medium, indicating that the addition of the inorganic salt inhibited the growth of the cells as a whole at this concentration. Among them, the inhibition effect of Ca ions is most remarkable, and the inhibition effect of Na ions is small. The inhibition of the growth of the cells may be caused by the collision between the added inorganic salt ions and the ions present in the original medium.

The red card skeleton powder obtained in example 14 was examined.

1.1 the red card skeleton powder is white or white-like powder observed, has no visible impurities and foreign matters, and is tasteless.

1.2 testing the pH value of the red card skeleton powder to be 5.0-7.5 by an acidimeter.

1.3 weighing 0.01g of sample, respectively adding into 100ml of water at 25 ℃ +/-2 ℃, 100ml of ethanol at 25 ℃ +/-2 ℃, 100ml of diethyl ether at 25 ℃ +/-2 ℃, 100ml of dilute sulfuric acid at 25 ℃ +/-2 ℃ and 100ml of 5% sodium hydroxide solution at 225 ℃ +/-2 ℃, strongly shaking for 30s every 5min, and observing the dissolution condition within 30min, wherein the sample is insoluble.

1.4, hydrolyzing the red card skeleton powder, performing paper chromatography separation, developing by using ninhydrin as a color developing agent, and identifying a sample as a red card skeleton by using a Meso-DAP standard product as a control.

1.5 precisely weighing appropriate amounts of ethanol and chloroform, quantitatively diluting with an internal standard solution (taking an appropriate amount of isopropanol, diluting with methanol to obtain a solution containing about 1.0mg of ethanol per 1 ml) to obtain a mixed solution containing about 1.0mg of ethanol and 1.0mg of chloroform per 1ml, precisely weighing 5ml, placing in a headspace bottle, and sealing to obtain a reference solution. A sample (200 mg) was taken and placed in a headspace bottle, and 5ml of the internal standard solution was added precisely, sealed, and shaken to dissolve it, thereby obtaining a sample solution. According to the residual solvent assay, DB-624(30 m.times.0.53 mm. times.2.5 μm) was used as a column, starting at 35 ℃ for 2 minutes, heating at 5 ℃ per minute to 80 ℃, further heating at 25 ℃ per minute to 200 ℃ for 2 minutes, detector temperature 220 ℃, injector temperature 200 ℃, headspace vial equilibration temperature 85 ℃ and equilibration time 20 minutes. And (3) respectively injecting the gas on the liquid after gas-liquid balance of the reference substance solution and the test sample solution into a gas chromatograph, recording the chromatogram, and calculating according to an external standard method by using peak area, wherein the residual quantity of ethanol is not higher than 5000ppm, and the residual quantity of chloroform is not higher than 60 ppm. .

1.6 the detailed operations were carried out in accordance with the microbiological examination protocol QFTL-YsS-03-030-. The result of the test was aseptic growth.

1.7 according to the method in GB/Z21738-2008, and simultaneously according to the characteristics of the red card framework, the detection is carried out by adopting a scanning electron microscope. And (3) placing the framework film of the red card in vacuum, drying to constant, working for 3min by using sputtering current of 4mV, spraying gold on the surface of the sample, and observing the operation of the fiber network structure by using a scanning electron microscope. The detection shows that the fiber diameter and the framework size are less than or equal to 1 micron.

1.8 the skeleton of the red carden consists of components such as arabinogalactan, mycolic acid and the like, wherein the content of the arabinose is 33 percent.

Accurately weighing an appropriate amount of arabinose control substances which are dried at 60 ℃ under reduced pressure for 1h, preparing arabinose into solutions containing 50 mug of arabinose, 60 mug of arabinose, 70 mug of arabinose, 80 mug of arabinose and 90 mug of arabinose per 1ml, accurately weighing 2ml of the solutions, placing the solutions in test tubes with plugs, respectively adding 0.5ml of 2.0% anthrone ethyl acetate solution and 4.0ml of concentrated sulfuric acid under the condition of ice bath cooling, shaking uniformly, preserving the temperature in a water bath at 80 ℃ for 30min, immediately cooling to room temperature by using running water, measuring the optical density at 625nm wavelength by using a spectrophotometry method (using pure water as a blank control instead of a standard solution), wherein the abscissa is the arabinose concentration, and the ordinate is the optical density value, and drawing a standard curve.

Accurately weighing 0.6-1.0 mg of sample into a test tube with a plug by using an analytical balance, and adding 4ml of distilled water for uniformly mixing. Then adding 1.0ml of 2% ethyl acetate anthrone and 8.0ml of concentrated sulfuric acid into ice water bath, shaking up, cooling, developing color in 80 deg.C water bath for 30min, cooling to room temperature with flowing water, measuring optical density at 625nm, and calculating polysaccharide content according to standard curve, wherein the polysaccharide content should not be less than 33%.

It should be understood that the above embodiments are only examples for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims

1. The preparation method of the red card skeleton powder is characterized by comprising the following steps:

2. The method for preparing a rbc framework powder according to claim 1, wherein in the first step, the activation process of nocardia is as follows: inoculating Nocardia genus into solid culture medium by streaking, and culturing at 28 deg.C for two days;

3. The method for preparing a red card skeleton powder of claim 1, wherein in the first step, the sterilization temperature is 115-118 ℃, and the sterilization time is 20 min.

4. The method for preparing red card skeleton powder of claim 1, wherein in the first step, the rotation speed of the shaking table is 160-320 r/min.

5. A method for preparing a red card skeleton powder according to claim 1, wherein in the first step, the carbon source of the liquid medium is glucose powder, and the nitrogen source is yeast powder; the pH of the liquid medium is 7-8.

6. The method for preparing a red card skeleton powder according to claim 1, wherein the temperature for culturing in the first step is 26 ℃.

7. The method for preparing a red card skeleton powder of claim 1, wherein in the first step, the inoculation amount of 50mL of liquid culture medium is 0.75 mL.

8. The method for preparing a red card skeleton powder according to claim 1, wherein in the second step, the rotation speed of the centrifugation is 4000r/min, and the time is 5 min.

9. The preparation method of the red card skeleton powder according to claim 1, wherein in the third step, the bacterial suspension is placed in a 5% KOH solution and is subjected to water bath at 100 ℃ for 40 min;

10. A red card skeleton powder prepared from the red card skeleton powder of any one of claims 1 to 9.