Method for the preparation of medicated leaven massa by pure strain fermentation

Abstract

The present invention relates to a method for the preparation of Medicated Leaven Massa by pure strain fermentation, characterized in that the Medicated Leaven Massa is prepared by fermentation under suitable conditions with Penicillium chrysogenum as a single fermentative strain and the culture medium is made of 6 ingredients, including flour (and/or wheat bran), Artemisia carvifolia, Polygonum flaccidum Meissn., Xanthium sibiricum Patr., Semen Armeniacae Amarum., and Vigna umbellata.

Claims

1. A method for preparing Medicated Leaven Massa, comprising: using Penicillium chrysogenum as a single strain for fermentation, wherein a culture medium for the fermentation is made from flour or a combination of flour and wheat bran, and Armeniacae semen amarum, Vigna umbellata, Artemisia annua Linn., Polygonum hydropiper L. and Xanthium sibiricum Patr., and performing the fermentation at a temperature of 2832 C., humidity of 7080%, and a culture time of 510 days.

2. The method for preparing Medicated Leaven Massa according to claim 1, further comprising: weighing Armeniacae semen amarum, Polygonum hydropiper L. and Xanthium sibiricum Patr. and extracting juice therefrom to prepare an adhesive for the fermentation; weighing wheat bran and flour with a ratio of 1:55:1, adding Armeniacae semen amarum powder and Vigna umbellata powder in a proportion of 37% of the weight of total wheat bran and flour, mixing the above materials together to form a solid culture medium for the fermentation; adding Penicillium chrysogenum into the solid culture medium in a proportion of 15 g of Penicillium chrysogenum per 100 g of the solid culture medium, and mixing well to make a soft material; forming the soft material into fermentative blocks; using the blocks for fermentation until a yellow skin is all over the surface of the blocks; and cutting the blocks into pieces of about 1 cm.sup.3, and drying to obtain the Medicated Leaven Massa.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is the standard curve of aflatoxin B1.

(2) FIG. 2 is Blast result of Penicillium chrysogenum sequence of the present invention and JF834167.1 of Gene Bank database.

DETAILED DESCRIPTION

(3) The present invention is further described below in conjunction with specific examples, and advantages and features of the present invention will become clearer with the description. However, these examples are only exemplary, without constituting any limitation to the scope of the present invention.

(4) It is to be understood by those skilled in the art that modifications or replacements may be made to the details and forms of the present technical solutions without departing from the spirit and scope of the present invention, but these modifications and replacements all fall into the protection scope of the present invention.

Example 1: Preparation of Medicated Leaven Massa by Laboratory Fermentation of Penicillium chrysogenum

(5) 25 g of dried Artemisia annua Linn., Polygonum hydropiper L., and Xanthium sibiricum Patr. each were weighed, distilled water 10, 8 and 6 times the amount was added and decocted 3 times for 40 min each time, and the water decoctions were combined, concentrated to 250 mL, transferred to a 500 mL sterilized conical flask, sealed, placed on a clean workbench and left to cool for use.

(6) 500 g of wheat bran, 500 g of flour and 40 g of Armeniacae semen amarum and Vigna umbellata powder each were weighed, held in a 1000 mL beaker which was sealed by 8 layers of gauze placed between kraft papers, sterilized under 103.4 kPa at 121.3 C. in a steam autoclave for 20 min, taken out immediately after the sterilization was completed, transferred to a clean workbench and left to cool for use.

(7) The medicine juice processed above and 20 g of Penicillium chrysogenum were mixed into the sterile culture medium, well admixed, made into a round-cake shape having a diameter of about 12 cm and a thickness of about 1 cm, held in a sterilized dish having a caliber of 18 cm, placed upside-down in an incubator at 28 C. with a relative humidity of 70%, cultured for 7 d until a yellow skin appeared, stopped from fermenting, taken out, cut into fermentative blocks sized about 1 cm.sup.3, dried and collected for storage, and mothproofing was to be noticed.

Example 2: Preparation of Medicated Leaven Massa with Penicillium Chrysogenum in Production

(8) 5 kg of fresh Artemisia annua Linn., Polygonum hydropiper L., and Xanthium sibiricum Patr. each were weighed and squeezed for fresh juice as an adhesive for use.

(9) 30 kg of wheat bran, 20 kg of flour and 2 kg of Armeniacae semen amarum and Vigna umbellata powder each were weighed, the fresh juice processed above and 100 g of Penicillium chrysogenum were mixed into the sterile culture medium, well admixed, made into 20128 cm fermentative blocks, stacked in an inverted T shape, covered by wet substances, cultured for 10 d at 30 C. with a relative humidity of 75% until a yellow skin appeared, stopped from fermenting, taken out, cut into fermentative blocks sized about 1 cm.sup.3 and dried to obtain it, and this method is suitable for production use.

Experimental Example 1: Examination of Minimal Inhibitory Concentration

(10) Medicated Leaven Massa produces the pharmaceutical effect of inhibiting enteric flora disturbance by an inhibitory mechanism, and thus the present experiments examined the raw materials for Medicated Leaven Massa fermentation, commercially available Medicated Leaven Massa and the Medicated Leaven Massa from pure strain fermentation with Penicillium chrysogenum for their inhibitory activities:

(11) Sample A: 10 g of dried Artemisia annua, Siberian Cocklebur and Polygonum hydropiper and 2 g of the powders of Armeniacae semen amarum and Vigna umbellata each were taken, distilled water 10, 8 and 6 times (v/m) the amount were added to extract 3 times respectively, for 1 h each time, and the extracts were combined, concentrated and then dried under reduced pressure for 72 h to obtain the extract of Sample A, i.e. the extract of raw materials for Medicated Leaven Massa without the addition of flour and wheat bran.

(12) Sample B: 100 g of commercially available Medicated Leaven Massa was taken, 95% ethanol 10, 8 and 6 times (v/m) were added to extract 3 times respectively, for 1 h each time, and the extracts were combined and subjected to rotary evaporation to recover the solvent, and then dried under reduced pressure for 72 h to obtain the extract of Sample B.

(13) Sample C was the Medicated Leaven Massa samples fermented with a single strain of Penicillium chrysogenum and fried respectively, 100 g of each sample was weighted and the extracted method was the same as that of Sample B.

(14) 1.0024 g of Sample A, 0.3750 g of Sample B and 0.3015 g of Sample C were weighted precisely and placed in 5 mL volumetric flasks respectively, 200 mL DMSO was added to promote the dissolution, and finally distilled water was added to volume to formulate test samples having concentrations of 200.5 mg/mL, 75.0 mg/mL and 60.3 mg/mL respectively; in addition, a 4% DMSO aqueous solution was formulated as a blank control.

(15) The minimal inhibitory concentration (MIC) was determined by serial dilution method on 96-well plate. The following solutions were added to a clean sterile 96-well plate: 100 L of nutrient broth culture medium was added to the 96-well plate; 100 L of the sample was added to the 1st well and The well was mixed with the culture medium, then 100 L of it was taken out and added to the 2nd well and so forth to the 10th well, and 100 L of it was taken out and disposed in a waste liquid of 75% ethanol; 100 L of nutrient broth culture medium was further added to the 11th well; 100 L of bacteria solution for experimental use was added to each well except the 12th well and well mixed, and the above processes were sterile operation.

(16) At this point, the n well corresponded to a medicine concentration of C.sub.0/2.sup.n+1 (C.sub.0 was the initial concentration), wherein the 11th well was a medicine blank and the 12th well was a bacteria blank, and each sample was tested 3 times at the same time. The results were observed after being cultured at a constant temperature of 37 C. for 18 h.

(17) The minimal bactericidal concentration (MBC) was determined by an agar-plate zoning method, each of the above bacteria/medicine mixed solutions without bacterial growth was inoculated on nutrient agar culture medium and cultured at a constant temperature of 37 C. for 24 h, and the bacterial growth was observed.

(18) Examination Results of the Inhibitory Effect

(19) Except that the DMSO blank group had no inhibitory effect, all the other three samples had an inhibitory effect at different degrees, wherein Sample A, the extract of raw materials for Medicated Leaven Massa except culture medium, had a relatively weaker inhibitory effect. After being subject to pure strain fermentation with a single strain, Sample C had a significantly inhibitory effect which higher than that of Sample B and commercially available Medicated Leaven Massa. The experiments showed that the raw materials for Medicated Leaven Massa had a significant inhibitory effect superior to that of the commercially available product after being fermented with Penicillium chrysogenum (Table 1-4).

(20) TABLE-US-00002 TABLE 1 Minimal inhibitory concentration (MIC) of Sample A Minimal Inhibition in the sample well with the inhibitory corresponding concentration (mg/mL) concentration Medicine Bacteria MIC Sample A 50.12 25.06 12.53 6.26 3.13 blank blank (mg/mL) Shigella dysenteriae + + + + 25.06 Escherichia coli + + + + 25.06 Staphylococcus albus + + + + 25.06 Staphylococcus citreus + + + + 25.06 Shigella flexneri + + + + 25.06 Staphylococcus aureus + + + + 25.06 Salmonella paratyphi B + + + + 25.06 -Streptococcus + + + + 25.06 haemolyticus Note: Sample A was the extract of raw materials for Medicated Leaven Massa, indicates no bacterial growth in this well, and + indicates bacterial growth in this well.

(21) TABLE-US-00003 TABLE 2 Minimal inhibitory concentration (MIC) of Sample B Minimal Inhibition in the sample well with the inhibitory corresponding concentration (mg/mL) concentration Medicine Bacteria MIC Sample B 18.75 9.38 4.69 2.35 1.18 blank blank (mg/mL) Shigella dysenteriae + + + 4.69 Escherichia coli + + + 4.69 Staphylococcus albus + + + 4.69 Staphylococcus citreus + + + 4.69 Shigella flexneri + + + 4.69 Staphylococcus aureus + + + 4.69 Salmonella paratyphi B + + + 4.69 -Streptococcus + + + 4.69 haemolyticus Note: Sample B was the extract of commercially available Medicated Leaven Massa, indicates no bacterial growth in this well, and + indicates bacterial growth in this well.

(22) TABLE-US-00004 TABLE 3 Minimal inhibitory concentration (MIC) of Sample C Minimal Inhibition in the sample well with the inhibitory corresponding concentration (mg/mL) concentration Medicine Bacteria MIC Sample C 15.08 7.54 3.77 1.84 0.94 blank blank (mg/mL) Shigella dysenteriae + + 1.84 Escherichia coli + + 1.84 Staphylococcus albus + + 1.84 Staphylococcus citreus + + + 3.77 Shigella flexneri + + 1.84 Staphylococcus aureus + + 1.84 Salmonella paratyphi B + + 1.84 -Streptococcus + + 1.84 haemolyticus Note: Sample C was the extract of the Medicated Leaven Massa fermented with a single strain of Penicillium chrysogenum, indicates no bacterial growth in this well, and + indicates bacterial growth in this well.

(23) TABLE-US-00005 TABLE 4 Minimal bactericidal concentration (MBC) Sample Strain Sample A Sample B Sample C Shigella dysenteriae 4.69 1.84 Escherichia coli 9.38 1.84 Staphylococcus albus 4.69 1.84 Staphylococcus citreus 4.69 3.77 Shigella flexneri 9.38 1.84 Staphylococcus aureus 4.69 1.84 Salmonella paratyphi B 4.69 1.84 -Streptococcus 4.69 1.84 haemolyticus Note: indicates no Minimal bactericidal concentration, Sample A was the extract of raw materials for Medicated Leaven Massa, Sample B was the extract of commercially available Medicated Leaven Massa, and Sample C was the extract of the Medicated Leaven Massa fermented with a single strain of Penicillium chrysogenum,

Experimental Example 2: Determination of the Content of Aflatoxin B1

(24) Pretreatment of the samples: the samples of the Medicated Leaven Massa fermented with a single strain of Aspergillus oryzae and Penicillium chrysogenum were pulverized and sieved with a 50-mesh sieve, respectively. 5 g of the two samples were weighted, 25 mL of sample extract was added, shaken for 10 min, and centrifuged at 4000 r/min for 5 min, 5 mL of the supernatant was taken, 10 mL of dichloromethane was added, shaken for 5 min, centrifuged and stratified, the lower layer of the dichloromethane phase was taken for use, 10 mL of dichloromethane was further added to the upper layer of the water phase, the extraction was repeated once, and the dichloromethane phases were combined. It was blow-dried with nitrogen at 50 C., 2 mL of sample extract was added to dissolve the volatiles, and 8 mL of sample diluent was further added for dilution to obtain the sample solution to be tested.

(25) Determination of absorbance: The required reagents were taken out from a refrigerated environment before the experiments, and balanced to room temperature (2025 C.). A required number of microplates were taken out, 50 L of the standard or the sample was added to the corresponding micro well, ABF1 enzyme label was added at 50 L/well, shaken gently and well mixed. After being covered by a covering film, the plate was placed in a dark environment at 25 C. for reaction for 30 min, the covering film was carefully lifted, the liquid within the wells was spin-dried, and it was washed adequately 5 times at an interval between each time of 30 s with a washing solution at 300 L/well, and patted to dry with absorbent paper. Substrate A solution at 50 L/well and then substrate B solution at 50 L/well were added, shaken gently and well mixed, and after being covered by a covering film, the plate was placed in a dark environment at 25 C. for reaction for 15 min. A stopping solution 50 L/well was added and shaken gently for well mixed, and the microplate reader was set at 450 nm to determine the OD value of each well.

(26) Determination Results of the Content of Aflatoxin B1

(27) (1) Determination of the Absorbance Percentage of the Standard

(28) The absorbance percentage of the standard equals the mean absorbance of the standard divided by the absorbance of the first standard (i.e. 0 standard) and then multiplied by 100%, that is, absorbance percentage (%)=B/B.sub.0100%.

(29) B stands for the mean absorbance of the standard solution.

(30) B.sub.0 stands for the mean absorbance of the 0 (ppb) sample solution.

(31) The measured absorbance percentages of the standard are seen in Table 5.

(32) TABLE-US-00006 TABLE 5 Absorbance percentages of the standard Standard Absorbance concentration Mean percentage B/B.sub.0 No. (ppb) absorbance B (%) B.sub.0 0 1.928 B.sub.1 0.1 1.696 87.97 B.sub.2 0.25 1.640 85.06 B.sub.3 0.5 1.576 81.74 B.sub.4 1.5 1.320 68.46 B.sub.5 5 0.472 24.48 Note: B0-B5 is the standards given in a test kit.

(33) (2) Plotting of the Standard Curve

(34) The standard curve was plotted with the absorbance percentage of the standard as the vertical ordinate and the concentrations of the aflatoxin B1 standard (ppb) as the horizontal ordinate (see FIG. 1).

(35) (3) Determination of the Absorbance Percentage of the Sample

(36) The absorbance percentage of the sample equals the mean absorbance of the samples divided by the absorbance of the first standard (i.e. 0 standard) and then multiplied by 100%, that is, absorbance percentage (%)=/B.sub.0100%.

(37) stands for mean absorbance of the sample solutions

(38) B.sub.0 stands for mean absorbance of the 0 (ppb) sample solution

(39) The calculation method of the absorbance percentage of the sample was the same as above, and the results are seen in Table 6.

(40) TABLE-US-00007 TABLE 6 Absorbance of the sample and content of aflatoxin B1 Absorbance Residue of percentage /B.sub.0 aflatoxin B1 Absorbance A (%) (g/kg) Fermented product 1.352 70.12 14.25 with Aspergillus oryzae Fermented product 1.664 86.31 1.63 with Penicillium chrysogenum

(41) As can be seen from the experimental results, the residue of aflatoxin B1 in the Medicated Leaven Massa fermented with Aspergillus oryzae was 14.25 g/kg, which was higher than the Chinese Pharmacopoeia standard 5 g/kg, the residue of aflatoxin B1 in the Medicated Leaven Massa fermented with Penicillium chrysogenum was 1.63 g/kg, which was lower than Chinese Pharmacopoeia standard 5 g/kg. Thereby it can be speculated that Aspergillus oryzae as an unwanted strain for Medicated Leaven Massa fermentation is the main source of aflatoxin.