CN115261276A - Method for improving activity of lactobacillus plantarum and preparation of composite microbial inoculum - Google Patents
Method for improving activity of lactobacillus plantarum and preparation of composite microbial inoculum Download PDFInfo
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The invention provides a method for improving the activity of lactobacillus plantarum, which comprises the following steps: (1) preparing an astragalus aqueous extract; (2) preparing an aqueous extract of hawthorn; (3) preparing an aqueous extract of the largehead atractylodes rhizome; (4) Mixing the obtained astragalus root aqueous extract, hawthorn aqueous extract and bighead atractylodes rhizome extract to obtain an aqueous extract mixed solution; (5) Inoculating lactobacillus plantarum into an MRS liquid culture medium for culture to obtain activated seed liquid; (6) Adding activated seed liquid into MRS liquid culture medium, adding 3% -5% water extract mixed solution, and co-culturing. The invention also provides a preparation method of the composite microbial inoculum. According to the invention, the astragalus, hawthorn and bighead atractylodes rhizome are added into lactobacillus plantarum for co-culture, so that the activity of the lactobacillus plantarum, namely the growth and reproduction capacity and the acid and bile salt resistance capacity are obviously improved; meanwhile, the composite microbial inoculum is prepared by drying the mixed solution into powder and mixing the powder with freeze-dried lactobacillus plantarum powder, and is used as a green feed additive.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a method for improving the activity of lactobacillus plantarum and a preparation method of a composite microbial inoculum.
Background
Lactobacillus plantarum is one of lactic acid bacteria, is an anaerobic or facultative anaerobic strain, is a straight or bent rod-shaped, single, sometimes paired or chained strain, and belongs to homofermentative lactic acid bacteria. The lactobacillus plantarum is one of probiotics strains which are published by the ministry of health of China and can be used for food, can survive in large quantities in intestinal tracts, maintain the flora balance in the intestinal tracts, promote the absorption of nutrient substances, have a certain immunoregulation effect, have a certain inhibition effect on pathogenic bacteria, and can be used as a probiotic feed additive for the breeding industry. However, after feeding, lactobacillus plantarum needs to be effectively resistant to the digestive system of animals, particularly to the influence of gastric acid and bile salt on the activity thereof, so that it is urgently needed to improve the growth and reproduction ability and the acid and bile salt resistance of the existing lactobacillus plantarum feeds.
Disclosure of Invention
The invention aims to provide a method for improving the activity of lactobacillus plantarum and a preparation method of a composite microbial inoculum, wherein the activity (namely, the growth reproductive capacity and the acid and bile salt resistance) of lactobacillus plantarum is remarkably improved by adding astragalus mongholicus, hawthorn and bighead atractylodes rhizome into the lactobacillus plantarum for co-culture, and the composite microbial inoculum is prepared by drying a mixed solution of the astragalus mongholicus, the hawthorn and the bighead atractylodes rhizome into powder and mixing the powder with freeze-dried lactobacillus plantarum powder and is used as a green feed additive for the breeding industry.
The invention adopts the following technical scheme to solve the technical problems:
a method for improving the activity of lactobacillus plantarum comprises the following steps:
(1) Boiling radix astragali slice with clear water, and filtering to obtain first leaching solution; mixing the filtered residue with clear water, boiling, and filtering to obtain a second leaching solution; mixing the leaching solutions obtained for the first and second times to obtain radix astragali water extract;
(2) Decocting the haw slices with clear water, filtering, decocting the residue twice, filtering, and combining the three decoctions to obtain the haw aqueous extract;
(3) Adding clear water into the white atractylodes rhizome, and soaking overnight; decocting twice, and filtering to obtain a crude drug solution; overnight, adding absolute ethyl alcohol every other day, and carrying out alcohol precipitation overnight; centrifuging the next day to obtain supernatant; recovering ethanol to obtain water extract of Atractylodis rhizoma;
(4) Mixing the obtained astragalus aqueous extract, hawthorn aqueous extract and bighead atractylodes rhizome extract according to the ratio of 1:1:1 to obtain an aqueous extract mixed solution;
(5) Taking lactobacillus plantarum by using an inoculating loop, inoculating the lactobacillus plantarum into an MRS liquid culture medium, and carrying out overnight culture in an incubator at 39 ℃ to obtain activated seed liquid;
(6) Adding 1% of the activated seed liquid into a fresh MRS liquid culture medium, then adding 3-5% of water extract mixed solution, and co-culturing at 39 ℃.
In a preferred embodiment of the present invention, in step (1), the astragalus membranaceus slices are boiled with clear water for 1 hour, and filtered to obtain a first leaching solution; mixing the filtered residue with clear water, boiling for 30min, and filtering to obtain a second leaching solution; mixing the first and second obtained leaching solutions to obtain radix astragali water extract.
As one preferable mode of the present invention, in the step (2), the hawthorn pieces and the clear water are decocted for 2 hours, the residues are decocted twice after filtration, the time is 1.5 hours and 1 hour respectively, and the three decocted liquids are combined after filtration, so as to obtain the hawthorn aqueous extract.
In a preferred embodiment of the present invention, in the step (3), clear water is added to the atractylodes macrocephala koidz, and the mixture is soaked overnight; decocting twice, each for 30min, and filtering to obtain a crude drug solution; overnight at 4 ℃, adding absolute ethyl alcohol every other day, and carrying out alcohol precipitation overnight at 4 ℃; centrifuging for 5min the next day, and collecting supernatant; recovering ethanol to obtain water extract of Atractylodis rhizoma.
As one of the preferable modes of the invention, in the step (1), the mass ratio of the astragalus membranaceus tablets to the clear water is 1:10, filtering by using four layers of gauze; the mass ratio of the residue to the clear water is 1:5; mixing the first leaching solution and the second leaching solution, and metering to 500mL;
in the step (2), the mass ratio of the hawthorn slices to the clear water is 1:10, filtering by using four layers of gauze; the mass ratio of the residue to the clear water is 1:10; merging the three decocted fluids, and fixing the volume to 500mL;
in the step (3), the proportion of the white atractylodes rhizome tablets to the clear water is 1:10, filtering by using four layers of gauze; adding absolute ethyl alcohol to make the concentration of the ethyl alcohol in the solution be 60%, and centrifuging at 4 ℃ and 11000rpm/min; and recovering ethanol by using a rotary evaporator to obtain the white atractylodes rhizome extract, and metering to 500mL.
In a preferred embodiment of the present invention, in the step (5), the lactobacillus plantarum strain preserved in one loop slant is extracted by using an inoculating loop, inoculated into 100mL of MRS liquid medium, and cultured overnight in an incubator at 39 ℃.
In a preferred embodiment of the present invention, in the step (6), the specific amount of the aqueous extract mixed solution is 3%.
A preparation method of a complex microbial inoculum comprises the following steps:
(1) Boiling radix astragali slice with clear water, and filtering to obtain first leaching solution; mixing the filtered residue with clear water, boiling, and filtering to obtain a second leaching solution; mixing the first and second obtained leaching solutions to obtain radix astragali water extract;
(2) Decocting fructus crataegi with clear water, filtering, decocting the residue twice, filtering, and mixing the three decoctions to obtain fructus crataegi water extract;
(3) Adding clear water into the white atractylodes rhizome, and soaking overnight; decocting twice, and filtering to obtain a raw liquid medicine; overnight, adding absolute ethyl alcohol every other day, and carrying out alcohol precipitation overnight; centrifuging the next day to obtain supernatant; recovering ethanol to obtain water extract of Atractylodis rhizoma;
(4) Mixing the obtained astragalus aqueous extract, hawthorn aqueous extract and bighead atractylodes rhizome extract according to the proportion of 1:1:1 to obtain a water extract mixed solution; then, carrying out spray drying on the water extract mixed solution to obtain water extract mixed powder;
(5) Taking lactobacillus plantarum by using an inoculating loop, inoculating the lactobacillus plantarum into an MRS liquid culture medium, and culturing overnight in an incubator at 39 ℃;
(6) After overnight culture, centrifugally collecting thalli, and washing for a plurality of times by using sterile normal saline to obtain lactobacillus plantarum bacterial sludge; adding a protective agent into the lactobacillus plantarum bacterial paste, pre-freezing, and freeze-drying in a freeze dryer; after the freeze drying is finished, grinding into powder;
(7) And (4) mixing the mixed powder of the water extract obtained in the step (4) with the lactobacillus plantarum freeze-dried powder obtained in the step (6) to obtain the compound microbial inoculum required by the target.
In a preferred embodiment of the present invention, in the step (4), the spray drying is performed under conditions of an inlet air temperature of 180 ℃ and a peristaltic pump speed of 7rpm/min.
In a preferred embodiment of the present invention, in the step (6), after the overnight culture is finished, centrifuging the mixture at 20 ℃ and 5000rpm/min for 5min by using a high-speed refrigerated centrifuge, collecting the bacterial cells, and washing the bacterial cells with sterile physiological saline for 2 times to obtain lactobacillus plantarum bacterial sludge; adding a protective agent with 20% of the volume of the fermentation liquor into the lactobacillus plantarum bacterial sludge, pre-freezing for 2 hours at the temperature of minus 80 ℃, and then placing the mixture into a freeze dryer for freeze drying for 24 to 36 hours; after freeze-drying, grinding into powder; wherein, the protective agent comprises the following components: lactose 9.05g/100mL, tryptone 8.79g/100mL, trehalose 11.21g/100mL.
In the invention, the adopted MRS liquid culture medium is a common culture medium in the field, and the components are not described again. The lactobacillus plantarum which is acted with the mixed solution of the aqueous extract can be any commercially available lactobacillus plantarum in the field.
The efficacy principle of astragalus, hawthorn and bighead atractylodes rhizome is as follows:
the astragalus contains more than 20 trace elements, more than 50 saponins and triterpenoids and more than 20 flavonoids. The astragalus polysaccharide is a polysaccharide extract of the root of astragalus, has the highest content in astragalus, has strong immunocompetence and is an important part for enhancing the immunity of organisms.
The hawthorn mainly contains inorganic salts, amino acids, organic acids and flavonoid compounds. Triterpenes are important components in organic acid compounds in hawthorn, and have important functions of tonifying heart, improving circulation, helping digestion and the like. The flavonoids compounds in the hawthorn have the effects of reducing blood pressure, resisting oxidation and the like, and are main bioactive components of the pharmacological action of the hawthorn.
The chemical components of the bighead atractylodes rhizome mainly comprise volatile oil and polysaccharide. Wherein, the atractylodes macrocephala polysaccharide is the main active ingredient of the atractylodes macrocephala, can influence the endocrine system, can effectively regulate the immune function, and has obvious effects of resisting oxidation and lowering blood pressure.
Compared with the prior art, the invention has the advantages that:
(1) The mixed solution of the water extract can improve the growth and reproduction capacity of the lactobacillus plantarum; researches show that astragalus polysaccharide in the astragalus extract and organic acid and flavonoid compounds in the hawthorn extract have the most obvious influence on the growth and reproduction capacity of lactobacillus plantarum;
(2) The water extract mixed solution can improve the acid resistance of the lactobacillus plantarum; researches show that astragalus polysaccharide in the astragalus extract and flavonoid compounds in the hawthorn extract have obvious improvement on the acid resistance of lactobacillus plantarum;
(3) The water extract mixed solution can improve the bile salt resistance of the lactobacillus plantarum; researches show that astragalus polysaccharide in the astragalus extract, organic acid and flavonoid compounds in the hawthorn extract and atractylodes polysaccharide in the atractylodes extract can improve the bile salt resistance of lactobacillus plantarum;
(4) The astragalus, the hawthorn and the bighead atractylodes rhizome belong to edible and medicinal plants for feeding, the lactobacillus plantarum belongs to a microorganism for feeding, and the composite microbial inoculum prepared by compounding the astragalus, the hawthorn and the bighead atractylodes rhizome can be used as a novel green feed additive to be developed and applied to the breeding industry.
Drawings
FIG. 1 is a graph showing the effect of mixed solutions of different concentrations of aqueous extracts on the growth and reproduction ability of Lactobacillus plantarum obtained in example 4;
FIG. 2 is a graph comparing the effect of different concentrations of aqueous extracts of Astragalus membranaceus and mixed solutions on the growth and reproduction ability of Lactobacillus plantarum in example 4;
FIG. 3 is a graph comparing the effect of different concentrations of aqueous extracts of Crataegus pinnatifida and mixed solutions on the growth and reproduction of Lactobacillus plantarum in example 4;
FIG. 4 is a graph comparing the effects of different concentrations of the Atractylodis rhizoma extract and the mixed solution on the growth and reproduction ability of Lactobacillus plantarum in example 4;
FIG. 5 is a graph showing the effect of a 3% aqueous extract mixture solution on the acid resistance of Lactobacillus plantarum in example 5;
FIG. 6 is a graph showing the effect of a single solution and a mixed solution on the acid resistance of Lactobacillus plantarum in example 5;
FIG. 7 is a graph showing the effect of the 3% mixed solution on the bile salt resistance of Lactobacillus plantarum in example 6;
FIG. 8 is a graph comparing the effect of a single solution and a mixed solution on the bile salt resistance of Lactobacillus plantarum in example 6;
FIG. 9 is a drawing showing a sample of the Lactobacillus plantarum freeze-dried powder and the aqueous extract mixed powder obtained in example 7 (in the drawing, drawing A shows the Lactobacillus plantarum freeze-dried powder, and drawing B shows the aqueous extract mixed powder).
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
The method for improving the activity of lactobacillus plantarum of the embodiment comprises the following steps:
(1) Boiling the astragalus slices with clear water (the mass ratio is 1; mixing the filtered residue with clear water (1:5 by mass ratio) again, boiling for 30min, filtering with four layers of gauze to obtain a second leaching solution; mixing the first and second obtained leaching solutions, and diluting to 500mL to obtain radix astragali water extract.
(2) Decocting the haw slices with clear water (the mass ratio is 1: 10) for 2 hours, filtering the haw slices with four layers of gauze, and decocting residues twice for 1.5 hours and 1 hour respectively; filtering, combining the three decocted liquids, and fixing the volume to 500mL to obtain the hawthorn aqueous extract.
(3) Adding clear water (the proportion of the white atractylodes rhizome tablets to the clear water is 1; decocting twice, each for 30min, and filtering with four layers of gauze to obtain the original liquid medicine; overnight at 4 ℃, adding absolute ethyl alcohol every other day to ensure that the concentration of the ethyl alcohol in the solution is 60 percent, and carrying out alcohol precipitation overnight at 4 ℃; centrifuging for 5min at 4 deg.C and 11000rpm/min the next day, and collecting supernatant; and recovering ethanol by using a rotary evaporator to obtain the white atractylodes rhizome extract, and fixing the volume to 500mL.
(4) Mixing the obtained astragalus aqueous extract, hawthorn aqueous extract and bighead atractylodes rhizome extract according to the ratio of 1:1:1 to obtain an aqueous extract mixed solution.
(5) The lactobacillus plantarum preserved in the slant of one ring is scratched by an inoculating ring, inoculated into 100mL of MRS liquid culture medium and cultured in an incubator at 39 ℃ overnight to obtain activated seed liquid.
(6) Adding 1% of the activated seed solution into fresh MRS liquid culture medium, adding 3% of water extract mixed solution, and co-culturing at 39 deg.C.
It should be noted that, in this embodiment, the adopted MRS liquid medium is a medium commonly used in the art, and the components thereof are not described again. The lactobacillus plantarum which is acted with the mixed solution of the aqueous extract can be any commercially available lactobacillus plantarum in the field.
Example 2
The method for improving the activity of lactobacillus plantarum in the embodiment has the steps basically the same as those in the embodiment 1, and mainly differs from the following steps: in the step (6), 1% of the activated seed solution is added into a fresh MRS liquid culture medium, and then 4% of the water extract mixed solution is added, and the mixture is co-cultured at the temperature of 39 ℃.
Example 3
The method for improving the activity of lactobacillus plantarum in this example basically comprises the same steps as in example 1, with the main differences that: in the step (6), 1% of the activated seed solution is added into a fresh MRS liquid culture medium, and then 5% of an aqueous extract mixed solution is added, and co-culture is carried out at the temperature of 39 ℃.
Experimental example 4
This example illustrates the effect of the aqueous extract mixture solution of the present invention on the growth and reproduction ability of Lactobacillus plantarum.
1. Action experiment of water extract mixed solution with different concentrations:
1. the experimental steps are as follows:
(1) Boiling the astragalus slices with clear water (the mass ratio is 1; mixing the filtered residue with clear water (1:5 by mass ratio) again, boiling for 30min, filtering with four layers of gauze to obtain a second leaching solution; mixing the leaching liquor obtained in the first and second times, and then metering to 500mL to obtain the astragalus aqueous extract.
(2) Decocting the haw slices with clear water (the mass ratio is 1: 10) for 2 hours, filtering the haw slices with four layers of gauze, and decocting residues twice for 1.5 hours and 1 hour respectively; filtering, combining the three decoctions, and diluting to 500mL to obtain fructus crataegi water extract.
(3) Adding clear water (the proportion of the white atractylodes rhizome tablets to the clear water is 1; decocting twice, each for 30min, and filtering with four layers of gauze to obtain the original liquid medicine; overnight at 4 ℃, adding absolute ethyl alcohol every other day to ensure that the concentration of the ethyl alcohol in the solution is 60 percent, and carrying out alcohol precipitation overnight at 4 ℃; centrifuging for 5min at 4 deg.C and 11000rpm/min the next day, and collecting supernatant; and recovering ethanol by using a rotary evaporator to obtain the white atractylodes rhizome extract, and metering to 500mL.
(4) Mixing the obtained astragalus aqueous extract, hawthorn aqueous extract and bighead atractylodes rhizome extract according to the ratio of 1:1:1 to obtain a water extract mixed solution.
(5) A loop of the slant-preserved Lactobacillus plantarum (purchased from Lactobacillus plantarum JYLP-326 of Jiayi, china family) was streaked with an inoculating loop, inoculated into 100mL of MRS liquid medium, and cultured overnight in an incubator at 39 ℃ to obtain an activated seed solution.
(6) Adding 1% of the above activated seed solution into fresh MRS liquid culture medium, adding mixed solution of 0,3%,5%, and 10% water extract, culturing at 39 deg.C, and measuring OD once every 2 hr 600 The value is obtained.
2. The experimental results are as follows: as shown in fig. 1.
FIG. 1 is a graph showing the effect of adding mixed aqueous extract solutions of different concentrations on the growth and reproduction of Lactobacillus plantarum in this example. As can be seen from FIG. 1, the growth and reproduction ability of Lactobacillus plantarum can be improved when 3% and 5% are added, wherein the improvement in growth and reproduction ability of Lactobacillus plantarum is most pronounced when 3% is added, and the growth and reproduction ability thereof is reduced when 10% is added.
Therefore, the lactobacillus plantarum growth and reproduction capacity can be effectively improved by adding the 3-5% water extract mixed solution, and the optimal concentration is 3%.
2. Control experiment for "single solution":
1. the experimental steps are as follows:
(1) Boiling the astragalus slices with clear water (the mass ratio is 1; mixing the filtered residue with clear water (1:5 by mass ratio) again, boiling for 30min, filtering with four layers of gauze to obtain a second leaching solution; mixing the first and second obtained leaching solutions, and diluting to 500mL to obtain radix astragali water extract.
(2) Decocting the haw slices with clear water (the mass ratio is 1: 10) for 2 hours, filtering the haw slices with four layers of gauze, and decocting residues twice for 1.5 hours and 1 hour respectively; filtering, combining the three decoctions, and diluting to 500mL to obtain fructus crataegi water extract.
(3) Adding clear water (the proportion of the white atractylodes rhizome tablets to the clear water is 1; decocting twice, each for 30min, and filtering with four layers of gauze to obtain the original medicinal liquid; standing overnight at 4 deg.C, adding anhydrous ethanol every other day to make ethanol concentration in the solution be 60%, and precipitating with ethanol at 4 deg.C overnight; centrifuging for 5min at 4 deg.C and 11000rpm/min the next day, and collecting supernatant; and recovering ethanol by using a rotary evaporator to obtain the white atractylodes rhizome extract, and fixing the volume to 500mL.
(4) A loop of the slant-preserved Lactobacillus plantarum (purchased from Lactobacillus plantarum JYLP-326 of Jiayi, china family) was streaked with an inoculating loop, inoculated into 100mL of MRS liquid medium, and cultured overnight in an incubator at 39 ℃ to obtain an activated seed solution.
(5) Adding 1% of the activated seed solution obtained in step (4) into fresh MRS liquid culture medium, adding 0,3%,5%,10%,20% of radix astragali aqueous extract, culturing at 39 deg.C, and measuring OD every 2h 600 The value is obtained.
(6) Adding 1% of the activated seeds obtained in the step (4) into a fresh MRS liquid culture mediumAdding 0,3%,5%,10% fructus crataegi water extract into the seed solution, culturing at 39 deg.C, and measuring OD every 2 hr 600 The value is obtained.
(7) Adding 1% of the activated seed solution obtained in step (4) into fresh MRS liquid culture medium, adding 0,3%,5%, and 10% of Atractylodis rhizoma water extract, culturing at 39 deg.C, and measuring OD once every 2h 600 The value is obtained.
2. The experimental results are as follows: as shown in fig. 2-4.
FIG. 2 shows the effect of adding different concentrations of aqueous extracts of Astragalus membranaceus and mixed solution on the growth and reproduction ability of Lactobacillus plantarum, wherein the OD value of the mixed solution is significantly higher than that of the aqueous extracts of Astragalus membranaceus. FIG. 3 shows the effect of adding aqueous extracts of Hawthorn fruit and mixed solution of different concentrations on the growth and reproduction ability of Lactobacillus plantarum, wherein the mixed solution has a significantly higher improvement on the growth and reproduction ability of Lactobacillus plantarum than the aqueous extracts of Hawthorn fruit. FIG. 4 is a graph showing the effect of adding different concentrations of Atractylodis rhizoma extract and mixed solution on the growth and reproduction ability of Lactobacillus plantarum, wherein the increase of the mixed solution was significantly higher than that of the Atractylodis rhizoma extract.
Therefore, the mixed solution added with the aqueous extract can improve the growth and reproduction capacity of the lactobacillus plantarum better than a single solution.
Experimental example 5
This example is intended to demonstrate the effect of the aqueous extract mixture solution of the present invention on the acid resistance of Lactobacillus plantarum.
1. Action experiment of water extract mixed solution:
1. the experimental steps are as follows:
(1) Boiling the astragalus slices with clear water (the mass ratio is 1; mixing the filtered residue with clear water (1:5 by mass ratio) again, boiling for 30min, filtering with four layers of gauze to obtain a second leaching solution; mixing the first and second obtained leaching solutions, and diluting to 500mL to obtain radix astragali water extract.
(2) Decocting the haw slices with clear water (the mass ratio is 1: 10) for 2 hours, filtering the haw slices with four layers of gauze, and decocting residues twice for 1.5 hours and 1 hour respectively; filtering, combining the three decoctions, and diluting to 500mL to obtain fructus crataegi water extract.
(3) Adding clear water (the proportion of the white atractylodes rhizome tablets to the clear water is 1; decocting twice, each for 30min, and filtering with four layers of gauze to obtain the original liquid medicine; overnight at 4 ℃, adding absolute ethyl alcohol every other day to ensure that the concentration of the ethyl alcohol in the solution is 60 percent, and carrying out alcohol precipitation overnight at 4 ℃; centrifuging for 5min at 4 deg.C and 11000rpm/min the next day, and collecting supernatant; and recovering ethanol by using a rotary evaporator to obtain the white atractylodes rhizome extract, and fixing the volume to 500mL.
(4) Mixing the obtained astragalus aqueous extract, hawthorn aqueous extract and bighead atractylodes rhizome extract according to the ratio of 1:1:1 to obtain an aqueous extract mixed solution.
(5) A loop of the slant-preserved Lactobacillus plantarum (purchased from Lactobacillus plantarum JYLP-326 of Jiayi, china family) was streaked with an inoculating loop, inoculated into 100mL of MRS liquid medium, and cultured overnight in an incubator at 39 ℃ to obtain an activated seed solution.
(6) Adding 1% of activated seed solution into fresh MRS liquid culture medium with pH values of 2, 3 and 7, respectively, culturing at 39 deg.C for 4 hr according to food retention time in stomach, and measuring OD 600 Values, as control. Meanwhile, adding 1% of inoculation amount of activated seed solution into fresh MRS liquid culture medium with pH values of 2, 3 and 7 respectively, and then adding 3% of water extract mixed solution; the food was incubated at 39 ℃ for 4 hours in an incubator according to the residence time in the stomach, and the OD was measured 600 Values were used as experimental groups.
2. The experimental results are as follows: as shown in fig. 5.
FIG. 5 is a graph showing the effect of adding a 3% aqueous extract mixture solution on the acid resistance of Lactobacillus plantarum in this example. As can be seen from fig. 5, when pH =2, pH =3, and pH =7, the OD value of lactobacillus plantarum was significantly increased after the 3% mixed solution was added, indicating that the 3% mixed solution could improve the acid resistance of lactobacillus plantarum.
Therefore, the acid resistance of the lactobacillus plantarum can be effectively improved by adding the mixed solution of the aqueous extract.
2. Control experiment for "single solution":
1. the experimental steps are as follows:
(1) Boiling the astragalus slices with clear water (the mass ratio is 1; mixing the filtered residue with clear water (1:5 by mass ratio) again, boiling for 30min, filtering with four layers of gauze to obtain a second leaching solution; mixing the first and second obtained leaching solutions, and diluting to 500mL to obtain radix astragali water extract.
(2) Decocting the haw slices with clear water (the mass ratio is 1: 10) for 2 hours, filtering the haw slices with four layers of gauze, and decocting residues twice for 1.5 hours and 1 hour respectively; filtering, combining the three decoctions, and diluting to 500mL to obtain fructus crataegi water extract.
(3) Adding clear water (the proportion of the white atractylodes rhizome tablets to the clear water is 1; decocting twice, each for 30min, and filtering with four layers of gauze to obtain the original liquid medicine; overnight at 4 ℃, adding absolute ethyl alcohol every other day to ensure that the concentration of the ethyl alcohol in the solution is 60 percent, and carrying out alcohol precipitation overnight at 4 ℃; centrifuging for 5min at 11000rpm/min at 4 ℃ the next day, and taking supernatant; and recovering ethanol by using a rotary evaporator to obtain the white atractylodes rhizome extract, and metering to 500mL.
(4) A loop of the slant-preserved Lactobacillus plantarum (purchased from Lactobacillus plantarum JYLP-326 of Jiayi, china family) was streaked with an inoculating loop, inoculated into 100mL of MRS liquid medium, and cultured overnight in an incubator at 39 ℃ to obtain an activated seed solution.
(5) Adding 1% of inoculation amount of activated seed liquid into fresh MRS liquid culture medium with pH values of 2, 3 and 7 respectively, and then adding 10% of astragalus aqueous extract; the food was incubated at 39 ℃ for 4 hours in an incubator according to the residence time in the stomach, and the OD was measured 600 Numerical values.
(6) Adding 1% of inoculation amount of activated seed liquid into fresh MRS liquid culture medium with pH values of 2, 3 and 7 respectively, and then adding 5% of hawthorn aqueous extract; the food was incubated at 39 ℃ for 4 hours in an incubator according to the residence time in the stomach, and the OD was measured 600 Numerical values.
(7) Adding 1% of inoculation amount of activated seed liquid into fresh MRS liquid culture medium with pH values of 2, 3 and 7 respectively, and then adding 3% of water extract of rhizoma Atractylodis Macrocephalae; the food was incubated at 39 ℃ for 4 hours in an incubator according to the residence time in the stomach, and the OD was measured 600 Numerical values.
2. The experimental results are as follows: as shown in fig. 6.
FIG. 6 shows the effect of the single solution and the mixed solution on the acid resistance of Lactobacillus plantarum. At pH =2, pH =3 and pH =7, the addition of the mixed solution has a greater influence on the improvement of the acid resistance of lactobacillus plantarum than the addition of the single solution.
Therefore, the acid resistance of the lactobacillus plantarum can be improved by adding the aqueous extract mixed solution in the invention compared with a single solution.
Experimental example 6
This example illustrates the effect of a mixed aqueous extract solution of the present invention on the bile salt tolerance of Lactobacillus plantarum.
1. Action experiment of water extract mixed solution:
1. the experimental steps are as follows:
(1) Boiling the astragalus slices with clear water (the mass ratio is 1; mixing the filtered residue with clear water (1:5 by mass ratio) again, boiling for 30min, filtering with four layers of gauze to obtain a second leaching solution; mixing the first and second obtained leaching solutions, and diluting to 500mL to obtain radix astragali water extract.
(2) Decocting the haw slices with clear water (the mass ratio is 1: 10) for 2 hours, filtering the haw slices with four layers of gauze, and decocting residues twice for 1.5 hours and 1 hour respectively; filtering, combining the three decocted liquids, and fixing the volume to 500mL to obtain the hawthorn aqueous extract.
(3) Adding clear water (the proportion of the white atractylodes rhizome tablets to the clear water is 1; decocting twice, each for 30min, and filtering with four layers of gauze to obtain the original liquid medicine; standing overnight at 4 deg.C, adding anhydrous ethanol every other day to make ethanol concentration in the solution be 60%, and precipitating with ethanol at 4 deg.C overnight; centrifuging for 5min at 11000rpm/min at 4 ℃ the next day, and taking supernatant; and recovering ethanol by using a rotary evaporator to obtain the white atractylodes rhizome extract, and fixing the volume to 500mL.
(4) Mixing the obtained astragalus aqueous extract, hawthorn aqueous extract and bighead atractylodes rhizome extract according to the ratio of 1:1:1 to obtain an aqueous extract mixed solution.
(5) A loop of the slant-preserved Lactobacillus plantarum (purchased from Lactobacillus plantarum JYLP-326 of Jiayi, china family) was streaked with an inoculating loop, inoculated into 100mL of MRS liquid medium, and cultured overnight in an incubator at 39 ℃ to obtain an activated seed solution.
(6) 1% inoculum of activated seed solution was added to fresh MRS broth, and 1mL of the mixed broth was collected as a blank. Meanwhile, adding 1% of inoculum size of activated seed solution into fresh MRS liquid culture medium containing 0.3% and 0.5% of bile salt, respectively collecting 1mL as a control group, adding 3% of water extract mixed solution, respectively collecting 1mL of mixed culture solution as an experimental group; the CFU and survival in bile salts were calculated from the time the food was in the stomach, incubated at 39 ℃ for 4h, dilution coated for each sample, and then incubated at 39 ℃ for 24h.
2. The experimental results are as follows: as shown in fig. 7.
FIG. 7 is a graph showing the effect of adding 3% of the mixed solution on the bile salt resistance of Lactobacillus plantarum in this example. As shown in FIG. 7, when 0.3% and 0.5% of bile salt was added, 3% of the mixed solution was further added. The survival rate of the lactobacillus plantarum is remarkably increased, which shows that the bile salt resistance of the lactobacillus plantarum can be improved by the 3% mixed solution.
Therefore, the aqueous extract mixed solution can effectively improve the bile salt resistance of the lactobacillus plantarum.
2. Control experiment for "single solution":
1. the experimental steps are as follows:
(1) Boiling the astragalus slices with clear water (the mass ratio is 1; mixing the filtered residue with clear water (1:5 by mass ratio) again, boiling for 30min, filtering with four layers of gauze to obtain a second leaching solution; mixing the leaching liquor obtained in the first and second times, and then metering to 500mL to obtain the astragalus aqueous extract.
(2) Decocting the haw slices with clear water (the mass ratio is 1: 10) for 2 hours, filtering the haw slices with four layers of gauze, and decocting residues twice for 1.5 hours and 1 hour respectively; filtering, combining the three decoctions, and diluting to 500mL to obtain fructus crataegi water extract.
(3) Adding clear water (the proportion of the white atractylodes rhizome tablets to the clear water is 1; decocting twice, each for 30min, and filtering with four layers of gauze to obtain the original medicinal liquid; overnight at 4 ℃, adding absolute ethyl alcohol every other day to ensure that the concentration of the ethyl alcohol in the solution is 60 percent, and carrying out alcohol precipitation overnight at 4 ℃; centrifuging for 5min at 4 deg.C and 11000rpm/min the next day, and collecting supernatant; and recovering ethanol by using a rotary evaporator to obtain the white atractylodes rhizome extract, and fixing the volume to 500mL.
(4) A loop of the slant-preserved Lactobacillus plantarum (purchased from Lactobacillus plantarum JYLP-326 of Jiayi, china family) was streaked with an inoculating loop, inoculated into 100mL of MRS liquid medium, and cultured overnight in an incubator at 39 ℃ to obtain an activated seed solution.
(5) 1% inoculum of activated seed solution was added to fresh MRS broth, and 1mL of the mixed broth was collected as a blank. Meanwhile, adding 1% inoculum size of activated seed liquid into fresh MRS liquid culture medium containing 0.3% and 0.5% bile salt, respectively collecting 1mL as a control group, adding 10% radix astragali water extract, and collecting 1mL of mixed culture solution as an experimental group; the CFU and survival in bile salts were calculated for each sample by incubation at 39 ℃ for 4h, based on the food residence time in the stomach, dilution coating, followed by incubation at 39 ℃ for 24h.
(6) 1% inoculum of activated seed solution was added to fresh MRS broth, and 1mL of the mixed broth was collected as a blank. Meanwhile, adding 1% inoculum size of activated seed liquid into fresh MRS liquid culture medium containing 0.3% and 0.5% bile salt, respectively collecting 1mL as a control group, adding 5% aqueous extract of fructus crataegi, and collecting 1mL of mixed culture solution as an experimental group; the CFU and survival in bile salts were calculated from the time the food was in the stomach, incubated at 39 ℃ for 4h, dilution coated for each sample, and then incubated at 39 ℃ for 24h.
(7) 1% inoculum of activated seed solution was added to fresh MRS broth, and 1mL of the mixed broth was collected as a blank. Meanwhile, adding 1% of inoculum size of activated seed solution into fresh MRS liquid culture medium containing 0.3% and 0.5% of bile salt, respectively collecting 1mL as a control group, adding 3% of rhizoma Atractylodis Macrocephalae aqueous extract, and collecting 1mL of mixed culture solution as an experimental group; the CFU and survival in bile salts were calculated for each sample by incubation at 39 ℃ for 4h, based on the food residence time in the stomach, dilution coating, followed by incubation at 39 ℃ for 24h.
2. The experimental results are as follows: as shown in fig. 8.
FIG. 8 is a graph showing the effect of the single solution and the mixed solution on the bile salt resistance of Lactobacillus plantarum in this example. As can be seen from FIG. 8, when 0.3% and 0.5% bile salts were added, the survival rate of Lactobacillus plantarum was slightly improved by adding the mixed solution compared to the single solution.
It is known that the mixed solution can improve the bile salt resistance of Lactobacillus plantarum more than the single solution.
Example 7
The preparation method of the complex microbial inoculum of the embodiment comprises the following steps:
(1) Boiling the astragalus slices with clear water (the mass ratio is 1; mixing the filtered residue with clear water (1:5 by mass ratio) again, boiling for 30min, filtering with four layers of gauze to obtain a second leaching solution; mixing the first and second obtained leaching solutions, and diluting to 500mL to obtain radix astragali water extract.
(2) Decocting the haw slices with clear water (the mass ratio is 1: 10) for 2 hours, filtering the haw slices with four layers of gauze, and decocting residues twice for 1.5 hours and 1 hour respectively; filtering, combining the three decoctions, and diluting to 500mL to obtain fructus crataegi water extract.
(3) Adding clear water (the proportion of the white atractylodes rhizome tablets to the clear water is 1; decocting twice, each for 30min, and filtering with four layers of gauze to obtain the original medicinal liquid; overnight at 4 ℃, adding absolute ethyl alcohol every other day to ensure that the concentration of the ethyl alcohol in the solution is 60 percent, and carrying out alcohol precipitation overnight at 4 ℃; centrifuging for 5min at 11000rpm/min at 4 ℃ the next day, and taking supernatant; and recovering ethanol by using a rotary evaporator to obtain the white atractylodes rhizome extract, and fixing the volume to 500mL.
(4) Mixing the obtained astragalus aqueous extract, hawthorn aqueous extract and bighead atractylodes rhizome extract according to the ratio of 1:1:1 to obtain a water extract mixed solution. And then, carrying out spray drying on the water extract mixed solution under the conditions that the air inlet temperature is 180 ℃ and the peristaltic pump speed is 7rpm/min to obtain water extract mixed powder.
(5) One-ring slant preserved lactobacillus plantarum was streaked with an inoculating ring, inoculated into 100mL of MRS liquid medium, and cultured overnight in an incubator at 39 ℃ for 24 hours.
(6) After the overnight culture is finished, centrifuging for 5min at 20 ℃ and 5000rpm/min by using a high-speed refrigerated centrifuge, collecting thalli, and washing for 2 times by using sterile normal saline to obtain lactobacillus plantarum bacterial sludge; adding a protective agent with 20% of the volume of the fermentation liquid into the lactobacillus plantarum bacterial sludge, pre-freezing for 2 hours at-80 ℃, and then placing the mixture into a freeze dryer for freeze drying for 24-36 hours, preferably 30 hours; after the freeze drying is finished, grinding into powder. Wherein, the protective agent comprises the following components: lactose 9.05g/100mL, tryptone 8.79g/100mL, trehalose 11.21g/100mL.
(7) Mixing the water extract mixed powder obtained in the step (4) with the lactobacillus plantarum freeze-dried powder obtained in the step (6) according to the ratio of 2:1 to obtain the compound bacterial agent required by the target.
Fig. 9 shows lactobacillus plantarum freeze-dried powder (a) and water extract mixed powder (B) obtained in this example.
The composite microbial inoculum of the mixed powder of the astragalus, the hawthorn and the bighead atractylodes rhizome and the lactobacillus plantarum freeze-dried powder can be obtained in the embodiment.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A method for improving the activity of lactobacillus plantarum, comprising the steps of:
(1) Boiling the astragalus slices with clear water, and filtering to obtain a primary leaching solution; mixing the filtered residue with clear water, boiling, and filtering to obtain a second leaching solution; mixing the first and second obtained leaching solutions to obtain radix astragali water extract;
(2) Decocting the haw slices with clear water, filtering, decocting the residue twice, filtering, and combining the three decoctions to obtain the haw aqueous extract;
(3) Adding clear water into the bighead atractylodes rhizome, and soaking overnight; decocting twice, and filtering to obtain a crude drug solution; overnight, adding absolute ethyl alcohol every other day, and carrying out alcohol precipitation overnight; centrifuging the next day to obtain supernatant; recovering ethanol to obtain water extract of Atractylodis rhizoma;
(4) Mixing the obtained astragalus aqueous extract, hawthorn aqueous extract and bighead atractylodes rhizome extract according to the ratio of 1:1:1 to obtain an aqueous extract mixed solution;
(5) Taking lactobacillus plantarum by using an inoculating loop, inoculating the lactobacillus plantarum into an MRS liquid culture medium, and carrying out overnight culture in an incubator at 39 ℃ to obtain activated seed liquid;
(6) Adding 1% of the activated seed solution into a fresh MRS liquid culture medium, adding 3% -5% of a water extract mixed solution, and co-culturing at 39 ℃.
2. The method for improving the activity of lactobacillus plantarum according to claim 1, wherein in step (1), the astragalus membranaceus slices are boiled with clear water for 1 hour, and filtered to obtain a first leaching solution; mixing the filtered residue with clear water, boiling for 30min, and filtering to obtain second extractive solution; mixing the first and second obtained leaching solutions to obtain radix astragali water extract.
3. The method for improving activity of lactobacillus plantarum as claimed in claim 1, wherein in step (2), the hawthorn slices are decocted with clear water for 2h, the residue is decocted twice after filtration for 1.5h and 1h respectively, and the three decoctions after filtration are combined to obtain the aqueous extract of hawthorn.
4. The method for improving the activity of lactobacillus plantarum according to claim 1, wherein in step (3), clear water is added to atractylodes macrocephala koidz, and the mixture is soaked overnight; decocting twice, each for 30min, and filtering to obtain a crude drug solution; standing overnight at 4 deg.C, adding anhydrous ethanol every other day, and precipitating with ethanol at 4 deg.C overnight; centrifuging for 5min the next day, and collecting supernatant; recovering ethanol to obtain water extract of Atractylodis rhizoma.
5. The method for improving the activity of lactobacillus plantarum according to claim 1, wherein in the step (1), the mass ratio of the astragalus membranaceus tablets to the clear water is 1:10, filtering by using four layers of gauze; the mass ratio of the residue to the clear water is 1:5; mixing the first leaching solution and the second leaching solution, and metering to 500mL;
in the step (2), the mass ratio of the haw flakes to the clear water is 1:10, filtering by using four layers of gauze; the mass ratio of the residue to the clear water is 1:10; merging the three decocted fluids, and fixing the volume to 500mL;
in the step (3), the proportion of the white atractylodes rhizome tablets to the clear water is 1:10, filtering by using four layers of gauze; adding absolute ethyl alcohol to make the concentration of the ethyl alcohol in the solution be 60%, and centrifuging at 4 ℃ and 11000rpm/min; and recovering ethanol by using a rotary evaporator to obtain the white atractylodes rhizome extract, and fixing the volume to 500mL.
6. The method for increasing the activity of Lactobacillus plantarum according to claim 1, wherein, in step (5), lactobacillus plantarum preserved in one loop slant was extracted with an inoculating loop, inoculated into 100mL of MRS liquid medium, and cultured overnight in an incubator at 39 ℃.
7. The method for improving the activity of lactobacillus plantarum according to claim 1, wherein in the step (6), the specific addition amount of the mixed solution of aqueous extract is 3%.
8. The preparation method of the composite microbial inoculum is characterized by comprising the following steps:
(1) Boiling the astragalus slices with clear water, and filtering to obtain a primary leaching solution; mixing the filtered residue with clear water, boiling, and filtering to obtain a second leaching solution; mixing the first and second obtained leaching solutions to obtain radix astragali water extract;
(2) Decocting fructus crataegi with clear water, filtering, decocting the residue twice, filtering, and mixing the three decoctions to obtain fructus crataegi water extract;
(3) Adding clear water into the bighead atractylodes rhizome, and soaking overnight; decocting twice, and filtering to obtain a crude drug solution; overnight, adding absolute ethyl alcohol every other day, and carrying out alcohol precipitation overnight; centrifuging the next day to obtain supernatant; recovering ethanol to obtain water extract of Atractylodis rhizoma;
(4) Mixing the obtained astragalus aqueous extract, hawthorn aqueous extract and bighead atractylodes rhizome extract according to the ratio of 1:1:1 to obtain an aqueous extract mixed solution; then, carrying out spray drying on the water extract mixed solution to obtain water extract mixed powder;
(5) Taking lactobacillus plantarum by using an inoculating loop, inoculating the lactobacillus plantarum into an MRS liquid culture medium, and culturing overnight in an incubator at 39 ℃;
(6) After overnight culture, centrifugally collecting thalli, and washing for a plurality of times by using sterile normal saline to obtain lactobacillus plantarum bacterial sludge; adding a protective agent into the lactobacillus plantarum bacterial paste, pre-freezing, and freeze-drying in a freeze dryer; after the freeze drying is finished, grinding into powder;
(7) And (4) mixing the mixed powder of the water extract obtained in the step (4) with the lactobacillus plantarum freeze-dried powder obtained in the step (6) to obtain the compound microbial inoculum required by the target.
9. The preparation method of the complex microbial inoculum according to claim 8, wherein in the step (4), the spray drying condition is that the air inlet temperature is 180 ℃ and the peristaltic pump speed is 7rpm/min.
10. The method for preparing a complex microbial inoculant according to claim 8, wherein in the step (6), after overnight culture is finished, a high-speed refrigerated centrifuge is used for centrifuging for 5min at 20 ℃ and 5000rpm/min, thalli are collected and washed for 2 times by using sterile normal saline to obtain lactobacillus plantarum bacterial sludge; adding a protective agent with the volume being 20% of that of the fermentation liquid into the lactobacillus plantarum bacterial sludge, pre-freezing for 2 hours at the temperature of minus 80 ℃, and then placing the mixture into a freeze dryer for freeze drying for 24 to 36 hours; after the freeze drying is finished, grinding into powder; wherein, the protective agent comprises the following components: lactose 9.05g/100mL, tryptone 8.79g/100mL, trehalose 11.21g/100mL.
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