CN115005264A - Method for inhibiting germination of spores of clostridium bifermentans - Google Patents
Method for inhibiting germination of spores of clostridium bifermentans Download PDFInfo
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Abstract
The invention discloses a method for inhibiting germination of spores of clostridium bifermentans. A method for inhibiting germination of spores of Clostridium bifermentans comprises treating Clostridium bifermentans with a reagent containing natural plant extract to inhibit germination of spores. According to the invention, the active substances are extracted from the mulberry leaves, the aloes or the liquorice to treat the spores of the clostridium bifidus in the presence of the germinant, so that a theoretical basis is provided for inhibiting the germination of the spores of the clostridium bifidus and controlling the pollution of the clostridium bifidus in the meat.
Description
Technical Field
The invention belongs to the field of foods, relates to a method for inhibiting germination of spores of clostridium bifermentans, and particularly relates to a method for inhibiting germination of spores of clostridium bifermentans under the condition that a germinant induces germination.
Background
The chicken breast is a very healthy meat product, has tender meat, delicious taste, rich nutrition and rich protein, and is popular with people because the vacuum-packaged chicken breast is convenient to eat and can be stored for a long time after being sterilized at high temperature and the color, the taste and the flavor of the meat are kept in the shelf life. However, the chicken breast is very easily polluted by various microorganisms because the chicken breast contains rich nutrient substances, most of the microorganisms can be killed after high-temperature sterilization, but some bacteria such as clostridium can generate spores with extremely strong stress resistance, so that the chicken breast can survive. The clostridium bifermentans is G + anaerobic bacteria capable of generating spores, has strong resistance to the outside, the anaerobic environment provided by the chicken breast after vacuum packaging is also just suitable for the growth of the clostridium bifermentans, and once the conditions are suitable, the spores of the clostridium bifermentans can germinate and grow to cause the meat to decay and deteriorate. And such spores are highly resistant to various stresses associated with food preservation methods, and therefore, it is difficult to directly kill the spores. However, if the germination of the spores is inhibited, the spores do not germinate and grow before eating, the pollution of the spores to the meat in the metabolic process can be avoided.
The plant resources in China are abundant, but most of the plant resources are not utilized, so that the plant resources are reasonably utilized, and active ingredients are extracted from the plant resources to be used as natural bacteriostatic agents, so that the natural bacteriostatic agent has a good development prospect in the future. The natural plant extract has the inhibiting effect on the germination and growth of spores on the physiological active substances, such as phenols, alkaloids, terpenoids and the like. Natural plant extracts can inhibit germination of spores by two aspects: (1) spore germination is inhibited by inhibiting or reacting with a nutrient receptor associated with germination. (2) By being adsorbed on the spores, the adsorption acts on the spores in the process of germinating the spores into vegetative cells, the structure of spore membranes is destroyed, DPA flows out, the heat resistance is reduced, and the internal structure is damaged, so that the growth of the spores is inhibited.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a method for inhibiting germination of spores of clostridium bifermentans.
It is another object of the present invention to provide a natural plant extract for inhibiting germination of spores of clostridium bifermentans.
It is yet another object of the present invention to provide the use of a natural plant extract for inhibiting germination of spores of clostridium bifermentans.
The purpose of the invention can be realized by the following technical scheme:
a method for inhibiting germination of spores of Clostridium bifermentans comprises treating Clostridium bifermentans with any one of the following reagents containing natural plant extracts for inhibiting germination of spores:
(1) and (3) mulberry leaf extract: pulverizing dried folium Mori, dissolving with 70-75% ethanol, ultrasonic extracting at 40-50 deg.C for 0.5-2 hr for 2-5 times, rotary evaporating to remove ethanol, and lyophilizing; pulverizing folium Mori, adding 75% ethanol at a volume of 1:10, ultrasonic extracting at 40 deg.C for 1 hr for 3 times, rotary evaporating to remove ethanol, and lyophilizing;
(2) aloe extract: pulverizing dried Aloe, dissolving with 70-75% ethanol, ultrasonic extracting at 40-50 deg.C for 0.5-2 hr for 2-5 times, rotary evaporating to remove ethanol, and lyophilizing; preferably, the aloe extract is obtained by pulverizing dried aloe, adding 75% ethanol at a volume of 1:10, dissolving, ultrasonic extracting at 45 deg.C for 1h for 3 times, rotary evaporating to remove ethanol, and lyophilizing;
(3) the Glycyrrhrizae radix extract is obtained by pulverizing Glycyrrhrizae radix, dissolving with 70-75% ethanol, ultrasonic extracting at 40-50 deg.C for 0.5-2 hr for 2-5 times, rotary evaporating to remove ethanol, and lyophilizing; preferably, the extract is obtained by pulverizing dried Glycyrrhrizae radix, adding 75% ethanol at a volume of 1:10, dissolving, ultrasonic extracting at 45 deg.C for 1h for 3 times, rotary evaporating to remove ethanol, and lyophilizing.
Preferably, the agent containing natural plant extracts further comprises a spore germination agent.
As a preferable mode of the invention, the spore germinant is selected from one or a combination of several of the following materials: d-glucose, D-fructose, D-galactose, D-xylose, L-alanine, L-aspartic acid, L-valine, L-proline, L-histidine, L-leucine, inosine and DPA.
As a further preferred feature of the present invention, the germinant is selected from the group consisting of DPA or a combination of L-alanine and inosine.
As a further preferable aspect of the present invention, the reagent containing natural plant extract is Tris-HCl buffer solution with pH 7.4, 25mM and containing L-alanine 120-180mmol/L + inosine 15-30mmol/L + natural plant extract; wherein when the natural plant extract is folium Mori extract, the concentration is 50-100 mg/mL; when the natural plant extract is aloe extract, the concentration is 25-100 mg/mL; when the natural plant extract is Glycyrrhrizae radix extract, the concentration is 3-7 mg/mL.
In a further preferred embodiment of the present invention, the reagent containing a natural plant extract is a Tris-HCl buffer solution containing DPA6-10mmol/L + natural plant extract at 25mM and pH 7.4; wherein when the natural plant extract is mulberry leaf extract, the concentration is 100 mg/mL; when the natural plant extract is aloe extract, the concentration is 25-100mg/mL, preferably 25-50 mg/mL; when the natural plant extract is Glycyrrhrizae radix extract, the concentration is 1.5-25mg/mL, preferably 1.5-7 mg/mL.
As a further preferred aspect of the present invention, the agent containing a natural plant extract is selected from any one of the following:
(1) Tris-HCl buffer at pH 7.4 at 25mM containing DPA6-10mmol/L + aloe vera extract 25 mg/mL;
(2) Tris-HCl buffer (25mM, pH 7.4) containing DPA6-10mmol/L + Glycyrrhiza extract 1.56 mg/mL.
Preferably, the method comprises the steps of mixing the reagent containing the natural plant extract with the clostridium bifidum spore suspension or the substance containing the clostridium bifidum spore, uniformly swirling, and putting the mixture into an anaerobic incubator at 37 ℃ for culture.
A natural plant extract for inhibiting germination of spores of Clostridium bifermentans is prepared by dissolving folium Mori powder, Aloe powder or Glycyrrhrizae radix powder with 70-75% ethanol, ultrasonic extracting at 40-50 deg.C for 0.5-2 hr for 2-5 times, rotary evaporating to remove ethanol, and lyophilizing.
The reagent containing the natural plant extract is characterized by being Tris-HCl buffer solution containing a spore germination agent and the natural plant extract.
Preferably, the reagent containing the natural plant extract is Tris-HCl buffer solution with the pH value of 7.4 and 25mM, which contains L-alanine 120-180mmol/L + inosine 15-30mmol/L + the natural plant extract; wherein when the natural plant extract is folium Mori extract, the concentration is 50-100 mg/mL; when the natural plant extract is aloe extract, the concentration is 25-100 mg/mL; when the natural plant extract is Glycyrrhrizae radix extract, the concentration is 3-7 mg/mL;
or the reagent containing the natural plant extract is Tris-HCl buffer solution with the pH value of 7.4 and the concentration of 25mM containing DPA6-10mmol/L + the natural plant extract; wherein when the natural plant extract is mulberry leaf extract, the concentration is 100 mg/mL; when the natural plant extract is aloe extract, the concentration is 25-100mg/mL, preferably 25-50 mg/mL; when the natural plant extract is Glycyrrhrizae radix extract, the concentration is 1.5-25mg/mL, preferably 1.5-7 mg/mL.
The natural plant extract and the reagent are applied to inhibition of germination of spores of clostridium bifermentans.
Has the advantages that:
according to the invention, mDS spore production culture medium is used for collecting spores of clostridium bifermentans to prepare spore suspension, and extracts of folium mori, aloe and liquorice are respectively used for inhibiting the germination of the spores of clostridium bifermentans. The result shows that the method for treating the spores of the clostridium bifidum can inhibit the spores of the clostridium bifidum from germinating, prevent the decay and toxic action of meat products caused by the germination and metabolism of residual spores, and provide certain theoretical guidance for the safety of the meat products.
Drawings
FIG. 1 inhibitory Effect of Mulberry leaf extract on spore germination of Clostridium bifermentans induced by 150mmol/L Ala +20mmol/L Ino (n ═ 3)
FIG. 2 inhibition curves of mulberry leaf extract on germination of Clostridium bifermentans spores induced by 150mmol/L Ala +20mmol/L Ino (n ═ 3)
FIG. 3 inhibitory Effect of Mulberry leaf extract on 8mmol/L DPA-induced germination of Clostridium bifermentans spores (n ═ 3)
FIG. 4 inhibition curve of mulberry leaf extract against 8mmol/L DPA-induced germination of Clostridium bifermentans spores (n ═ 3)
FIG. 5 inhibitory Effect of Aloe Vera extract on spore germination of Clostridium bifermentans induced by 150mmol/L Ala +20mmol/L Inosine (n ═ 3)
FIG. 6 inhibition curves of aloe vera extract on 150mmol/L Ala +20mmol/L Inosine induced germination of Clostridium bifermentans spores (n ═ 3)
FIG. 7 inhibition of 8mmol/L DPA-induced germination of Clostridium bifermentans spores by Aloe extract (n ═ 3)
FIG. 8 inhibition curves of aloe vera extract on 8mmol/L DPA-induced germination of Clostridium bifermentans spores (n ═ 3)
FIG. 9 inhibitory Effect of Glycyrrhiza extract on spore germination of Clostridium bifermentans induced by 150mmol/L Ala +20mmol/L Inosine (n ═ 3)
FIG. 10 inhibition curves of Glycyrrhiza extract against 150mmol/L Ala +20mmol/L Inosine induced germination of Clostridium bifermentans spores (n ═ 3)
FIG. 11 inhibitory Effect of Glycyrrhiza extract on 8mmol/L DPA-induced germination of Clostridium bifermentans spores (n ═ 3)
FIG. 12 inhibition curve of Glycyrrhiza extract against 8mmol/L DPA-induced germination of Clostridium bifermentans spores (n ═ 3)
Detailed Description
Materials and reagents
Clostridium bifermentans: isolated from "yellow professor chicken breast" (Nanjing yellow professor food science and technology Co., Ltd.).
Mulberry leaf, aloe, and licorice were purchased from Xianchao Zaokang drugstore (Nanjing).
The reinforced clostridium culture medium, the reinforced clostridium agar, the L-Ala, the inosine and the DPA are all purchased from Nanjing butyl Beibei biological technology Limited; NaCl was purchased from national drug group chemical reagents, Inc.
Second, instrument and equipment
Autoclave HVE-50 Hirayama, Japan; biochemical incubator B1-150A Schurbaky instruments and Equipment (Shanghai) Co., Ltd; high speed refrigerated centrifuge Allegra-64R, Beckman, USA; constant temperature water bath HH-W Ginkard City Hengfeng apparatus manufacturing Co., Ltd; innova series ultra-low temperature vertical refrigerator NEW BRUNSWICK Inc. of UK; desk type pH meter FE20 Mettler Toledo, Switzerland; AUY120 electronic analytical balance, shimadzu corporation, japan; biosafety cabinet SG403A biosafety cabinet SG403A, Baker corporation; upright fluorescence microscope scope. A1, Carl Zeiss, Germany; the multifunctional microplate reader M2e USA MD company.
Thirdly, the index measuring method comprises the following steps:
(1) preparation of spore suspension
0.1mL of the activated bacterial solution was inoculated into 10mL of fresh RCM medium and anaerobically cultured at 37 ℃ for 18 hours. Then, 0.1mL of the bacterial suspension was applied to Modified Duncan-Strong (mDS) sporulation medium agar, anaerobically cultured at 37 ℃ for 7d, sporulated with cold sterile water, centrifuged (7000rpm, 15min, 4 ℃), the supernatant was decanted, the lower precipitate was left, and washed with cold sterile water 4 times. And (3) placing the spore suspension in a refrigerator at 4 ℃, heating the spore suspension in a water bath at 80 ℃ for 20min after 24h, killing vegetative cells in the spore suspension, centrifuging the spore suspension, removing supernatant, and repeating the operation for 2-3 times. The spores were > 95% pure by microscopy and the spore suspension concentration was adjusted with sterile water to an OD600 of about 1.
mds (modified Duncan strong) medium agar: weighing 15g of tryptone, 4g of yeast powder, 10g of disodium hydrogen phosphate, 4g of raffinose, 1g of sodium thioglycolate and 15g of agar powder, dissolving in 1L of distilled water, and keeping the pH value at 7.5. Autoclaving at 121 deg.C for 15 min.
(2) Determination of spore germination rate
Selecting 10 2mL centrifuge tubes, dissolving folium Mori/Aloe/Glycyrrhrizae radix extract lyophilized powder with sterilized 25mM Tris-HCl buffer solution (pH 7.4) containing germinant to make the mother solution concentration be 100mg/mL, filtering with 0.45 μm organic filter membrane after completely dissolving, and diluting the mother solution to 10 concentrations by two-fold dilution method, wherein each concentration is set in three groups. Mixing 500 μ L folium Mori/Aloe/Glycyrrhrizae radix extract diluent with 500 μ L spore suspension, vortex uniformly, placing into 37 deg.C anaerobic incubator for culturing for 1h, placing 200 μ L each group in ELISA plate, measuring OD600 before and after culture, and calculating germination rate. Spore germination (%) was expressed as:
(X 0 -X 1 )÷X 0 ×100％。
in the formula: x 0 OD600, X of spores before germination treatment 1 OD600 of spores after 1h of germination treatment.
(3) Measurement of spore germination inhibition Curve
Selecting 2, 1, 1/2 XOIC folium Mori/Aloe/Glycyrrhrizae radix extract, measuring OD600 of 0h, 1h, 2h, 3h, 4h according to the method of (3), and plotting inhibition curve of spore germination.
Example 1 inhibition of spore germination of Clostridium bifermentans induced by 150mmol/L Ala +20mmol/L Inosine by Mulberry leaf extract
1.1 preparation of Mulberry leaf extract
Taking mulberry leaf powder, adding 75% ethanol in a volume of 1:10, carrying out ultrasonic extraction for 1h at 40 ℃, extracting for 3 times, removing ethanol by rotation, and freeze-drying to obtain dry powder.
1.2 influence of Mulberry leaf extract on germination Rate of Clostridium bifermentans spores
The mother liquor of mulberry leaf extract (100mg/mL) was diluted to ten concentrations with Tris-HCl buffer (25mM, pH 7.4) containing 150mmol/L Ala +20mmol/L liposine as a germinant, and the spore suspensions of Clostridium bifermentans induced by 150mmol/L Ala +20mmol/L Inosine as a germinant were treated separately and the treatment was repeated three times.
The ten concentrations of the mulberry leaf extract have an inhibiting effect on germination of clostridium bifidus spores induced by a germinant, and the germination of the clostridium bifidus spores can be completely inhibited when the concentrations are more than or equal to 50mg/mL, so that the minimum inhibitory concentration (OIC) is 50mg/mL (figure 1).
1.3 inhibition curve of mulberry leaf extract on germination of spores of clostridium bifermentans
The 2, 1, 1/2 XOIC mulberry leaf extract is selected to treat Clostridium bifidum spores, the 2, 1 XOIC mulberry leaf extract can better inhibit germination of the Clostridium bifidum spores within 4 hours, the 1/2 XOIC mulberry leaf extract cannot completely inhibit in the initial 1 hour, spores have partial germination, and then germination stops (figure 2).
Example 2 inhibition of 8mmol/L DPA-induced germination of Clostridium bifermentans spores by Mulberry leaf extract
2.1 influence of Mulberry leaf extract on germination Rate of Clostridium bifermentans spores
The mulberry leaf extract mother liquor (100mg/mL) was diluted to ten concentrations with Tris-HCl buffer (25mM, pH 7.4) containing 8mmol/L DPA of germinant, and the spore suspensions of Clostridium bifermentans induced by 8mmol/L DPA of germinant were treated separately and the treatment was repeated three times.
All concentrations of the mulberry leaf extract inhibited spore germination induced by DPA (8mmol/L), but even at 200mg/mL, it did not completely inhibit spore germination of Clostridium bifermentans, with very weak spores (FIG. 3).
2.2 inhibition curve of mulberry leaf extract to germination of spores of Clostridium bifermentans
200mg/mL, 100mg/mL and 50mg/mL of mulberry leaf extract are selected to treat the spores of Clostridium bifermentans, at 200mg/mL, the spores of Clostridium bifermentans germinate within the initial 3 hours, at 100mg/mL and 50mg/mL, the spores of Clostridium bifermentans germinate within the initial 1 hour, then germination stops, and the germination inhibiting effect of the three concentrations decreases with the decrease of the concentration (figure 4).
As can be seen from examples 1 and 2, all concentrations of the mulberry leaf extract had inhibitory effects on germination of spores induced by germinants when the lyophilized powder of the mulberry leaf extract was treated by dissolving the mulberry leaf extract in 25mM Tris-HCl buffer (pH 7.4) containing 150mmol/L Ala +20mmol/L Inosine. When the concentration is more than or equal to 50mg/mL, the germination of spores can be completely inhibited, so that the minimum spore germination inhibiting concentration is 50mg/mL, and the clostridium bifermentans treated by the concentration does not germinate within 4 h. When the lyophilized powder of mulberry leaf extract was dissolved in 25mM Tris-HCl buffer (pH 7.4) containing 8mmol/L of DPA, all concentrations of the mulberry leaf extract inhibited spore germination induced by DPA (8mM) in the same manner.
Example 3 inhibition of spore germination of Clostridium bifermentans induced by 150mmol/L Ala +20mmol/L Inosine by Aloe extract
3.1 preparation of Aloe extract
Taking aloe powder, adding 75% ethanol in a volume of 1:10, performing ultrasonic extraction at 40 ℃ for 1h, extracting for 3 times, removing ethanol by rotation, and performing freeze-drying to obtain dry powder.
3.2 Effect of Aloe extract on Germination Rate of Clostridium bifermentans spores
Aloe extract stock solution (100mg/mL) was diluted to ten concentrations with Tris-HCl buffer (25mM, pH 7.4) containing germinant 150mmol/L Ala +20mmol/L liposine, and the spore suspensions of Clostridium bifermentans induced by germinant 150mmol/L Ala +20mmol/L Inosine were treated separately and the treatment was repeated three times.
The ten concentrations of the aloe extract have an inhibition effect on germination of clostridium bifidum spores induced by germinants, and when the concentrations are more than or equal to 50mg/mL, the germination of the clostridium bifidum spores can be completely inhibited, so that the minimum inhibitory concentration (OIC) is 50mg/mL (figure 5).
3.3 inhibition Curve of Aloe extract on spore germination of Clostridium bifermentans
The 2, 1, 1/2 xoic aloe extracts were selected for clostridium bifidum spores treatment, the 2, 1 xoic aloe extracts inhibited clostridium bifidum spore germination well within 4 hours, the 1/2 xoic aloe extracts treatment, clostridium bifidum spores did not completely inhibit germination within the first 2 hours, but germination ceased after two hours (fig. 6).
Example 4 inhibition of 8mmol/L DPA-induced spore germination of Clostridium bifermentans by Aloe extract
4.1 Effect of Aloe extract on Germination Rate of Clostridium bifermentans spores
Aloe extract stock solution (100mg/mL) was diluted to ten concentrations with Tris-HCl buffer (25mM, pH 7.4) containing 8mmol/L DPA as germinant, and the clostridium bifermentans spore suspensions induced by 8mmol/L DPA as germinant were treated separately and the treatment was repeated three times.
Except for the case that the concentration of the aloe extract is 0.19mg/mL, the aloe extract cannot inhibit spore germination of clostridium bifermentans induced by DPA (8mmol/L), all the other concentrations can inhibit the spore germination induced by DPA completely, and the concentration is more than or equal to 25mg/mL, the dosage of the aloe extract is less than that of spore germination induced by 150mmol/L Ala +20mmol/L Inosine, and the minimum inhibitory concentration (OIC) is 25mg/mL (figure 7).
4.2 inhibition Curve of Bacillus bifidus Germination by Aloe extract
The 2, 1, 1/2 xoic aloe extracts were selected for treatment of clostridium bifidum spores, the 2, 1 xoic aloe extracts still inhibited spore germination well within 4 hours, the 1/2 xoic aloe extracts did not completely inhibit germination within the first 2 hours, after which germination ceased (fig. 8).
As can be seen from examples 3 and 4, when aloe extract lyophilized powder was treated by dissolving aloe extract lyophilized powder in 25mM Tris-HCl buffer (pH 7.4) containing 150mmol/L Ala +20mmol/L Inosine, all concentrations of aloe extract had inhibitory effect on germination of spores induced by germination, and aloe extract at a concentration of 50mg/mL or more could completely inhibit germination of spores, so that the minimum inhibitory concentration of spores was 50mg/mL, and neither Clostridium bifermentans treated at this concentration germinated within 4 h. When aloe extract lyophilized powder was dissolved in 25mM Tris-HCl buffer (pH 7.4) containing 8mmol/L DPA, all concentrations of aloe extract were able to inhibit DPA (8mM) -induced spore germination in the same manner, and at a concentration of 25mg/mL or more, DPA-induced spore germination could be completely inhibited within 4 hours, using an amount less than 150mmol/L Ala +20mmol/L Inosine-induced spore germination.
Example 5 inhibition of spore germination of Clostridium bifermentans induced by 150mmol/L Ala +20mmol/L Inosine by Glycyrrhiza extract
5.1 preparation of Glycyrrhiza extract
Taking licorice powder, adding 75% ethanol in a volume of 1:10, carrying out ultrasonic extraction at 40 ℃ for 1h, extracting for 3 times, removing ethanol by rotation, and freeze-drying to obtain dry powder.
5.2 Effect of Glycyrrhiza extract on germination Rate of Clostridium bifermentans spores
Mother liquor of licorice extract (100mg/mL) was diluted to ten concentrations with Tris-HCl buffer (25mM, pH 7.4) containing germinant 150mmol/L Ala +20mmol/L lignine, and spore suspensions of clostridium bifermentans induced by germinant 150mmol/L Ala +20mmol/L Inosine were treated separately, and the treatment was repeated three times.
The ten concentrations of the licorice extract all have an inhibiting effect on germination of clostridium bifidus spores induced by a germinant, and when the concentrations are more than or equal to 3.12mg/mL, the germination of the clostridium bifidus spores can be completely inhibited, so that the minimum inhibitory concentration (OIC) is 3.12mg/mL (figure 9).
5.3 inhibition Curve of Glycyrrhiza extract on germination of spores of Clostridium bifermentans
The 2, 1, 1/2 XOIC licorice extract was selected to treat Clostridium bifidum spores, the 2, 1 XOIC licorice extract inhibited well the germination of Clostridium bifidum spores within 4 hours, the 1/2 XOIC licorice extract did not completely inhibit within the first 1h, and the spores germinated slightly (FIG. 10).
Example 6 inhibition of 8mmol/L DPA-induced spore germination of Clostridium bifermentans by Glycyrrhiza extract
6.1 Effect of Glycyrrhiza extract on germination Rate of Clostridium bifermentans spores
The mother liquor of licorice extract (100mg/mL) was diluted to ten concentrations with Tris-HCl buffer (25mM, pH 7.4) containing 8mmol/L DPA as a germinant, and the spore suspensions of clostridium bifermentans induced by 8mmol/L DPA as a germinant were treated separately and the treatment was repeated three times.
All concentrations of the licorice extract inhibited DPA (8mmol/L) induced spore germination, and at concentrations greater than or equal to 1.56mg/mL, completely inhibited DPA induced spore germination at a use level less than 150mmol/LAla +20mmol/L Inosine induced spore germination, at which the minimum inhibitory spore germination concentration (OIC) was 1.56mg/mL (FIG. 11).
6.2 inhibition Curve of Glycyrrhiza extract on germination of spores of Clostridium bifermentans
When 2, 1, 1/2 XOIC licorice extracts were selected to treat Clostridium bifermentans spores, the 2, 1 XOIC licorice extracts still inhibited spore germination well within 4 hours, and the 1/2 XOIC licorice extracts did not completely inhibit germination within the first 2 hours (FIG. 12).
As can be seen from examples 5 and 6, all concentrations of the licorice extract had inhibitory effect on germination of germinants induced spores when the licorice extract lyophilized powder was treated by dissolving it in 25mM Tris-HCl buffer (pH 7.4) containing 150mmol/L Ala +20mmol/L Inosine. When the concentration is more than or equal to 3.12mg/mL, the germination of spores can be completely inhibited, so that the minimum spore germination inhibiting concentration is 3.12mg/mL, and the clostridium bifidus treated by the concentration does not germinate within 4 h. When the licorice extract freeze-dried powder is dissolved in 25mM Tris-HCl buffer (pH 7.4) containing 8mmol/L of DPA, under the same method, all concentrations of the licorice extract can inhibit spore germination induced by DPA (8mM), and when the concentration is more than or equal to 1.56mg/mL, the licorice extract can completely inhibit spore germination induced by DPA, and the use amount of the licorice extract freeze-dried powder is less than that of spore germination induced by 150mmol/L of Ala +20mmol/L of Inosine.
Claims (10)
1. A method for inhibiting spore germination of clostridium bifidum is characterized in that the clostridium bifidum is treated by using a reagent containing a natural plant extract to inhibit spore germination:
(1) and (3) mulberry leaf extract: pulverizing dried folium Mori, dissolving with 70-75% ethanol, ultrasonic extracting at 40-50 deg.C for 0.5-2 hr for 2-5 times, rotary evaporating to remove ethanol, and lyophilizing; preferably, mulberry leaves are crushed, 75% ethanol is added according to the volume of 1:10, ultrasonic extraction is carried out for 1h at 40 ℃, extraction is carried out for 3 times, ethanol is removed by rotary evaporation, and freeze-drying is carried out to obtain the mulberry leaf extract;
(2) aloe extract (E): is prepared by pulverizing dried Aloe, dissolving with 70-75% ethanol, ultrasonic extracting at 40-50 deg.C for 0.5-2 hr for 2-5 times, rotary evaporating to remove ethanol, and lyophilizing; preferably, the aloe extract is obtained by pulverizing dried aloe, adding 75% ethanol at a volume of 1:10, dissolving, ultrasonic extracting at 45 deg.C for 1h for 3 times, rotary evaporating to remove ethanol, and lyophilizing;
(3) the Glycyrrhrizae radix extract is obtained by pulverizing Glycyrrhrizae radix, dissolving with 70-75% ethanol, ultrasonic extracting at 40-50 deg.C for 0.5-2 hr for 2-5 times, rotary evaporating to remove ethanol, and lyophilizing; preferably, the extract is obtained by pulverizing dried Glycyrrhrizae radix, adding 75% ethanol at a volume of 1:10, dissolving, ultrasonic extracting at 45 deg.C for 1h for 3 times, rotary evaporating to remove ethanol, and lyophilizing.
2. The method of claim 1, wherein the agent comprising a natural plant extract further comprises a spore germinant.
3. The method of claim 2, wherein said spore germinant is selected from the group consisting of: d-glucose, D-fructose, D-galactose, D-xylose, L-alanine, L-aspartic acid, L-valine, L-proline, L-histidine, L-leucine, inosine, and DPA; the germinant is preferably selected from the group consisting of DPA or a combination of L-alanine and inosine.
4. The method as claimed in claim 3, wherein the reagent containing natural plant extract is Tris-HCl buffer with pH 7.4 containing L-alanine 120-180mmol/L + inosine 15-30mmol/L + natural plant extract at 25 mM; wherein when the natural plant extract is folium Mori extract, the concentration is 50-100 mg/mL; when the natural plant extract is aloe extract, the concentration is 25-100 mg/mL; when the natural plant extract is Glycyrrhrizae radix extract, the concentration is 3-7 mg/mL.
5. The method according to claim 3, wherein the natural plant extract-containing reagent is 25mM Tris-HCl buffer pH 7.4 containing DPA6-10mmol/L + natural plant extract; wherein when the natural plant extract is mulberry leaf extract, the concentration is 100 mg/mL; when the natural plant extract is aloe extract, the concentration is 25-100mg/mL, preferably 25-50 mg/mL; when the natural plant extract is Glycyrrhrizae radix extract, the concentration is 1.5-25mg/mL, preferably 1.5-7 mg/mL.
6. The method according to any one of claims 1 to 5, comprising mixing the natural plant extract-containing agent with a Clostridium bifidum spore suspension or a Clostridium bifidum spore-containing material, vortexing the mixture, and culturing the mixture in an anaerobic incubator at 37 ℃.
7. A natural plant extract for inhibiting germination of spores of Clostridium bifermentans is prepared by dissolving folium Mori powder, Aloe powder or Glycyrrhrizae radix powder with 70-75% ethanol, ultrasonic extracting at 40-50 deg.C for 0.5-2 hr for 2-5 times, rotary evaporating to remove ethanol, and lyophilizing.
8. A reagent comprising the natural plant extract of claim 7, wherein said reagent is Tris-HCl buffer containing spore germinant and the natural plant extract of claim 7.
9. The reagent as claimed in claim 8, wherein the reagent containing natural plant extract is Tris-HCl buffer solution with pH 7.4 containing L-alanine 120-180mmol/L + inosine 15-30mmol/L + natural plant extract at 25 mM; wherein when the natural plant extract is folium Mori extract, the concentration is 50-100 mg/mL; when the natural plant extract is aloe extract, the concentration is 25-100 mg/mL; when the natural plant extract is Glycyrrhrizae radix extract, the concentration is 3-7 mg/mL;
or the reagent containing the natural plant extract is Tris-HCl buffer solution which contains DPA6-10mmol/L + natural plant extract and has the pH value of 7.4 and 25 mM; wherein when the natural plant extract is mulberry leaf extract, the concentration is 100 mg/mL; when the natural plant extract is aloe extract, the concentration is 25-100mg/mL, preferably 25-50 mg/mL; when the natural plant extract is Glycyrrhrizae radix extract, the concentration is 1.5-25mg/mL, preferably 1.5-7 mg/mL.
10. Use of the natural plant extract of claim 7, the agent of claim 8 or 9 for inhibiting germination of clostridium bifidum spores.
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