CN115895963B - Preparation method of rhizoma atractylodis stem and leaf extract and application of rhizoma atractylodis stem and leaf extract in promoting proliferation of lactobacillus plantarum - Google Patents
Preparation method of rhizoma atractylodis stem and leaf extract and application of rhizoma atractylodis stem and leaf extract in promoting proliferation of lactobacillus plantarum Download PDFInfo
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Abstract
The invention discloses application of an atractylis ovata stem and leaf extract in promoting lactobacillus plantarum proliferation, and also discloses a preparation method of the atractylis ovata stem and leaf extract, which belongs to the technical field of atractylis ovata stem and leaf extract preparation.
Description
Technical Field
The invention relates to the technical field of preparation of atractylis ovata stem and leaf extracts, in particular to a preparation method of atractylis ovata stem and leaf extracts for promoting proliferation of lactobacillus plantarum.
Background
Rhizoma Atractylodis is stem and leaf of Atractylodes lancea or rhizome part of stem and leaf of Atractylodes lancea in Compositae. Modern pharmacological researches show that rhizoma atractylodis is used as a medicinal material, has unique chemical components and remarkable pharmacological activity, has the effects of resisting ulcer, arrhythmia, inflammation, bacteria, liver protection, blood pressure reduction and the like, and is widely applied. The stem and leaf of the rhizoma atractylodis is used as a non-medicinal part of the rhizoma atractylodis to be discarded as garbage, so that the resource is seriously wasted. Research shows that the stem and leaf of rhizoma atractylodis has the functions of anti-inflammatory, bacteriostasis and antioxidation.
Lactobacillus plantarum is a lactic acid bacterium commonly found in butter, meat and many vegetable fermented products, and is also commonly found in the human gastrointestinal tract, and has many health care effects: ① Has a certain immunoregulatory effect; ② Has inhibiting effect on pathogenic bacteria; ③ Lowering serum cholesterol levels and preventing cardiovascular disease; ④ Maintaining the balance of flora in the intestinal tract; ⑤ Promoting nutrient absorption; ⑥ Alleviating lactose intolerance; ⑦ Inhibit the formation of tumor cells, etc. Therefore, the method has important significance in promoting the proliferation of lactobacillus plantarum. However, the stem and leaf of Atractylodes lancea as waste in the production process of Atractylodes lancea has not been researched and developed yet, and whether it can promote the proliferation of Lactobacillus plantarum is unclear.
Therefore, the extraction of the rhizoma atractylodis extract and the development of the functions thereof are technical problems which are needed to be solved by the person skilled in the art.
Disclosure of Invention
In view of the above, the invention discovers that the rhizoma atractylodis stem and leaf extract can promote the proliferation of lactobacillus plantarum, optimize the extraction condition of the rhizoma atractylodis stem and leaf extract and furthest improve the extraction efficiency of the rhizoma atractylodis stem and leaf extract
In order to achieve the above purpose, the present invention adopts the following technical scheme:
application of rhizoma Atractylodis stem and leaf extract in promoting lactobacillus plantarum proliferation is provided.
Preferably, the rhizoma atractylodis stem and leaf extract promotes the content of total protein and lactic acid produced by lactobacillus plantarum fermentation, improves the growth rate of lactobacillus plantarum and promotes the self-aggregation capability of lactobacillus plantarum.
As the invention conception same as the technical scheme, the invention also claims a preparation method of the rhizoma atractylodis stem and leaf extract for promoting the proliferation of lactobacillus plantarum, which comprises the following steps: in the liquid-material ratio 1:50-1: 40. extracting stem and leaf extract of rhizoma Atractylodis at 45-50deg.C, pH4-5, ethanol concentration 50-55%, ultrasonic power 500-600w and ultrasonic time 25-30 min.
Compared with the prior art, the invention discovers the effect of promoting the proliferation of lactobacillus plantarum for the first time, so that the purity and the extraction rate of the lactobacillus plantarum are important for better promoting the proliferation of the lactobacillus plantarum.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the effect of ethanol concentration on extraction yield of rhizoma Atractylodis stem and leaf extract for promoting lactobacillus plantarum proliferation;
FIG. 2 shows the effect of feed liquid comparison on extraction yield of rhizoma Atractylodis stem and leaf extract for promoting Lactobacillus plantarum proliferation;
FIG. 3 is the effect of ultrasonic power on extraction yield of rhizoma Atractylodis stem and leaf extract for promoting Lactobacillus plantarum proliferation;
FIG. 4 shows the effect of pH on the extraction yield of rhizoma Atractylodis stem and leaf extract for promoting Lactobacillus plantarum proliferation;
FIG. 5 is a graph showing the effect of ultrasonic temperature on extraction rate of rhizoma Atractylodis stem and leaf extract for promoting Lactobacillus plantarum proliferation;
FIG. 6 shows the effect of ultrasound time on extraction yield of rhizoma Atractylodis stem and leaf extract for promoting Lactobacillus plantarum proliferation.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
According to the embodiment of the invention, a single-factor liquid-to-material ratio (A), an ultrasonic temperature (B), pH (C) and an ethanol concentration (D) which have obvious influence on the extraction rate of the stem and leaf extract of the rhizoma atractylodis for promoting the proliferation of lactobacillus plantarum are selected, and the extraction rate of the extract and the optimal extraction condition for promoting the growth of lactobacillus plantarum are inspected: selecting ethanol concentration (0, 45, 50, 55, 60, 65%) liquid-to-material ratio (10, 20, 30, 40, 50 mL/g), ultrasonic time (20, 25, 30, 40 min), ultrasonic temperature (25, 35, 40, 45, 50, 55, 60 ℃), ultrasonic power (300, 400, 500, 600, 700 w), pH (4, 5,6,7,8, natural) optimization test;
wherein C in the abscissa represents Lactobacillus plantarum treated by adding rhizoma Atractylodis stem and leaf
Experimental procedures and results are described in the examples below.
Example 1 investigation of the Effect of ethanol concentration on Lactobacillus plantarum growth Rate, self-aggregation Capacity, total protein content, lactic acid content, acetic acid content and extraction Rate
1) Exploring the influence of ethanol concentration on extraction rate of rhizoma atractylodis stem and leaf extract
15G of rhizoma atractylodis stems and leaves are weighed each time and respectively put into 5 1000mL ultrasonic bottles, and ultrasonic conditions are as follows: setting ethanol concentration of 0, 45, 50, 55, 60 and 65% at constant temperature of 50deg.C under ultrasonic power of 500w for 20min with natural pH, performing ultrasonic extraction, and calculating extraction rate; the calculation formula is as follows: extraction = m 2/m1 x 100%.
2) Exploring the influence of the rhizoma atractylodis stem and leaf extracts extracted under different ethanol concentrations in the step 1) on the proliferation of lactobacillus plantarum
(1) Preparation of Lactobacillus plantarum suspension
After the Lactobacillus plantarum frozen at-80 ℃ is activated, the Lactobacillus plantarum frozen stock is inoculated into MRS culture medium according to 1 percent and cultured for 24 hours at 37 ℃ and 200 rpm. The bacterial liquid is regulated to 1X 10 9 CFU/mL for standby.
(2) Lactobacillus plantarum count
The rhizoma Atractylodis stem and leaf extract is prepared into 200mg/mL mother liquor, and added into 40mL MRS culture medium with 0.22 μm needle sterile filter to obtain final concentration of 7.5mg/mL. Lactobacillus plantarum was inoculated at 1% inoculum size, incubated at 37℃at 200rpm for 24h. After culturing, the bacterial liquid is subjected to gradient dilution under aseptic operation, the temperature is 37 ℃, the time is 48 hours, and the plate count is carried out.
3) The influence of the atractylis lancea stem and leaf extract extracted under the condition of different ethanol concentrations in the step 1) on the self-aggregation capability of lactobacillus plantarum is explored
Taking 5mL of the cultured bacterial liquid, putting the bacterial liquid into a sterile centrifuge tube, determining that the absorbance at 600nm is A 0 when the bacterial liquid is measured for 0h, standing and culturing at 37 ℃ for 2h, carefully sucking the supernatant, and determining that the absorbance at 600nm is A 2.
Self-aggregation ability (%) = (a 0-A2)/A0 ×100%
4) Exploring the influence of the total protein content of lactobacillus plantarum of the rhizoma atractylodis stem and leaf extract extracted under different ethanol concentrations in the step 1)
And (3) taking the cultured bacterial liquid, and extracting the total protein by using a bacterial protein extraction kit (a middle Creatai bacterial protein extraction kit RTD 8101). Centrifuging 2mL of bacterial liquid at 8000rpm for 3min at normal temperature, removing supernatant, and collecting bacterial precipitate. Preparing a buffer solution: 10. Mu.L PMSF, 10. Mu.L lysozyme and 1. Mu.L DNase I were added to each mL extraction buffer, and the mixture was kept at 4℃until it was ready for use (preparation at present). 1mL of buffer solution is added into each tube of thalli, and the thalli are blown uniformly until no bacterial mass exists. Incubation was carried out at 37℃for 1h, during which time the flick centrifuge tube accelerated lysis. After completion of the cleavage, the mixture was centrifuged at 13000rpm at 4℃for 5 minutes, and the crude enzyme solution was collected from the supernatant. The total protein content was measured according to BCA kit method (bi yun tian total protein content measurement kit P10012).
5) Exploring the influence of the lactobacillus plantarum which is extracted from the stems and leaves of the rhizoma atractylodis under different ethanol concentrations on the content of lactic acid and acetic acid;
The culture broth was centrifuged at 8000rpm for 5min, and the supernatant was carefully aspirated and filtered through a 0.22 μm needle filter for HPLC analysis. Detection column: kromasil 100-5-C18; column temperature: 25 ℃; detecting a wavelength; 210nm; mobile phase: methanol: k 2HPO4 (0.01 mol/L, pH=2.7) 3:97; flow rate: 0.8mL/min; sample injection amount: 20. Mu.L.
The results are shown in figure 1, and show that the ethanol concentration is 50-55%, and the proliferation promoting effect and extraction rate of rhizoma Atractylodis stem and leaf extract for promoting proliferation of lactobacillus plantarum are good, so that the ethanol concentration is selected to be 50-55%.
Example 2 investigation of the effect of feed liquid ratio on Lactobacillus plantarum growth Rate, self-aggregation Capacity, total protein content, lactic acid content, acetic acid content and extraction Rate
1) Exploring the influence of liquid-liquid ratio on extraction rate of rhizoma atractylodis stem and leaf extract
15G of rhizoma atractylodis stems and leaves are weighed each time and respectively put into 5 1000mL of ultrasonic products, and ultrasonic conditions are as follows: ultrasonic power 500w and constant temperature 50 ℃, ethanol concentration 50%, ultrasonic time 20min, pH being natural, the ratio of the set feed liquid is 1:10,1:20,1:30,1:40,1:50, performing ultrasonic extraction.
2) The effect of the extract of the stems and leaves of the rhizoma atractylodis extracted under different feed liquid ratios in the step 1) on the proliferation of lactobacillus plantarum is examined, and the method is the same as the step 2) in the example 1.
3) Exploring the influence of the atractylis ovata stem and leaf extracts extracted under different feed liquid ratios in the step 1) on the self-aggregation capability of lactobacillus plantarum; as in step 3) of example 1.
4) The influence of the total protein content of lactobacillus plantarum which is extracted from the stem and leaf extract of rhizoma atractylodis under different feed-liquid ratios in the step 1) is explored, and the method is the same as that in the step 4) of the example 1.
5) Exploring the influence of the lactobacillus plantarum which is extracted from the stems and leaves of the rhizoma atractylodis under different feed-liquid ratios in the step 1) on the content of lactic acid and acetic acid; as in step 5) of example 1.
The results are shown in fig. 2, and the feed liquid ratio is 1:50-1: within the range of 40, the rhizoma atractylodis stem and leaf extract for promoting the proliferation of lactobacillus plantarum has better proliferation promoting effect and extraction rate; so the material-liquid ratio is 1:50-1:40.
Example 3 investigation of the Effect of ultrasonic Power on Lactobacillus plantarum growth Rate, self-aggregation Capacity, total protein content, lactic acid content, acetic acid content and extraction Rate
1) Influence of ultrasonic power on extraction rate of rhizoma atractylodis stem and leaf extract for promoting proliferation of lactobacillus plantarum
15G of rhizoma atractylodis stems and leaves are weighed each time and respectively put into 5 1000mL of ultrasonic products, and ultrasonic conditions are as follows: constant temperature 50 ℃, ethanol concentration 50%, feed-to-liquid ratio 1:50, ultrasonic time 20min, natural pH, setting ultrasonic power 300, 400, 500, 600, 700w, respectively, and performing ultrasonic extraction.
2) The effect of the stem and leaf extract of Atractylodes lancea on the proliferation of Lactobacillus plantarum, which was extracted under the different powers of the above step 1), was examined in the same manner as in step 2) of example 1.
3) The influence of the atractylis lancea stem and leaf extracts extracted under different powers in the step 1) on the self-aggregation capability of lactobacillus plantarum is examined, and the method is the same as that in the step 3) of the example 1.
4) The influence of the total protein content of lactobacillus plantarum produced by the atractylis stem and leaf extracts extracted under different powers in the step 1) is explored, and the method is the same as that in the step 4) of the example 1.
5) The influence of the lactobacillus plantarum on the lactic acid and acetic acid production content of the rhizoma atractylodis stem and leaf extract extracted under different powers in the step 1) is explored, and the method is the same as that in the step 5) of the example 1.
The result is shown in figure 3, which shows that the ultrasonic power is in the range of 500-600w, and the proliferation promoting effect and the extraction rate of the rhizoma atractylodis stem and leaf extract for promoting the proliferation of lactobacillus plantarum are better. So that the ultrasonic power is selected to be 500-600w
Example 4 investigation of the effect of pH on Lactobacillus plantarum growth Rate, self-aggregation Capacity, total protein content, lactic acid content, acetic acid content and extraction Rate
1) Influence of pH on extraction rate of rhizoma Atractylodis stem and leaf extract for promoting proliferation of Lactobacillus plantarum
15G of rhizoma atractylodis stems and leaves are weighed each time and respectively put into 5 1000mL of ultrasonic products, and ultrasonic conditions are as follows: ultrasonic power 500w, constant temperature 50 ℃, ethanol concentration 50%, feed-liquid ratio 1:50, ultrasonic extracting for 20min, wherein the pH values are respectively set to be 4,5,6,7 and 8, and natural pH values.
2) The effect of the stem and leaf extract of Atractylodes lancea on the proliferation of Lactobacillus plantarum, which is extracted at different pH values in the above step 1), was examined in the same manner as in step 2) of example 1.
3) The effect of the above-mentioned atractylis lancea stem and leaf extracts extracted at different pH in step 1) on the self-aggregating ability of Lactobacillus plantarum was examined, as in step 3) of example 1.
4) The influence of the total protein content of lactobacillus plantarum produced by the rhizoma atractylodis stem and leaf extracts extracted under different pH values in the step 1) is explored, and the method is the same as that in the step 4) of the example 1.
5) The influence of the lactobacillus plantarum on the lactic acid and acetic acid production content of the rhizoma atractylodis stem and leaf extract extracted under different pH values in the step 1) is explored, and the method is the same as that in the step 5) of the example 1.
The result is shown in figure 4, which shows that the rhizoma atractylodis stem and leaf extract with the pH value in the range of 4-5 has better proliferation promoting effect and extraction rate. Therefore, the pH value is selected to be 4-5.
Example 5 investigation of the Effect of ultrasound temperature on Lactobacillus plantarum growth Rate, self-aggregation Capacity, total protein content, lactic acid content, acetic acid content and extraction Rate
1) Influence of ultrasonic temperature on extraction rate of rhizoma Atractylodis stem and leaf extract for promoting proliferation of lactobacillus plantarum
15G of rhizoma atractylodis stems and leaves are weighed each time and respectively put into 5 1000mL of ultrasonic products, and ultrasonic conditions are as follows: ultrasonic power 500w, ethanol concentration 50%, feed liquid ratio 1: and 50, carrying out ultrasonic extraction at ultrasonic temperatures of 25, 35, 40, 45, 50, 55 and 60 ℃ for 20 min.
2) The effect of the above step 1) on the proliferation of Lactobacillus plantarum was examined on the extract of stems and leaves of Atractylodes lancea which was extracted at different ultrasonic temperatures, as in step 2) of example 1.
3) The influence of the atractylis stem and leaf extracts extracted at different ultrasonic temperatures in the step 1) on the self-aggregation capability of lactobacillus plantarum is explored, and the method is the same as that in the step 3) of the example 1.
4) The influence of the total protein content of lactobacillus plantarum produced by the atractylis stem and leaf extracts extracted at different ultrasonic temperatures in the step 1) is explored, and the method is the same as that in the step 4) of the example 1.
5) Exploring the influence of the above step 1) on lactic acid and acetic acid production contents of Lactobacillus plantarum by rhizoma Atractylodis stem and leaf extracts extracted at different ultrasonic temperatures, the same as step 5 of example 1
The result is shown in figure 5, which shows that the proliferation promoting effect and the extraction rate of the rhizoma atractylodis stem and leaf extract for promoting the proliferation of lactobacillus plantarum are better when the ultrasonic power is within the range of 45-50 ℃. Therefore, the ultrasonic temperature is selected to be 45-50 ℃.
Example 6 investigation of the Effect of ultrasound time on Lactobacillus plantarum growth Rate, self-aggregation Capacity, total protein content, lactic acid content, acetic acid content and extraction Rate
1) Influence of ultrasonic time on extraction rate of rhizoma Atractylodis stem and leaf extract for promoting proliferation of lactobacillus plantarum
15G of rhizoma atractylodis stems and leaves are weighed each time and respectively put into 5 1000mL of ultrasonic products, and ultrasonic conditions are as follows: ultrasonic power 500w, constant temperature 50 ℃, ethanol concentration 50%, feed-liquid ratio 1: and 50, setting the ultrasonic time to be 20, 30, 35 and 40min respectively, and carrying out ultrasonic extraction.
2) The effect of the stem and leaf extract of Atractylodes lancea on the proliferation of Lactobacillus plantarum, which is extracted under different ultrasound times in the above step 1), was examined as in the step 2) of example 1.
3) The influence of the atractylis stem and leaf extracts extracted under different ultrasonic time in the step 1) on the self-aggregation capability of lactobacillus plantarum is explored, and the method is the same as the step 3) in the embodiment 1.
4) The influence of the total protein content of lactobacillus plantarum which is extracted from the stem and leaf extract of rhizoma atractylodis under different ultrasonic time in the step 1) is explored, and the method is the same as the step 4) of the example 1.
5) The influence of the lactobacillus plantarum on the lactic acid and acetic acid production content of the rhizoma atractylodis stem and leaf extract extracted under different ultrasonic time in the step 1) is explored, and the method is the same as the step 5 of the example 1.
The results are shown in FIG. 6, and show that the ultrasonic time is 25-30min, and the proliferation promoting effect and extraction rate of rhizoma Atractylodis stem and leaf extract for promoting proliferation of lactobacillus plantarum are good, so that the ultrasonic time is 25-30min.
From the above results, the optimal extraction conditions were found to be: liquid-to-material ratio 1:50-1:40, the ultrasonic temperature is 45-50 ℃, the pH is 4-5, the ethanol concentration is 50-55%, the ultrasonic power is 500-600w, the ultrasonic time is 25-30min, and the extraction rate of the rhizoma atractylodis stem and leaf extract for promoting the proliferation of lactobacillus plantarum can reach more than 25%.
Example 7
In the liquid-material ratio 1:50-1: 40. extracting rhizoma Atractylodis stem and leaf extract and rhizoma Atractylodis extract at 45-50deg.C, pH4-5, ethanol concentration 50-55%, ultrasonic power 500-600w and ultrasonic time 25-30min, and calculating growth promotion rate (%), self aggregation ability (%), LA and AA of lactobacillus plantarum; see table 1.
TABLE 1
| Detection index | Rhizoma Atractylodis extract | Rhizoma Atractylodis stem and leaf extract |
| Growth promotion rate (%) | 118.78 | 143.66 |
| Self aggregation ability (%) | 28.68 | 38.14 |
| LA | 19.29 | 33.47 |
| AA | 33.82 | 39.10 |
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (2)
1. The application of the atractylis ovata stem and leaf extract in promoting the proliferation of lactobacillus plantarum is characterized in that the atractylis ovata stem and leaf extract is prepared by the following steps: in the liquid-material ratio 1:50-1: 40. extracting rhizoma Atractylodis stem and leaf extract at 45-50deg.C, pH4-5, ethanol concentration 50-55%, ultrasonic power 500-600 w and ultrasonic time 25-30 min.
2. The use according to claim 1, wherein the atractylis stem and leaf extract promotes lactobacillus plantarum fermentation to produce total protein, acetic acid and lactic acid content, increases lactobacillus plantarum growth rate, and promotes lactobacillus plantarum self-aggregation ability.
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| CN109497132A (en) * | 2018-11-08 | 2019-03-22 | 江南大学 | A kind of compound multiplication agent promoting lactobacillus plantarum high efficiently multiplying in newborn matrix system |
| CN110840924A (en) * | 2019-11-01 | 2020-02-28 | 中国农业科学院饲料研究所 | Application of rhizoma atractylodis stem and leaf extract |
| CN110923186A (en) * | 2019-12-31 | 2020-03-27 | 南昌诺汇医药科技有限公司 | Lactobacillus plantarum proliferation agent |
| CN114152705A (en) * | 2021-12-06 | 2022-03-08 | 中国农业科学院饲料研究所 | Rhizoma atractylodis stem and leaf HPLC fingerprint quality evaluation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109497132A (en) * | 2018-11-08 | 2019-03-22 | 江南大学 | A kind of compound multiplication agent promoting lactobacillus plantarum high efficiently multiplying in newborn matrix system |
| CN110840924A (en) * | 2019-11-01 | 2020-02-28 | 中国农业科学院饲料研究所 | Application of rhizoma atractylodis stem and leaf extract |
| CN110923186A (en) * | 2019-12-31 | 2020-03-27 | 南昌诺汇医药科技有限公司 | Lactobacillus plantarum proliferation agent |
| CN114152705A (en) * | 2021-12-06 | 2022-03-08 | 中国农业科学院饲料研究所 | Rhizoma atractylodis stem and leaf HPLC fingerprint quality evaluation method |
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| 超声-微波协同提取苍术中苍术素的工艺;戈振凯;赵永恒;杨彩霞;邱婧然;;医药导报;20180401(04);75-77 * |
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