CN115251094B - Method for extracting indocalamus leaf antibacterial active extract for food packaging - Google Patents
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- 239000000284 extract Substances 0.000 title claims abstract description 79
- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 52
- 241000755540 Indocalamus Species 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 32
- 235000013305 food Nutrition 0.000 title claims abstract description 13
- 238000004806 packaging method and process Methods 0.000 title claims abstract description 12
- 238000000605 extraction Methods 0.000 claims abstract description 42
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 30
- 241000607764 Shigella dysenteriae Species 0.000 claims abstract description 20
- 229940007046 shigella dysenteriae Drugs 0.000 claims abstract description 20
- 239000002904 solvent Substances 0.000 claims abstract description 20
- 241000293871 Salmonella enterica subsp. enterica serovar Typhi Species 0.000 claims abstract description 18
- 238000010992 reflux Methods 0.000 claims abstract description 10
- 238000012795 verification Methods 0.000 claims abstract description 10
- 238000012360 testing method Methods 0.000 claims description 56
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- 241000894006 Bacteria Species 0.000 claims description 34
- 239000000243 solution Substances 0.000 claims description 28
- 230000001580 bacterial effect Effects 0.000 claims description 24
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- 238000012258 culturing Methods 0.000 claims description 10
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- 239000003242 anti bacterial agent Substances 0.000 claims description 5
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 30
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 abstract description 30
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 abstract description 26
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 abstract description 20
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- 229930182566 Gentamicin Natural products 0.000 description 13
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- 229960002518 gentamicin Drugs 0.000 description 9
- 229960000583 acetic acid Drugs 0.000 description 8
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- 229940079593 drug Drugs 0.000 description 7
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- 230000003385 bacteriostatic effect Effects 0.000 description 6
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 5
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- 150000004676 glycans Chemical class 0.000 description 5
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- 229920001282 polysaccharide Polymers 0.000 description 5
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- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
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- 229930003935 flavonoid Natural products 0.000 description 4
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 240000007594 Oryza sativa Species 0.000 description 3
- 235000007164 Oryza sativa Nutrition 0.000 description 3
- 229930013930 alkaloid Natural products 0.000 description 3
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- 230000002421 anti-septic effect Effects 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 3
- -1 flavonoid compounds Chemical class 0.000 description 3
- 150000002215 flavonoids Chemical class 0.000 description 3
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- 239000004480 active ingredient Substances 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
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- 239000002585 base Substances 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
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- 239000002024 ethyl acetate extract Substances 0.000 description 2
- 229930003944 flavone Natural products 0.000 description 2
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- 229910052573 porcelain Inorganic materials 0.000 description 2
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- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 2
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 description 1
- 241000223602 Alternaria alternata Species 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 241000193388 Bacillus thuringiensis Species 0.000 description 1
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 1
- 235000017491 Bambusa tulda Nutrition 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- 238000009631 Broth culture Methods 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
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- 244000082204 Phyllostachys viridis Species 0.000 description 1
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- 241000894120 Trichoderma atroviride Species 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
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- 239000004599 antimicrobial Substances 0.000 description 1
- 239000006286 aqueous extract Substances 0.000 description 1
- 229940097012 bacillus thuringiensis Drugs 0.000 description 1
- 239000011425 bamboo Substances 0.000 description 1
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- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
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- 238000005485 electric heating Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 229930182486 flavonoid glycoside Natural products 0.000 description 1
- 150000007955 flavonoid glycosides Chemical class 0.000 description 1
- 150000007946 flavonol Chemical class 0.000 description 1
- HVQAJTFOCKOKIN-UHFFFAOYSA-N flavonol Natural products O1C2=CC=CC=C2C(=O)C(O)=C1C1=CC=CC=C1 HVQAJTFOCKOKIN-UHFFFAOYSA-N 0.000 description 1
- 235000011957 flavonols Nutrition 0.000 description 1
- 239000005003 food packaging material Substances 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical group 0.000 description 1
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
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- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000003716 rejuvenation Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
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- 238000000926 separation method Methods 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/40—Liliopsida [monocotyledons]
- A01N65/44—Poaceae or Gramineae [Grass family], e.g. bamboo, lemon grass or citronella grass
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Plant Pathology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Agronomy & Crop Science (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Food Preservation Except Freezing, Refrigeration, And Drying (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a indocalamus leaf antibacterial activity extract for food packaging and an extraction process, wherein 9 extraction solvents of water, pH4 hydrochloric acid solution, pH4 acetic acid solution and pH10 ammonia water solution, 30%, 60%, 80%, 95% ethanol and ethyl acetate are respectively adopted, and three extraction methods of an ultrasonic method, a reflux method and an extraction method are adopted to obtain different indocalamus leaf extracts; by adopting an improved oxford standard method for antibacterial activity verification, the indocalamus leaf water extract has the advantages of good antibacterial effect on bacillus subtilis, pseudomonas aeruginosa, shigella dysenteriae and salmonella typhi, remarkable heat stability and acid stability, wide application prospect and extremely high use value when being used as a food additive, cosmetics and daily chemical raw materials.
Description
Technical Field
The invention relates to the technical field of indocalamus leaf processing, in particular to indocalamus leaf antibacterial activity extract for food packaging and an extraction method.
Background
Pathogenic bacteria and spoilage bacteria have serious influence on human health and food safety, and excessive use of chemical synthesis preservatives such as sorbic acid, benzoic acid and the like has certain food safety problems; the wide use of antibiotics in human production and life not only causes drug residue pollution, but also causes different degrees of drug resistance for some pathogenic bacteria. Therefore, plant-source natural antiseptic bacteriostats are increasingly favored by people.
The rice dumpling leaf is a natural food packaging material which is popular with people, and is mainly the leaf of Indocalamus (Indocalamus Nakai) plant in the Indocalamus genus of the Gramineae bamboo subfamily, namely Indocalamus leaf. The indocalamus has more than 20 kinds, is mainly distributed in the south area of the Yangtze river basin, has rich resources, and has at least 131 families of rice dumpling leaf production enterprises in Hunan. From the food packaging perspective, the indocalamus leaf extract has certain antiseptic and antibacterial effects, and is a good natural antiseptic and antibacterial agent source of plant sources.
Disclosure of Invention
Based on the recognition and utilization of indocalamus She Chuantong, common pathogenic bacteria (shigella dysenteriae and salmonella dysenteriae) and putrefying bacteria (bacillus subtilis and pseudomonas aeruginosa) are used as test bacteria, broadleaf indocalamus leaves are used as materials, an oxford cup method is adopted, and the extraction process and the antibacterial activity of the extract are researched by measuring the diameter of a bacteriostasis ring.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows: the extraction method of indocalamus leaf antibacterial activity extract for food packaging comprises the following steps:
a first step of: preparing a bacterial suspension:
selecting strains, inoculating strain culture medium, culturing at 34-40deg.C for 20-26 hr to rejuvenate strains, and taking bacterial suspension with proper dilution ratio according to 8-12 times dilution method; inoculating the bacterial suspension into a bacterial suspension culture medium, and culturing for 20-26 hours at the temperature of 34-40 ℃ to obtain bacterial suspension;
and a second step of: preparation of a bacteriostasis culture medium:
1) Preparation of the bottom layer Medium
Pouring the antibiotic verification culture medium into a bottle filled with distilled water, heating and dissolving, placing into different culture dishes, and cooling and solidifying to obtain a bottom culture medium;
2) Preparation of fungus layer culture medium
The bottom culture medium prepared in the step 1) is filled into a sterile bottle according to the mass parts, and then bacterial suspension is inoculated and mixed uniformly to obtain a bacterial layer culture medium;
and a third step of: preparation of culture test fungus plate
Adding the fungus layer culture medium prepared in the step 2) into the bottom layer culture medium in the step 1), quickly horizontally automatically and uniformly tiling, cooling and solidifying, and culturing at 36-37 ℃ for 15-16h to obtain a culture test fungus plate;
fourth step: indocalamus leaf treatment
Cleaning indocalamus leaves to be detected, drying at 50-55 ℃, crushing, sieving with a 42-target standard sieve, and packaging for later use;
fifth step: preparation of the extract
1) Extracting a solvent: weighing folium indocalami tessellati powder of certain quality, adding 12 times of extraction solvent, refluxing for 15min, soaking for 20-26h, and repeating for 2 times for 20-30 min;
2) An extract: mixing the 2 times of extractive solutions of 1), rotary evaporating, and concentrating under reduced pressure to obtain folium indocalami tessellati extract.
The invention adopts three extraction methods of ultrasonic method, reflux method and leaching method to obtain different extracts of indocalamus leaf; through comparative researches, the indocalamus leaf water extract has remarkable antibacterial effect on bacillus subtilis, pseudomonas aeruginosa, shigella dysenteriae and salmonella typhi, and has good thermal stability and acid stability, wide application prospect and extremely high use value.
Drawings
FIG. 1 is a graph of factors and levels of the orthogonal test of the present invention.
FIG. 2 is a table showing the growth of the test bacteria of the present invention on 4 media.
FIG. 3 shows the bacteriostatic effect (zone diameter/mm) of different solvent extracts of indocalamus leaf according to the present invention.
FIG. 4 is a graph of the bacteriostatic effect (diameter of zone of inhibition/mm) of the gentamicin standard series of the invention against 4 test bacteria.
FIG. 5 is a graph showing the antibacterial effect (diameter of zone of inhibition/mm) of indocalamus leaf extract according to the different extraction methods of the present invention.
FIG. 6 is a graph of results of orthogonal experiments versus analysis (zone diameter/mm) according to the present invention.
FIG. 7 is a graph of the minimum inhibitory concentration (zone diameter/mm) of the optimal extract according to the present invention.
FIG. 8 shows the effect of temperature and pH on the bacteriostatic effect of the optimal extract (zone diameter/mm) according to the invention.
Detailed Description
The invention is further described below, with preferred embodiments of the invention being:
up to now, the research on indocalamus leaves at home and abroad mainly focuses on the nutrition matters and element contents thereof, and the extraction, separation and test of active substances such as volatile oil, total flavone, polysaccharide and the like, wherein flavonoids, polysaccharide, phenolic acids, terpenes, alkaloids and the like have strong antibacterial and bactericidal effects, the research on the corrosion and bacteria inhibition of extracts is still insufficient, the literature is very limited, and the research method is limited. Lijun super et al studied the inhibition of indocalamus leaf extract on tobacco pathogenic fungi; sun Kekui Indocalamus leaf polysaccharide extraction and purification and inhibition effects on escherichia coli, bacillus subtilis, bacillus licheniformis and staphylococcus aureus are studied; le Wei and Ji Ruidong, etc., have studied the extraction of total flavonoids of indocalamus leaves and the antibacterial effect on bacteria (staphylococcus aureus, escherichia coli) and fungi (candida albicans, saccharomyces cerevisiae and penicillium); he Zhou et al studied the effect of indocalamus leaf extract on the activity of staphylococcus aureus and escherichia coli; deng Longxing the influence of the crude indocalamus leaf extract on the growth rate of trichoderma atroviride and alternaria alternata is studied; sun Jia et al extract and isolate the indocalamus leaf antibacterial components with 95% ethanol solution, and found that 10 of these compounds have antibacterial activity against Staphylococcus aureus, bacillus thuringiensis, escherichia coli and Pseudomonas aeruginosa. The antibacterial literature research methods are single, the filter paper method is adopted, the adsorption of the filter paper has no quantitative index, the specificity of the culture medium is low, the test strains are not too many, the pseudomonas aeruginosa, the shigella dysenteriae and the salmonella typhi are not covered, the bacterial film is manually coated on the culture medium by using a coating rod, the bacterial film is difficult to be uniform, the antibacterial effect is not quantitatively compared with that of antibiotics, and the research method is very limited.
In the embodiment, bacillus subtilis, pseudomonas aeruginosa, shigella dysenteriae and salmonella typhi are used as test strains to carry out antibacterial activity extraction on indocalamus leaves, and the main raw materials and equipment of the embodiment are as follows:
leaf of Indocalamus latiflorus is obtained from Hongjiang area of Huaihua, crushed by crusher, sieved by a 42-target standard sieve, and sample powder is sealed with plastic bag for use; oxford cup (8 mm. Times.6 mm. Times.10 mm); cell culture dish, 100mm.
The test bacteria, bacillus subtilis (Bacillus subtilis) CMCC (B) 63501, pseudomonas aeruginosa [ Pseudomonas aeruginoda (Schroeter) Migula ] ATCC9027-1, shigella dysenteriae (Shigella dysenteriae) CMCC (B) 51252 and salmonella typhi (S.tyrpi) CMCC (B) 50071 are all 1-generation bacterial porcelain beads and are stored at-80 ℃.
Reagents, antibiotic assay Medium No. 1 (pH 6.5-6.6), antibiotic assay Medium No. 2 (pH 7.8-8.0), antibiotic assay Medium No. 3 (pH 6.5-6.6) and antibiotic assay Medium No. 4 (pH 7.8-8.0), qingdao high technology Industrial park, haibo Biotechnology Co., ltd; nutrient agar medium and nutrient broth medium, beijing land bridge technologies Co., ltd; gentamicin (national drug standard substance, batch No. 130326-201616, national food and drug verification institute) was diluted with sterile water to 0.01, 0.02, 0.03, 0.04, 0.05mg/mL standard series solutions.
95% ethanol (AR), hydrochloric acid (GR), ammonia (GR), glacial acetic Acid (AR), ethyl acetate (chromatographic purity), and 0.85% physiological saline.
FD-15-T250A type high-speed pulverizer for Chinese medicinal materials, shanghai Minkou area naval vessel industry and trade company; s220 pH meter, meltrele-tolido instruments (Shanghai) limited; RE-52AA rotary evaporator, shanghai elegant Biochemical instruments, inc.; LMQ.C-80E type vertical sterilizer, new Hua medical instruments Co., ltd; 1374 type II A2 biosafety cabinet, simer Feier instruments Co., ltd; DW-HL388 type ultra-low temperature refrigerator, mitsubishi low temperature technology Co., ltd; SPX-150 type biochemical incubator, shanghai Jin Huike electronic Co., ltd; 9960B ultrasonic cleaner, tianjin Kobe photoelectric technology Co., ltd; GZX-9140MBE electric heating constant temperature drying oven, test instruments Inc. of Tianjin.
The extraction method of the embodiment comprises the following steps:
a first step of: test strain rejuvenation and preparation of bacterial suspension
Taking 1 test bacillus subtilis, pseudomonas aeruginosa, shigella dysenteriae and salmonella typhi strain porcelain beads, respectively inoculating 100mL of nutrient broth culture medium, culturing at 36.5 ℃ for 24h to rejuvenate strains, taking bacterial suspensions with proper dilution factors according to a 10-time dilution method, respectively taking 1mL of bacterial suspensions, inoculating into nutrient agar culture medium, culturing at 36.5 ℃ for 24h, measuring bacterial concentration of the bacterial suspensions by colony counting, and taking the bacterial suspension with the concentration of about 1 multiplied by 10 7 The cfu/mL bacterial suspension is used for bacteriostasis test;
and a second step of: selection of bacteriostasis test Medium
1) Preparation of the bottom layer Medium
29.5g of antibiotic verification medium No. 1 (pH 6.5-6.6), antibiotic verification medium No. 2 (pH 7.8-8.0), antibiotic verification medium No. 3 (pH 6.5-6.6) and antibiotic verification medium No. 4 (pH 7.8-8.0) are respectively weighed, heated and dissolved in a triangular flask containing 1000mL of distilled water, autoclaved at 115 ℃ for 30min, and about 20mL of each is taken in different culture dishes, cooled and solidified to obtain 4 bottom culture medium plates;
2) Preparation of a fungus layer Medium
Taking 16 sterile jars of 200mL, dividing into 4 groups, sequentially adding 1) prepared antibiotic assay culture medium No. 1 (pH6.5-6.6), antibiotic assay culture medium No. 2 (pH7.8-8.0), antibiotic assay culture medium No. 3 (pH6.5-6.6) and antibiotic assay culture medium No. 4 (pH7.8-8.0) into each 100mL, sequentially adding 1mL of 1X 10 concentration 7 cfu/mL of 4 bacterial suspensions of bacillus subtilis, pseudomonas aeruginosa, shigella dysenteriae and salmonella typhi are uniformly mixed to prepare 16 different bacterial layer culture mediums which are rapidly inoculated when the culture mediums are hot;
and a third step of: production and culture of test bacteria plates
Sequentially adding 5mL of the culture mediums of the bacillus subtilis, the pseudomonas aeruginosa, the shigella dysenteriae and the salmonella typhi in the step 2) corresponding to the bottom culture medium into the 4 plates in the step 1) while the culture mediums are hot, rapidly and horizontally automatically and uniformly laying, cooling and solidifying to obtain 16 different test bacteria plates, culturing the test bacteria plates at 36.5 ℃ for 15-16h, and observing the growth conditions of the test bacteria on the different culture mediums;
fourth step: indocalamus leaf treatment
Cleaning fresh indocalamus leaves, drying at 50-55 ℃, crushing, sieving with a 42-target standard sieve, and packaging for later use;
fifth step: preparation of the extract
Different solvent extractions: weighing folium indocalami tessellati powder 10g, adding 100mL of extraction solvent into a 250mL triangular flask, refluxing for 15min, leaching for 24h, performing ultrasonic treatment for 30min, repeating for 2 times, combining 2 times of extraction solutions, performing rotary evaporation, concentrating under reduced pressure, and fixing volume to 10mL, wherein each milliliter of test solution contains 1g of dry sample, namely sample solution containing 1g/mL of crude drug, and extracting folium indocalami tessellati extract by using different solvents.
Extraction by different methods: weighing 3 parts of indocalamus leaf powder, 10g of each part, selecting an extraction solvent, respectively extracting for 2 times by using three methods of an ultrasonic method (frequency 40KHZ, power 400W, temperature 45 ℃ and extraction time 30 min), a reflux method (reflux temperature 100 ℃ and extraction time 60 min) and an extraction method (room temperature and extraction time 24 h) according to a feed-liquid ratio of 1:10, performing rotary evaporation and reduced pressure concentration on the extraction solution, and preparing sample solution containing 1g/mL of crude drug, thereby obtaining extracts of different methods of the same solvent.
Sixth step: bacteriostasis test
The antibacterial test is carried out on the extract prepared in the fifth step by using 4 test bacteria of bacillus subtilis, pseudomonas aeruginosa, shigella dysenteriae and salmonella typhi as indicator bacteria, using the diameter of a bacteriostasis circle as an evaluation standard and using a gentamicin standard series as a contrast and using an oxford cup method. Preparing a test fungus plate by using a screened antibiotic detection culture medium according to a second step method, lightly horizontally placing 4 sterile oxford cups in the plate by using sterile forceps, adding 0.283mL of extract or reference substance into the oxford cups, enabling the test solution not to overflow the oxford cups in operation, simultaneously performing reagent blank and parallel tests, culturing for 15-16h in a biochemical incubator at 36.5 ℃, observing the antibacterial effect, measuring the diameter of an antibacterial ring, and calculating the average value.
Selecting three factors of feed liquid ratio, ultrasonic time and ultrasonic times, respectively represented by A, B, C, and setting feed liquidThe ratio of the ultrasonic wave to the ultrasonic wave is 1:10, 1:12 and 1:16, the ultrasonic times are 2, 3 and 4, the ultrasonic time is 35, 45 and 55min, and the three factor levels are shown in the figure 1 and are shown as L 9 (3 3 ) And (3) extracting indocalamus leaf antibacterial extract through orthogonal test, preparing a sample solution containing 1g/mL of crude drug, carrying out antibacterial test on the test extract according to the sixth step, measuring the diameter of an antibacterial circle, comparing antibacterial effects, finding out optimal extraction technological parameters, and obtaining the optimal extract.
Determination of minimum inhibitory concentration:
the Minimum Inhibitory Concentration (MIC) was determined using a double dilution method. The optimal extract obtained from the above test initially contained crude drug concentration 1.00g/mL, was diluted ten times with sterile water to obtain 0.100g/mL of a test solution, and then diluted by double dilution to obtain 5 extract series solutions of 0.0500, 0.0250, 0.0125, 0.00625, 0.00312 g/mL. And (3) performing a bacteriostasis test on the optimal extract series solution according to the sixth step, and determining the lowest bacteriostasis concentration by taking a bacteriostasis zone as an assessment index.
Taking 30mL of the optimal extract test solution, equally dividing into 6 parts, respectively filling into sterile headspace bottles, sealing, respectively treating 3 parts in a constant temperature drying oven at 60, 80 and 100 ℃ for 35min, and cooling to room temperature; and 2 parts of hydrochloric acid solution and ammonia water solution are respectively used for adjusting the pH to 4 and 10, the rest 1 part of untreated solution is used for carrying out a bacteriostasis test on the test solution before and after the treatment according to the sixth step, and the diameter of a bacteriostasis circle is used as an evaluation index to analyze the stability of the optimal extract.
In the second step method, 4 test bacteria of bacillus subtilis, pseudomonas aeruginosa, shigella dysenteriae and salmonella typhi were cultured with 4 antibiotic assay media under the same conditions, and the growth of the test bacteria is shown in fig. 2.
The results in FIG. 2 show that antibiotic assay Medium No. 4 (pH 7.8-8.0) simultaneously meets the growth requirements of 4 test bacteria, so it was selected as the indocalamus leaf extract bacteriostasis test medium.
The indocalamus leaf has different chemical structures of flavone, polysaccharide, phenolic acid, terpenes, alkaloid and other components, and has different polarities. The flavonol and flavonoid glycoside have larger polarity, are easily dissolved in water, diluted ethanol, ethyl acetate and the like, the flavonoid aglycone has small polarity, is difficult to dissolve in water, is easily dissolved in methanol, ethanol, ethyl acetate and the like, and natural flavonoid compounds mostly exist in glycoside forms; polysaccharides are soluble in water, but not true solutions, and insoluble in organic solvents such as alcohols; the terpenoid has medium polarity and small polarity, is dissolved in organic solvents such as chloroform, ethyl acetate and the like, the terpene glycoside is easy to dissolve in water, and the terpene lactone is soluble in alkali; phenolic acid is dissolved in water; most of alkaloids are insoluble in water, soluble in organic solvents such as chloroform and ethanol, and their salts are readily soluble in water. Therefore, the test selects water, pH4 hydrochloric acid solution, pH4 acetic acid solution, pH10 ammonia water solution, ethanol with different concentrations and ethyl acetate as extraction solvents, and the polarity of the solvents is as follows: water, pH4 hydrochloric acid solution and pH10 aqueous ammonia solution > ethanol > ethyl acetate.
According to the fifth step method, 9 kinds of extraction solvents are used to obtain 9 kinds of indocalamus leaf extracts. The antibacterial test was performed on 4 test bacteria according to the sixth step using 9 solvent indocalamus latiflorus leaf extracts of 1#, 2#, 3#, 4#, 5#, 6#, 7#, 8#, 9#, respectively, water, a pH4 hydrochloric acid solution, a pH4 acetic acid solution, a pH10 ammonia water solution, 30% (v+v) ethanol, 60% (v+v) ethanol, 80% (v+v) ethanol, 95% (v+v) ethanol, and ethyl acetate, and the results were shown in FIG. 3; the antibacterial effect of the control is shown in figure 4.
The results in fig. 3 show that the 3# extract has the strongest bacteriostatic effect, namely a pH4 acetic acid solution extract, followed by 1# and 9# extracts, water and ethyl acetate respectively; the total inhibition zone value of the reagent blank pH4 acetic acid solution is the largest, which shows that the pH4 acetic acid solution has the strongest inhibition effect, and the reagent blank only has no inhibition effect by water, pH4 hydrochloric acid solution and pH10 ammonia water solution; although the total inhibition zone value of the pH4 acetic acid solution extract is the maximum and is 81.5mm, the blank total inhibition zone value of the acetic acid solution reagent is the maximum and is 73.2mm, and the inhibition effect of the extract cannot be truly reflected; the antibacterial effects of the water and ethyl acetate extracts are not much different and are 66.1mm and 65.4mm respectively, but the total antibacterial circle value of the ethyl acetate blank is 34.2mm, and the antibacterial effect of the extract can not be truly reflected by the ethyl acetate extract; therefore, under the condition of ultrasonic extraction, the water extract has remarkable antibacterial effect on 4 test bacteria, and water is the best extraction solvent. Meanwhile, the indocalamus leaf water extract is better than the alcohol extract.
The results of fig. 4 show that gentamicin has remarkable antibacterial effect on 4 test bacteria, and the correlation coefficient of the antibacterial result curve equation of the standard series is greater than 0.95, which shows that the antibacterial effect of gentamicin on each test bacteria has positive correlation with the concentration in the selected concentration range.
Comparing fig. 3 and fig. 4, the antibacterial effect of the water extract on bacillus subtilis and salmonella typhi is equivalent to 0.01g/mL gentamycin, the antibacterial effect on pseudomonas aeruginosa is equivalent to 0.05g/mL gentamycin, the antibacterial effect on shigella dysenteriae is >0.05g/mL gentamycin, and the total antibacterial effect on 4 test bacteria is equivalent to 0.03g/mL gentamycin.
The oxford cup method bacteriostasis test adopted in the test is more scientific, and the result is reliable. Firstly, the selected culture medium is an antibiotic verification culture medium, and the specificity is strong; secondly, the prepared fungus layer culture medium is uniform, 5mL of fungus suspension is quantitatively added to each dish of bottom layer culture medium, the fungus amount is sufficient, and the fungus layer horizontally automatically flows and spreads, so that the thickness is uniform; furthermore, 0.283mL of test solution is accurately added into each oxford cup, so that the quantitative determination is accurate and no overflow exists.
Four representative extraction solvents of water, 60% ethanol, pH4 hydrochloric acid aqueous solution and pH10 ammonia water solution are adopted, 3 extraction methods of an ultrasonic method, a reflux method and a common leaching method are selected according to the fifth step, antibacterial active substances in indocalamus leaves are extracted, and the antibacterial effect of each extract on test bacteria is shown in figure 5.
The result of FIG. 5 shows that when the extraction solvent is water, the total inhibition zone of the extract obtained by the ultrasonic method is the largest and is 67.4mm; when the extraction solvent is 60% ethanol, the total inhibition zone of the extract obtained by the reflux method is 49.7mm at maximum; when the extraction solvent is a pH4 hydrochloric acid aqueous solution, the total inhibition zone of the extract obtained by a reflux method is 49.2mm at maximum; when the extraction solvent is pH10 ammonia water solution, the total inhibition zone of the extract obtained by the ultrasonic method is maximum, which is 54.3mm. It can be seen that water ultrasound is the best extraction method.
Selecting factors and levels of Table 1 according to L by using water as extraction solvent 9 (3 3 ) Orthogonal test design, preparing indocalamus leaf extract, performing bacteriostasis test on 4 test bacteria according to the sixth step, and analyzing under each extraction conditionThe antibacterial effect of the obtained extract is shown in FIG. 6.
The results of FIG. 6 show that from the very poor R value analysis, the B factor is three>Factor A>Factor C, namely the primary and secondary order of factors affecting the extraction effect of antibacterial substances, is the number of times of ultrasound>Feed-to-liquid ratio>Ultrasound time, number of ultrasound is the main factor. From the diameter analysis of the total inhibition zone, the inhibition effect of the No. 8 extract is best, and the condition is optimal, so that the No. 8 test A 3 B 2 C 1 (feed-liquid ratio 1:16, ultrasonic times 3 times and ultrasonic time 35 min) is the optimal extraction process parameter, and the extract obtained under the process condition is the optimal extract. Compared with FIG. 3, the diameter of the inhibition zone of the optimal extract No. 8 on the bacillus subtilis is 16.1mm, which is equivalent to the inhibition effect of 0.01mg/mL gentamicin; the diameter of a bacteria inhibition zone for salmonella typhi is 18.3mm, which is equivalent to the bacteria inhibition effect of gentamicin of 0.04 mg/mL; the diameters of the inhibition zones of the bacteria for pseudomonas aeruginosa and shigella dysenteriae are 23.3 and 21.8 respectively, and are both larger than 0.05mg/mL of gentamicin, and the total inhibition effect of the bacteria for 4 test bacteria is also larger than 0.05mg/mL of gentamicin.
Because the inner diameter of the oxford cup is 6mm, the extract with the diameter of the inhibition zone larger than 6mm has an antibacterial effect. The diameter of the inhibition zone is more than 6mm and less than 8mm, the inhibition zone is low-sensitivity, the diameter of the inhibition zone is more than 8mm and less than 14mm, the inhibition zone is moderate-sensitivity, the diameter of the inhibition zone is more than 14mm and less than 20mm, the inhibition zone is high-sensitivity, and the inhibition zone is extremely-sensitivity, and the inhibition zone is more than 20 mm. With reference to this standard, bacillus subtilis and salmonella typhi are highly sensitive to the optimal extracts, and pseudomonas aeruginosa and shigella dysenteriae are both extremely sensitive to the optimal extracts.
The lowest inhibitory concentration of the best extract was determined by the method for determining the lowest inhibitory concentration, and the results are shown in FIG. 7.
The results in FIG. 7 show that the optimal extract initially contained crude drug at a concentration of 1.000g/mL (1 g dry leaf per mL of test solution) had a Minimum Inhibitory Concentration (MIC) of 0.025g/mL for Bacillus subtilis and Shigella dysenteriae and 0.05g/mL for Pseudomonas aeruginosa and Salmonella typhi.
Thermal stability and acid-base stability test
When the indocalamus leaf is applied to food packaging, the indocalamus leaf is mainly used as an inner packaging material of steamed foods, such as rice dumplings, and the inner environment of the human stomach and small intestine respectively shows acidity and alkalinity, so the thermal stability and the acid-base stability of indocalamus leaf extracts are selected to be examined. The antibacterial effect of the optimal extract before and after treatment at 60deg.C, 80deg.C, 100deg.C, pH4 and pH10 is shown in figure 8.
The results in FIG. 8 show that the optimal extract has insignificant changes in the antibacterial effect on 4 test bacteria after heat treatment at 60, 80, 100 ℃ and treatment with hydrochloric acid solution to pH4, indicating that the antibacterial activity is stable. However, after adjusting the pH to 10 with ammonia, the extract has no bacteriostatic effect, indicating that the active ingredients in the aqueous extract are chemically changed under alkaline conditions, and the bacteriostatic active ingredients may be mainly phenolic hydroxyl and carboxylic acid substances, which are to be further studied.
The method shows that the indocalamus leaf water extract for food packaging has obvious antibacterial effect on bacillus subtilis, pseudomonas aeruginosa, shigella dysenteriae and salmonella typhi, and is the optimal extract; ultrasonic methods are the most effective extraction methods; the optimal extraction process parameters are as follows: the feed liquid ratio is 1:16, the ultrasonic times are 3 times, and the ultrasonic time is 35min; the optimal extract has the Minimum Inhibitory Concentration (MIC) of 0.025g/mL for bacillus subtilis and shigella dysenteriae, the Minimum Inhibitory Concentration (MIC) of 0.05g/mL for pseudomonas aeruginosa and salmonella typhi, good thermal stability and acid stability, wide application prospect and great use value. The above-mentioned embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, so that all changes made in the shape and principles of the present invention are covered by the scope of the present invention.
Claims (1)
1. A method for extracting active substances for bacteriostasis by using an extraction process of indocalamus leaf bacteriostasis active extract for food packaging is characterized in that: the antibacterial object is shigella dysenteriae and salmonella typhi, and the method comprises the following steps:
a first step of: preparing a bacterial suspension:
selecting strains, inoculating a strain liquid culture medium, culturing at 36-37deg.C for 20-26 hr to rejuvenate the strains, and taking a strain suspension with proper dilution ratio according to 8-12 times dilution method; inoculating the bacterial suspension into a solid culture medium, culturing for 20-26 hours at 36-37 ℃, and counting to obtain bacterial suspension with accurate concentration;
and a second step of: preparation of a bacteriostasis culture medium:
1) Preparation of the bottom layer Medium
Pouring the antibiotic verification culture medium into a bottle filled with distilled water, heating and dissolving, placing into different culture dishes, and cooling and solidifying to obtain a bottom culture medium;
2) Preparation of fungus layer culture medium
The bottom culture medium prepared in the step 1) is filled into a sterile bottle according to the mass parts, and then bacterial suspension is inoculated and mixed uniformly to obtain a bacterial layer culture medium;
and a third step of: preparation of test bacteria plate
Adding the fungus layer culture medium prepared in the step 2) into the bottom layer culture medium in the step 1), quickly horizontally automatically flowing and uniformly tiling, and cooling and solidifying to obtain a culture test fungus plate;
fourth step: indocalamus leaf treatment
Cleaning indocalamus leaves to be detected, drying at 50-55 ℃, crushing, sieving with a 42-target standard sieve, and packaging for later use;
fifth step: preparation of the extract
1) Extracting a solvent: weighing folium indocalami tessellati powder of certain mass, adding ultrapure water of 12 times of mass, soaking, refluxing for 15min, leaching for 20-26h, ultrasonic treating for 20-30min, filtering, and repeating for 2 times;
2) An extract: mixing the 2 times of extractive solutions of 1), rotary evaporating, and concentrating under reduced pressure to obtain folium indocalami tessellati extract;
sixth step: antibacterial activity verification of extract
Using test bacteria of shigella dysenteriae and salmonella typhi as indicator bacteria, using the diameter of a bacteriostasis circle as an evaluation standard, using an antibiotic standard series as a contrast, and using an improved oxford cup method to carry out a bacteriostasis test on the extract prepared in the fifth step; preparing a test bacteria plate according to the second and third steps, lightly horizontally placing 4 sterile oxford cups in the plate by using sterile forceps, adding an extract or a reference substance into the oxford cups, simultaneously performing reagent blank and parallel test, culturing for 15-20 h at 36-37 ℃, observing the antibacterial effect, and measuring the diameter of an antibacterial ring.
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