CN114982792B - Extraction process, bacteriostasis and application of total flavonoids in agricultural wastes - Google Patents
Extraction process, bacteriostasis and application of total flavonoids in agricultural wastes Download PDFInfo
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Classifications
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- 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
-
- 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
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B4/00—General methods for preserving meat, sausages, fish or fish products
- A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
- A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
- A23B4/20—Organic compounds; Microorganisms; Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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
-
- 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical & Material Sciences (AREA)
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- Plant Pathology (AREA)
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- Mycology (AREA)
- Biotechnology (AREA)
- Agronomy & Crop Science (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Pest Control & Pesticides (AREA)
- Food Science & Technology (AREA)
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Abstract
The invention relates to the technical field of agricultural waste utilization, in particular to an extraction process, bacteriostasis and application of total flavonoids in wild rice stem bracts. The extraction process of the wild rice stem bract total flavonoids comprises the following steps: cellulase addition amount is 0.70%, enzymolysis time is 60min, ultrasonic power is 704W, ultrasonic time is 47min, ethanol concentration is 60%, and feed-liquid ratio is 1:40g/mL; the Minimum Inhibitory Concentration (MIC) of the wild rice stem bract total flavonoids to escherichia coli is 2.5mg/mL, and the MIC to staphylococcus aureus is 1.25mg/mL; the wild rice stem bract total flavone solution can be used as a fresh-keeping agent for chilled fresh pork.
Description
Technical Field
The invention relates to the technical field of agricultural waste utilization, in particular to an extraction process of total flavonoids in wild rice stem bracts and application thereof.
Background
Wild rice stem, ancient wild rice, is a perennial root vegetation, belonging to the genus of Gramineae wild rice, native to China and southeast Asia. Cane shoots are a plant of both medicinal and edible origin, the edible part of which is its enlarged fleshy stem. As food, caulis Zizaniae Caduciflorae has high nutritive value, and is rich in protein, fat and saccharideVitamins, minerals, etc., and also contains various amino acids essential to human body; as a medicinal material, cane shoots are sweet and slightly cold in taste and have the effects of dispelling heat, promoting the production of body fluid, quenching thirst, promoting urination, removing dampness and promoting diuresis, so that the cane shoots have the reputation of 'ginseng in water'. The wild rice stem plant is high and large, the biological yield is high, the biological quality of the wild rice stem bract accounts for 50-70% of the total mass of the wild rice stem plant, and each 667m 2 The fresh bract produced in the same year of the double-cropping cane shoots is more than 5000 kg. As calculated in this way, the fresh quality of the wild rice stem bracts in Jiangsu province for one year is between 35 and 49 ten thousand tons. In the harvesting season of cane shoots, a large amount of cane shoots are abandoned on villages, roads, river sides and the like to become pollutants, and some of the cane shoots are directly burnt to cause serious environmental pollution and also become sites for breeding diseases and insect pests.
Therefore, comprehensive development and utilization of wild rice stem bract resources are the problems and difficulties which need to be solved in the wild rice stem industry at present. Jiang Bairong (China livestock and poultry industry, 2013 (7): 3) tries to feed the wild rice stem bracts as silage to ruminants such as cattle and sheep, and is considered as a better agricultural waste treatment measure, however, the energy utilization rate is lower only after livestock pass through the abdomen and return to the field, and in fact, the agricultural waste is also rich in functional components with development value.
Disclosure of Invention
The first aim of the invention is to provide an extraction process of total flavonoids of wild rice stem bracts (TFZB), which screens out optimal parameters of the total flavonoids of wild rice stem bracts (TFZB) cellulase in cooperation with ultrasonic auxiliary alcohol extraction through a single factor test and a response surface method, and provides references for extracting functional components of similar agricultural wastes.
The second purpose of the invention is to provide the application of the wild rice stem bract Total Flavone (TFZB) extracted by the extraction method, which has better inhibition effect on escherichia coli and staphylococcus aureus and has great application prospect.
The third purpose of the invention is to provide the cold fresh pork preservative prepared by the TFZB provided by the extraction method.
According to a first aspect of the present invention, the present invention provides an extraction process of total flavonoids of wild rice stem bracts (TFZB), in order to achieve the above technical purposes, the specific technical scheme is as follows:
a method for extracting total flavonoids from wild rice stem bracts comprises the following steps:
(1) Cleaning the collected fresh wild rice stem bracts, drying to constant weight, crushing, sieving, soaking a certain amount of wild rice stem bract powder in a buffer solution of disodium hydrogen phosphate-citric acid water, and then adding cellulase for enzymolysis.
(2) After enzyme is added in the step (1), the enzymolysis is carried out for 40 to 90 minutes;
(3) After enzymolysis, inactivating enzyme at high temperature in water bath, adding a certain amount of absolute ethyl alcohol and deionized water, and performing ultrasonic-assisted extraction;
(4) And (3) ultrasonically extracting the sample in the step (3) for 20-70 min.
Preferably, the mesh number of the crushed and sieved powder in the step (1) is 40 mesh; the pH value of the buffer solution of disodium hydrogen phosphate-citric acid water is=5, and the solid-liquid ratio is 1g/40mL; the amount of cellulase added is 0.30 to 0.80% (mass/volume ratio), and more preferably 0.70% (mass/volume ratio).
The addition amount of the cellulase has a great influence on the extraction rate of total flavonoids in the bracts of the cane shoots. In a certain enzymolysis time, along with the increase of the enzyme addition amount, the enzymolysis rate is accelerated, the cellulase can react with more substrates, so that flavonoid substances are fully exposed, the subsequent alcohol extraction and ultrasonic extraction are facilitated, excessive enzymes have adhesive performance, a dissolution channel of the flavonoid can be blocked, and the increase of the enzyme amount can also cause competition among enzymes or even inhibition. For this reason, the cellulase addition amount in the present invention is 0.30% to 0.80%, preferably 0.70% (mass/volume ratio).
Preferably, step (2) is enzymatically hydrolyzed at 37℃for 60min. Along with the prolongation of the enzymolysis time, the enzyme and the substrate can fully react, and the cell wall structure of the wild rice stem bract is degraded under the action of the cellulase, so that the flavonoid compounds are easier to dissolve and diffuse into the ethanol solution, and if the enzymolysis time is too long, part of the flavonoid compounds are oxidized due to long-time heating. Therefore, the enzymolysis time of the invention is 40 min-90 min, preferably 60min.
Preferably, after the enzymolysis in the step (3), the enzyme is inactivated in a water bath at 90 ℃ for 10min, a certain amount of absolute ethyl alcohol and deionized water are added to adjust the final concentration of the ethanol in the system to 60V%, and finally the extraction is carried out under the conditions of 55 ℃ and ultrasonic power of 300W-800W.
When the ultrasonic power is lower, the cavitation effect generated by the ultrasonic wave is small, so that the dissolution of flavonoid compounds is reduced, along with the increase of the ultrasonic power, the kinetic energy of each molecule of the extracting solution is increased, the cavitation effect of the ultrasonic wave is enhanced, the dissolution speed of the flavonoid is accelerated, the ultrasonic power is too high, the molecular motion is too severe, the reaction between the flavonoid molecules and other components is enhanced, the flavonoid is damaged, and the extraction rate is reduced. Thus, the ultrasonic power in the present invention is determined to be 300W to 800W, and preferably the ultrasonic power is 704W.
Preferably, the ultrasonic extraction time in step (4) is 47min. Along with the extension of ultrasonic extraction time, flavonoids in the wild rice stem bracts are gradually accumulated and increased, but the flavonoids are destroyed by long-time heating extraction. Therefore, the ultrasonic extraction time is determined to be 20 min-70 min in the invention,
the invention compares the TFZB extraction rate and the flavone content under four methods of alcohol extraction (namely no enzyme and ultrasonic treatment), cellulase auxiliary alcohol extraction (the addition amount of cellulase is 0.70 percent, enzymolysis is carried out for 60 minutes and no ultrasonic treatment), ultrasonic auxiliary alcohol extraction (no enzyme treatment and 704W ultrasonic treatment are carried out for 47 minutes) and cellulase synergistic ultrasonic auxiliary alcohol extraction (the addition amount of cellulase is 0.70 percent, enzymolysis is carried out for 60 minutes and 704W ultrasonic treatment is carried out for 47 minutes). Preferably, the extraction rate of TFZB and the flavone content of the extract are respectively (1.92+/-0.02)% and (150+/-1.25) mg/g by adopting a cellulase and ultrasonic assisted alcohol extraction method. (FIG. 1)
According to a second aspect of the invention, the invention provides an application of total flavonoids of wild rice stem bracts, which has a good inhibition effect on escherichia coli and staphylococcus aureus, and the specific technical scheme is as follows:
(1) The method is adopted to extract the total flavonoids TFZB of the wild rice stem bracts, the TFZB powder is obtained by freeze drying after concentration, a certain amount of TFZB powder is weighed, a small amount of 60% ethanol solution is added for dissolution assistance, then TFZB solutions with different concentrations are prepared, escherichia coli (E.coli ATCC 25922) and staphylococcus aureus (S.aureus ATCC 25923) are used as test strains, and the MIC of the test strains for the escherichia coli is measured to be 2.5mg/mL, and the MIC for the escherichia coli is measured to be 1.25mg/mL.
According to a third aspect of the invention, the invention provides the cold fresh pork preservative prepared by the TFZB provided by the extraction method.
The specific technical scheme is as follows:
peeling fresh pork foreleg meat, uniformly dividing the pork foreleg meat into small blocks with the weight of 35 g+/-5 g, adding 240 samples, spraying different treatment liquids (sterile water, sodium benzoate solution with the concentration of 5.0mg/mL, wild rice stem bract flavone solution with the concentration of 0.5mg/mL and wild rice stem bract flavone solution with the concentration of 1.0 mg/mL) on the pork samples,
each treatment liquid was sprayed with 12 samples (approximately 1mL of liquid per 100 square cm), 4 storage times, 3 replicates. The pork treated as above was sealed with food grade plastic preservative film (GB/T10457-2021), stored in a refrigerator at 4deg.C+ -0.5deg.C, and sampled at days 0, 3, 6, and 9 of storage, respectively, for determination of sensory score, pH, TVB-N content, metMb% content, and total colony count.
Preferably, the fresh-keeping agent for the cold pork is 1.0mg/mL of wild rice stem bract flavone solution.
Compared with the prior art, the invention has the beneficial effects that:
(1) the extraction rate of the wild rice stem bract total flavonoids is obviously improved by the aid of the cellulase and the ultrasonic method.
(2) The MIC of the wild rice stem bract general flavone provided by the invention to escherichia coli is 2.5mg/mL, and the MIC to staphylococcus aureus is 1.25mg/mL, so that the wild rice stem bract general flavone can be used as an environmental killer.
(3) The cane shoot bract total flavone provided by the invention can be used as a fresh-keeping agent for cold fresh pork after being prepared into a solution, and especially when the concentration is 1.0mg/mL, the fresh-keeping effect is better than that of sodium benzoate solution of 5.0 mg/mL.
Drawings
FIG. 1 is a graph showing total flavone extraction rate and flavone content of wild rice stem bracts by different extraction methods, wherein each system is respectively 1: alcohol extraction; 2: a cellulase assisted alcohol extraction process; 3: ultrasonic assisted alcohol extraction; 4: cellulase and ultrasonic wave assisted alcohol extraction method are cooperated to obtain corresponding results;
fig. 2 is an electron microscope observation of the extraction residue of the bracts of zizania latifolia, wherein (a): alcohol extraction; (b): a cellulase assisted alcohol extraction process; (c): ultrasonic assisted alcohol extraction; (d): cellulase and ultrasonic wave assisted alcohol extraction method are cooperated to obtain corresponding results;
FIG. 3 is a graph showing the effect of cellulase addition on the extraction rate of total flavonoids in wild rice stem bracts;
FIG. 4 is a graph showing the influence of enzymolysis time on the extraction rate of total flavonoids in wild rice stem bracts;
FIG. 5 is a graph showing the effect of ultrasonic power on the extraction rate of total flavonoids in wild rice stem bracts;
FIG. 6 is a graph showing the effect of ultrasonic time on the extraction rate of total flavonoids from wild rice stem bracts;
FIG. 7 is a response surface contour plot;
FIG. 8 is a graph showing pH change during storage of different treatment groups of chilled fresh pork;
FIG. 9 is a graph showing the change in sensory scores during storage of different treatment groups of chilled fresh pork;
FIG. 10 is a graph showing the variation of TVB-N values during storage of different treatment groups of chilled fresh pork;
FIG. 11 is a graph showing the% value change of MetMb during storage of different treatment groups of chilled fresh pork;
FIG. 12 is a graph showing the total number of colonies during storage of chilled fresh pork from different treatment groups.
Among the averages in fig. 3 to 6, the differences are insignificant (P > 0.05) when there is one letter marked the same, and the differences are significant (P < 0.05) when there is a different letter marked the same. A, B, C, D in FIGS. 8-12 shows that the difference is insignificant (P > 0.05) for all the same letters marked with one letter and significant (P < 0.05) for all the different letters marked with the same letter in the same storage time. a. b, c, d represent the same process, the differences are insignificant (P > 0.05) for all the same letter marked, and the differences are significant (P < 0.05) for all the different letters marked.
Detailed Description
The wild rice stem bracts are collected in the vegetable market and beside the farmland at the periphery of the developing area of the northeast of the ordinary China in 2021, the cold fresh pork is provided by the large Tang agricultural development Co, the analytically pure rutin standard is purchased from Shanghai source leaf biotechnology Co, other chemically pure chemicals and cellulose are purchased from Shanghai biological engineering Co, and the test strain is stored in the biological and food engineering institute microbiology laboratory of the ordinary China Jiangsu China. All experiments were three-fold, data expressed as mean ± SD, statistical analysis of data using t-test or ANOVA analysis, P <0.05 considered statistical differences.
Example 1 extraction Process of Total flavonoids from Zizania latifolia bracts (alcohol extraction method)
Cleaning the collected fresh wild rice stem bracts, drying to constant weight, crushing, sieving with a 40-mesh sieve, soaking a certain amount of wild rice stem bract powder in disodium hydrogen phosphate-citric acid buffer solution (pH=5, solid-liquid ratio of 1g/40 mL), adding a certain amount of absolute ethyl alcohol and deionized water to adjust the final concentration of the ethyl alcohol of a system to 60% (volume ratio), extracting at 80 ℃ for 60min, cooling, concentrating, and centrifuging to obtain a total flavone crude extract. Taking a certain amount of crude flavone extract, measuring the total flavone content, and calculating to obtain the total flavone extraction rate of (0.35+/-0.01)%. After freeze drying, 1.0g of the crude extract of flavone is weighed, the flavone content in the crude extract is measured after dissolution, and the total flavone content in the crude extract is (132+/-1.04) mg/g (figure 1).
The extraction rate calculation formula is as follows:
wherein: c is total flavone concentration (mg/mL) in the wild rice stem bract flavone crude extract
V is the volume of crude extract (mL)
M is the mass (mg) of the wild rice stem bract powder
The calculation formula of the total flavone content is as follows:
wherein: c is total flavone concentration (mg/mL) in the crude extract solution
V is the volume of the solution (mL)
M is the mass (g) of the freeze-dried powder of the crude extract
Example 2 extraction Process of Total flavonoids from Zizania latifolia bracts (enzyme-assisted alcohol extraction)
Cleaning the collected fresh wild rice stem bracts, drying to constant weight, crushing, sieving with a 40-mesh sieve, soaking a certain amount of wild rice stem bract powder in disodium hydrogen phosphate-citric acid buffer solution (pH=5, solid-liquid ratio of 1g/40 mL), adding 0.4% (mass-volume ratio) cellulase, carrying out enzymolysis at 37 ℃ for 60min, inactivating enzyme in a water bath at 90 ℃ for 10min, adding a certain amount of absolute ethyl alcohol and deionized water, regulating the final concentration of the ethyl alcohol of a system to 60% (volume ratio), finally extracting at 80 ℃ for 60min, cooling, concentrating, and centrifuging to obtain the total flavone crude extract. And (3) measuring the total flavone content in a certain amount of flavone crude extract, and calculating to obtain the total flavone extraction rate (0.49+/-0.01)%, wherein the total flavone content in the crude extract is (133+/-1.21) mg/g (shown in figure 1).
Example 3 extraction Process of Total flavonoids from Zizania latifolia bracts (ultrasound-assisted alcohol extraction)
Cleaning the collected fresh wild rice stem bracts, drying to constant weight, crushing, sieving with a 40-mesh sieve, soaking a certain amount of wild rice stem bract powder in disodium hydrogen phosphate-citric acid buffer solution (pH=5, solid-liquid ratio of 1g/40 mL), adding a certain amount of absolute ethyl alcohol and deionized water to adjust the final concentration of the ethanol of the system to 60%, performing ultrasonic treatment at 55 ℃ for 50min under the condition of ultrasonic power of 700W, cooling, concentrating, and centrifuging to obtain a total flavone crude extract. And (3) measuring the total flavone content in a certain amount of flavone crude extract, and calculating to obtain the total flavone extraction rate (1.32+/-0.02)%, wherein the total flavone content in the crude extract is (142+/-1.25) mg/g (shown in figure 1).
Example 4 extraction Process of Total flavonoids from Zizania latifolia bracts (enzymatic synergistic ultrasound assisted alcohol extraction)
Cleaning the collected fresh wild rice stem bracts, drying to constant weight, crushing, sieving with a 40-mesh sieve, soaking a certain amount of wild rice stem bract powder in disodium hydrogen phosphate-citric acid buffer solution (pH=5, solid-liquid ratio of 1g/40 mL), adding 0.7% of cellulase, performing enzymolysis at 37 ℃ for 60min, performing water bath enzyme deactivation at 90 ℃ for 10min, adding a certain amount of absolute ethyl alcohol and deionized water to adjust the final concentration of the ethanol of the system to 60%, performing ultrasonic treatment at 55 ℃ under the condition of ultrasonic power of 700W for 50min, cooling, concentrating, and centrifuging to obtain the total flavone crude extract. And (3) measuring the total flavone content in a certain amount of flavone crude extract, and calculating to obtain the total flavone extraction rate (1.84+/-0.02)%, wherein the total flavone content in the crude extract is (149+/-1.15) mg/g (shown in figure 1).
Based on example 4, the influence of cellulase consumption, enzymolysis time, ultrasonic power and time on the extraction rate of the wild rice stem bracts is studied by a single-factor experimental method, and is shown in figures 3-6 respectively.
The bract residues extracted by the four methods in examples 1-4 are dried to constant weight at 60 ℃, a proper amount of residues are taken out by tweezers on dissociated mica sheets, and observation under a scanning electron microscope after metal spraying and film plating shows that when TFZB is extracted by adopting an alcohol extraction method, the bract cell skeleton is relatively intact, and the damage of ethanol to the skeleton fiber structure is small (figure 2 a); after the enzymolysis and ultrasonic technology is introduced, the cell skeleton of the wild rice stem bract is destroyed, some fiber structures are broken, and pore diameters with different sizes appear on the cell skeleton (figures 2b and 2 c); when the enzymolysis and ultrasonic technology are combined on the basis of alcohol extraction, the bract cytoskeleton is seriously damaged, uniform larger pore diameters appear on the skeleton, so that the contact area of a solid phase and a liquid phase is increased, the penetrating power of a solvent is also increased, and the components such as flavone in cells are thoroughly dissolved out, so that the dissolution amount is increased. Preferably, the cellulase is used for assisting the alcohol extraction method in cooperation with ultrasonic waves. (FIG. 2 d)
Example 5 extraction Process of Total flavonoids from Zizania latifolia bracts (response surface optimized enzyme synergistic ultrasound)
Based on example 4, experiments were designed using the Box-Behnken method in Design-Expert V8.0.6.1 software. Selecting cellulase dosage (X) 1 ) Enzymolysis time (X) 2 ) Ultrasonic power (X) 3 ) And ultrasound time (X 4 ) The 4 variables are independent variables, the extraction rate of the wild rice stem bract flavone is taken as a response value, the low, medium and high test levels of each independent variable are respectively-1, 0 and 1, the coding and the results are shown in table 1, and the analysis of variance results are shown in table 2. And (5) analyzing by Design-Expert V8.0.6.1 software to obtain a quadratic polynomial regression equation of the extraction rate of the wild rice shoot bract flavone on the coding independent variable.
Y＝1.92+0.027X 1 -0.037X 2 +0.014X 3 -0.10X 4 +7.5×10 -3 X 1 X 2 -2.5×10 -3 X 1 X 3 +0.035X 1 X 4 -0.055X 2 X 3 -0.085X 2 X 4 -0.040X 3 X 4 -0.28X 1 2 -0.41X 2 2 -0.36X 3 2 -0.18X 4 2
As can be seen from the contour diagrams of Table 2 and FIG. 7, P of the model<0.0001, the linear relationship between model variable and 4 independent variables was very significant, i.e. the method was reliable. The mismatch term p= 0.2046>0.05, is not obvious, shows that the fitting condition of the equation is good, and can be used for predicting and analyzing the extraction rate of the wild rice stem bract flavone. R is R 2 = 0.9755, which indicates that only 2.45% of the tests cannot be determined by the power of the equation, and that no other significant factors have been affected in the test, and that conditions are appropriate. For model y=f (X 1 ,X 2 ,X 3 ,X 4 ) Performing analysis of variance, one term X 4 Quadratic term X 1 2 、X 2 2 、X 3 2 、X 4 2 Has extremely obvious influence on test response value, and one-time item X 2 And interaction item X 2 X 4 (fig. 7 d) has a significant effect on the response value, and the rest items are not significant, namely the ultrasonic time and the enzymolysis time and the interaction of the ultrasonic time and the enzymolysis time have a large effect on the extraction rate of the wild rice shoot bracts.
TABLE 1 test combinations and results of Box-Behnken method design
TABLE 2 response surface model analysis of variance
The optimal technological conditions for extracting the wild rice stem bract flavone are 0.703% of cellulase addition amount, 59.820min of enzymolysis time, 703.700W of ultrasonic power and 47.139min of ultrasonic time, the optimal extraction conditions are modified to be 0.7% of cellulase addition amount, 60min of enzymolysis time, 704W of ultrasonic power and 47min of ultrasonic time in consideration of practical operation feasibility, 3 repeated verification experiments are carried out under the conditions, and the average value of flavone extraction rates is (1.922 +/-0.024)%, and the total flavone content in the crude extract is (150+/-1.25) mg/g (figure 1).
EXAMPLE 6 antibacterial Properties of Total flavonoids of Zizania latifolia bracts
2.0g of wild rice stem bract flavone powder extracted under the optimal extraction condition after correction in example 5 is weighed, dissolved with a small amount of 60V% ethanol, and distilled water is added to make the final concentration of the wild rice stem bract flavone powder 20mg/mL. Adding 100ul of sterile beef extract peptone liquid culture medium into 2-10 holes of sterile 96-well plate, respectively adding 100ul of prepared 20mg/mL of wild rice stem bract total flavone solution into hole 1 and hole 2, mixing hole 2, sucking 100ul of solution into hole 3, mixing hole 3, sucking 100ul of solution into hole 4, diluting 2 times sequentially until adding into hole 10, sucking 100ul of solution after mixing, discarding 100ul of solution, adding 100ul of solution (Escherichia coli and Staphylococcus aureus in logarithmic phase) into 1-10 holes, thus TFZB final concentration in 1-10 holes is 10000, 5000, 2500 … … 39.0625, 19.53125ug/mL sequentially, repeating each treatment for 3 times, and immediately placing into enzyme-labeling instrument to determine OD of each hole 600 Values and recorded data, and then the 96-well plate is placed in a constant temperature and humidity incubator at 37 ℃ for culture24h, the OD of each well of the cell plate was again determined 600 Values were recorded and OD before and after incubation was calculated for each well 600 Difference, i.e. DeltaOD 600 OD is not less than 0% before and after culturing 600 The minimum concentration of TFZB solution less than or equal to 0.05 can be judged as the Minimum Inhibitory Concentration (MIC).
Gram-negative bacteria such as Escherichia coli (ATCC 25922) and gram-positive bacteria such as Staphylococcus aureus (ATCC 25923) are used as test strains, and ΔOD of wild rice stem bract flavone at different mass concentrations 600 See table 3 below, respectively.
TABLE 3 ΔOD of E.coli and Staphylococcus aureus suspensions at different mass concentrations of wild rice shoot bract flavone 600
As can be seen from the data in Table 3, when the mass concentration of the wild rice stem bract flavone is 2.5mg/mL, the ΔOD of the E.coli bacterial suspension is 600 0.048 of<0.05, delta OD of Staphylococcus aureus when the mass concentration of wild rice shoot bract flavone is 1.25mg/mL 600 Is 0.040<0.05, the MIC of wild rice shoot bract flavone for Escherichia coli is 2.5mg/mL, and the MIC for Staphylococcus aureus is 1.25mg/mL.
Example 7 preparation of chilled fresh pork preservative
Weighing Zizania latifolia bract total flavone powder, dissolving with a small amount of 60V% ethanol water solution, and preparing into 0.5mg/mL and 1mg/mL with distilled water to obtain two kinds of Zizania latifolia bract total flavone spray solutions with different concentrations.
Peeling fresh pork foreleg meat, uniformly dividing the pork foreleg meat into small blocks with the weight of 35 g+/-5 g, adding 240 samples, spraying different treatment liquids (sterile water, sodium benzoate solution with the concentration of 5.0mg/mL, cane shoot bract total flavone solution with the concentration of 0.5mg/mL and cane shoot bract total flavone solution with the concentration of 1.0 mg/mL) on the pork samples,
each treatment liquid was sprayed with 12 samples (approximately 1mL of liquid per 100 square cm), 4 storage times, 3 replicates. Sealing the processed pork with food-grade plastic preservative film (GB/T10457-2021), storing in a refrigerator at 4+/-0.5 ℃, sampling at 0, 3, 6 and 9 days of storage, and measuring sensory score, pH value, volatile basic state nitrogen (TVB-N) content, high-iron myoglobin percentage (MetMb%) content and total colony count, wherein the results show that the fresh-keeping effect is ordered: 1.0mg/mL of cane shoot bract flavone solution >5.0mg/mL of sodium benzoate solution >0.5mg/mL of cane shoot bract flavone solution > sterile water.
The foregoing examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the foregoing examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the present invention should be made therein and are intended to be equivalent substitutes within the scope of the present invention.
Claims (3)
1. A method for extracting total flavonoids from wild rice stem bracts comprises the following steps:
(1) Cleaning the collected fresh wild rice stem bracts, drying to constant weight, crushing, sieving, soaking a certain amount of wild rice stem bract powder in a buffer solution of disodium hydrogen phosphate-citric acid water, and then adding cellulase for enzymolysis;
(2) After enzyme is added in the step (1), the enzymolysis is carried out for 60min at 37 ℃;
(3) After enzymolysis, inactivating enzyme at high temperature in water bath, adding a certain amount of absolute ethyl alcohol and deionized water, and performing ultrasonic-assisted extraction;
(4) Ultrasonically extracting the sample in the step (3) for 47 min;
the mesh number of the crushed and sieved powder in the step (1) is 40 meshes; the pH of the buffer solution of disodium hydrogen phosphate-citric acid water is=5, and the solid-to-liquid ratio is 1g/40mL; the addition amount of the cellulase is 0.70 percent (mass-volume ratio);
and (3) after enzymolysis, inactivating enzyme in a water bath at 90 ℃ for 10min, adding a certain amount of absolute ethyl alcohol and deionized water to adjust the final concentration of the ethanol in the system to 60V%, and finally extracting at 55 ℃ under the condition that the ultrasonic power is 704W.
2. The application of the wild rice stem bract total flavone is used for preparing medicines for inhibiting escherichia coli and staphylococcus aureus; the cane shoot bract total flavone is prepared by the method of claim 1;
coli @E. coli ATCC 25922) and Staphylococcus aureusS. aureusATCC 25923) as a test strain, and the pair thereof was determinedE. coliHas a MIC of 2.5mg/mL forS. aureusHas a MIC of 1.25mg/mL.
3. The application of the wild rice stem bract total flavone is used for preparing a cold fresh pork preservative; the cane shoot bract total flavone is prepared by the method of claim 1; the fresh pork preservative is 1.0mg/mL of wild rice stem bract flavone solution.
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