CN112998181A - High-activity sweet sorghum polyphenol and preparation method and application thereof - Google Patents
High-activity sweet sorghum polyphenol and preparation method and application thereof Download PDFInfo
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- CN112998181A CN112998181A CN202110289996.2A CN202110289996A CN112998181A CN 112998181 A CN112998181 A CN 112998181A CN 202110289996 A CN202110289996 A CN 202110289996A CN 112998181 A CN112998181 A CN 112998181A
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- polyphenol
- sweet sorghum
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- 235000013824 polyphenols Nutrition 0.000 title claims abstract description 134
- 150000008442 polyphenolic compounds Chemical class 0.000 title claims abstract description 129
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3481—Organic compounds containing oxygen
- A23L3/349—Organic compounds containing oxygen with singly-bound oxygen
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses high-activity sweet sorghum polyphenol and a preparation method and application thereof. The preparation method comprises the following steps: 1) cleaning sweet sorghum stalks, squeezing to obtain juice, mixing the juice and the residues, adding an acidic ethanol solution, mixing, and leaching to obtain a polyphenol crude extract; 2) mixing the polyphenol coarse extract with AlCl3+ZnCl2Mixing the composite precipitant, and precipitating to obtain complex of polyphenol and precipitant; 3) washing the complex with acidic ethanol, centrifuging the obtained washing solution, removing supernatant, mixing the washed precipitate with HCl solution, extracting, and collecting the extract of the organic layer; 4) concentrating the extract, standing at room temperature, drying, and dissolving in water to obtain purified concentrated solution of polyphenol; 5) and (4) freeze-drying the polyphenol purified concentrated solution to obtain the polyphenol of sweet sorghum stalks. The invention adopts the composite ion precipitator to purify the polyphenol of the sweet sorghum stalks, so that the polyphenol has stronger antioxidant activity and can be applied to the research field of anti-food-borne microorganisms.
Description
Technical Field
The invention relates to high-activity sweet sorghum polyphenol and a preparation method and application thereof, belonging to the field of food additive research.
Background
At present, many of the antioxidants and preservatives widely used in food products are artificially synthesized, such as Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), and tert-butylhydroquinone (TBHQ). The synthetic antioxidants are not used as much or as little as possible because they are easily toxic, inflammatory and even tumor-inducing and harmful to the human body. Plant polyphenols from natural sources have obvious antioxidant effect, are often used as antioxidants, bacteriostats, preservatives and the like, and are widely applied to various fields of foods, medicines, nutrition and health care and the like. In recent years, a great deal of research is carried out on various plant polyphenol substances at home and abroad, however, the market of natural plant antioxidants is far from being saturated, and a large promotion space still exists, but the industrial extraction of plant polyphenols always faces the problems of low extraction efficiency and poor antioxidant activity, and further industrial application of the plant polyphenols is influenced.
Crops are good sources of plant active substances, such as tannin, anthocyanin, phenolic acid, phytosterol and the like, and are potential sources of antioxidants, phenols, cholesterol-lowering and other nutrient substances. Sweet sorghum belongs to the genus C4The energy-source crops have the characteristics of salt and alkali resistance, drought resistance, high sugar concentration, high biomass accumulation and the like, can be planted in marginal lands such as saline-alkali soil, sandy beach and the like, and mainly comprise substances such as sucrose, glucose, fructose, cellulose, hemicellulose, lignin, protein, starch, pectin and the like. At present, the utilization of sweet sorghum stalks is mainly focused on the development of some bulk products, such as fermentation for producing biochemicals, preparation of high fructose corn syrup, production of livestock and poultry feed and compost and the like, however, the research reports focus on the utilization of high sugar components and cellulose components in sweet sorghum, but the report of comprehensive utilization of abundant polyphenols in the sweet sorghum stalks is not provided, so that the efficient extraction of the polyphenols in the sweet sorghum stalks is realized, the comprehensive utilization rate of the sweet sorghum is remarkably improved, and the problem that the industrial chain of the sweet sorghum is urgently to be solved is extended.
Currently, polyphenols from different plant sources have different extraction methods, such as hexane extraction, methanol extraction, and water extraction. In order to further improve the extraction rate of polyphenol, some physical auxiliary extraction methods are also widely applied to the extraction process of plant polyphenol, such as high-pressure auxiliary extraction, microwave auxiliary extraction, ultrasonic auxiliary extraction, enzyme auxiliary extraction, supercritical carbon dioxide extraction and the like. For high-sugar crops, ultrasonic-assisted acid ethanol extraction is the main way to obtain polyphenol. Considering the high sugar concentration of sweet sorghum and the abundance of other components (such as pigments, proteins, fats, and waxes), these components may be incorporated into the sweet sorghum polyphenols during extraction, thereby affecting the quality of the extracted polyphenols. Therefore, the sweet sorghum polyphenol extracted by the ultrasonic-assisted acid ethanol needs to be further purified by combining other technologies.
Disclosure of Invention
The invention aims to provide high-activity sweet sorghum polyphenol and a preparation method and application thereof. The invention takes sweet sorghum stalks as raw materials to extract polyphenol compounds, and the prepared high-activity sweet sorghum polyphenol is applied to the research of anti-food-borne microorganisms.
The preparation method of the high-activity sweet sorghum polyphenol provided by the invention comprises the following steps:
1) cleaning sweet sorghum stalks, squeezing to obtain juice, mixing the juice and the residues, adding an acidic ethanol solution, mixing, adjusting the pH of a system, leaching, and performing suction filtration after leaching to obtain a polyphenol crude extract;
2) mixing the polyphenol coarse extract with AlCl3+ZnCl2Mixing the composite precipitant, adjusting pH, standing for precipitation, and separating to obtain complex of polyphenol and precipitant;
3) washing the complex by using the acidic ethanol, centrifuging the obtained washing liquid, removing supernatant, mixing the washed precipitate with HCl solution, extracting by using ethyl acetate, and collecting the extract liquor of an organic layer;
4) concentrating the extract, standing at room temperature for drying, and then adding water for dissolving to obtain a purified polyphenol concentrated solution;
5) and (3) freeze-drying the purified concentrated solution of the polyphenol to obtain the powdery polyphenol of the sweet sorghum stalks.
In the method, the acidic ethanol can be prepared by adding 1.6mol/L HCl and absolute ethanol into deionized water, wherein the volume percentage of the 1.6mol/L HCl can be 2%, and the volume percentage of the absolute ethanol can be 60%;
the mass volume ratio of the juice and residue mixture to the acidic ethanol is 1g:5mL, namely the solid-to-liquid ratio;
in the step 1), a hydrochloric acid buffer solution is adopted to adjust the pH value of the system to 5.0-6.0;
the leaching conditions in step 1) are as follows: heating in water bath at 30 deg.C; the ultrasonic treatment is carried out under ultrasound, and the power of the ultrasonic wave can be 600W; the leaching time can be 4 hours;
the leaching is carried out in a high shear emulsifier.
In the invention, the ultrasonic instrument adopted by the ultrasonic is Shaanxi Kaideli KDL-60022R;
the high-shear emulsifying machine of the emulsifying machine is an Anhui poly-chromium machine SG-101, a long hole working head is used, and the rotating speed is 500 rpm.
In the method, the step 1) further comprises the following steps, and the filter residue is repeatedly subjected to the following processes for 3 times: adding an acidic ethanol solution into the filter residue, mixing, adjusting the pH of the system, leaching, and performing suction filtration after leaching to obtain filter residue and polyphenol crude extraction diluent; and then combining the polyphenol crude extract diluent with the polyphenol crude extract for the next operation.
In the above method, the AlCl is3+ZnCl2The mass-volume ratio of the composite precipitator to the coarse polyphenol extracting solution from the sweet sorghum stalks can be 1: 20;
the AlCl3+ZnCl2AlCl in composite precipitator3And ZnCl2The mass ratio of (b) may be 1: 2.
In the method, in the step 2), the pH is adjusted to 6.0; with 1mol/L NaHCO3Adjusting the pH value of the solution;
the standing time can be 15-30 min;
the centrifugation rate can be 6000r/min, the temperature can be 20 ℃, and the centrifugation time can be 15 min.
In the invention, the adopted centrifugal machine can be a Hunan Kaida DL7M-12L centrifugal machine.
In the above method, the mass-to-volume ratio of the complex of polyphenol and precipitant in step 3) to the acidic ethanol may be 1g:5 mL;
the speed of the centrifugation can be 6000r/min, the temperature can be 20 ℃, and the centrifuge used for the centrifugation can be a Hunan Kaida DL7M-12L centrifuge;
the mass-to-volume ratio of the washed precipitate to the HCl solution can be 1g:5 mL;
the concentration of the HCl solution can be 1 mol/L;
the number of extractions may be 3.
In the method, the concentration in the step 4) adopts reduced pressure distillation; the temperature of the reduced pressure distillation can be 40 ℃;
the drying temperature can be 40 ℃ and the drying time can be 6 h.
In the invention, the rotary evaporator adopted by reduced pressure distillation is specifically Qingdao poly-wound JC-ZF-RE5000, and the rotating speed is set to be 50 r/min;
the electrothermal blowing drying oven adopted for drying is specifically Shanghai Ganyi DHG-9203A, and the set temperature can be 40 ℃.
In the above method, in step 5), the freeze-drying process is as follows: pre-freezing at-80 deg.C for 18h, and freeze-drying at-60 deg.C under vacuum degree of 2Pa for 36 h.
In the invention, the vacuum drier used for freeze drying is specifically Shanghai Yu bright YY-2000 FDA.
The invention also provides the polyphenol prepared by the method.
In the invention, the sweet sorghum stem polyphenol is analyzed according to the first 20% substances detected by a non-target metabolome, and phenolic acids in the sweet sorghum stem polyphenol comprise gallic acid, gentisic acid, p-hydroxybenzoic acid, ferulic acid, caffeic acid, chlorogenic acid and o-coumaric acid.
The sweet sorghum stem polyphenol has high antioxidant activity.
The sweet sorghum stem polyphenol can be applied to the research field of anti-food-borne microorganisms. Specifically, the cell membrane of the food-borne microorganism is damaged when the food-borne microorganism (escherichia coli CMCC44102 and staphylococcus aureus CMCC26003) is killed obviously, so that the protein and nucleic acid are leaked.
The invention has the following advantages:
1) the polyphenol compounds are extracted from the sweet sorghum stalks, the by-products in the sweet sorghum stalks are changed into valuable, the comprehensive utilization rate of the sweet sorghum is improved, the industrial chain of the sweet sorghum is extended, and the added value of the sweet sorghum product is obviously increased.
2) The acidic ethanol can gently and effectively extract polyphenol compounds in sweet sorghum stalks under the action of ultrasonic waves and an emulsifying instrument, and then composite metal ions which are cheap and easy to obtain are used as a precipitating agent, so that polyphenol is complexed with the metal ions under acidic or neutral conditions, and the metal ions can remove a large amount of impurities such as polysaccharide and the like through precipitation, so that the degradation effect of the impurities on the polyphenol substances is greatly weakened, the problems of low extraction efficiency, poor antioxidant activity and the like in the extraction process are solved, the yield is remarkably increased, and the polyphenol compounds are ensured to have strong antioxidant activity.
3) The method has the advantages of simple equipment, convenient operation, rapid separation and no environmental pollution, and the obtained polyphenol compound maintains the structure and biological activity of the principle.
4) The sweet sorghum stem polyphenol produced by the invention has the characteristics of high safety, high yield, good product quality and low cost, has high antioxidant activity, is applied to the research field of food-borne microorganisms, and can obviously kill food-borne microorganisms (escherichia coli CMCC44102 and staphylococcus aureus CMCC26003) to cause the damage of cell membranes of the food-borne microorganisms, thereby causing the leakage of protein and nucleic acid.
Drawings
Fig. 1 is a spectrogram of sweet sorghum stalk polyphenol obtained in example 1 of the present invention detected according to non-target metabolome, in which fig. 1(a) is a negative ion detection mode, and fig. 1(b) is a positive ion detection mode.
FIG. 2 is a graph showing the results of measuring the total antioxidant capacity of sweet sorghum polyphenols obtained in example 1 and comparative examples 1-2 of the present invention, wherein the bar graph of acidic ethanol in combination with complex ion extraction is the result of example 1, the bar graph of acidic ethanol in combination with single ion extraction is the result of comparative example 1, and the bar graph of acidic ethanol extraction is the result of comparative example 2.
Fig. 3 is a graph showing the lethal effect of sorghum saccharatum polyphenols obtained in example 1 of the present invention on food-borne microorganisms, wherein (a) in fig. 3 is a graph showing the survival rate of food-borne microorganisms at different concentrations of sorghum saccharatum polyphenols, and (B) is a count showing the survival rate of food-borne microorganisms at different concentrations of sorghum saccharatum polyphenols.
FIG. 4 shows the leakage of sweet sorghum polyphenol from example 1 of the present invention to food-borne microbial proteins and nucleic acids, and in FIG. 4, (A) shows the results of protein leakage and (B) shows the results of nucleic acid leakage.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Examples 1,
1. Collecting freshly harvested sweet sorghum stems from a sweet sorghum planting base, juicing and crushing, taking 500g of the mixture of the sweet sorghum stems, adding 2.5L of 60% acidic ethanol (containing 2% by volume of 1.6mol/L HCl) solution, and adjusting the pH value to 5.0-6.0 by using a buffer solution. Then placing the mixture in a water bath at 30 ℃ for heating, and stirring and leaching for 4 hours under 600W ultrasonic waves. After the extraction, the extraction is carried out while the solution is hot, and filter residues are washed for 2 times by acidic ethanol.
2. Taking 1L of diluted solution of the polyphenol crude extract, adding 50g AlCl at room temperature3+ZnCl2Composite precipitator (mass ratio AlCl)3:ZnCl21:2) (i.e. precipitant: mass-to-volume ratio of sweet sorghum stalks 1:20) precipitating polyphenol with 1mol/L NaHCO3Adjusting pH to 6.0, standing for 30min, precipitating completely, centrifuging at 6000r/min for 15min, and discarding supernatant to obtain complex of polyphenol and precipitant.
3. Taking 100g of complex of polyphenol and precipitator, adding 500mL of 60% acidic ethanol for washing, fully mixing washing solutions, immediately centrifuging for 15min under the condition of 6000r/min, discarding supernatant, and repeatedly washing for 3 times; to the washed precipitate was added 500mL of 1mol/L HCl solution to completely dissolve it. Then, ethyl acetate of the same volume was added to the solution to extract the mixture for 3 times.
4. Taking 500mL of extract, carrying out rotary evaporation at 40 ℃ to obtain a polyphenol concentrated solution, and recovering ethyl acetate; pouring the polyphenol concentrated solution into an aluminum box, standing at room temperature for drying, and adding a small amount of deionized water for redissolving to obtain the polyphenol purified concentrated solution.
5. And (3) placing the obtained purified concentrated solution of the polyphenol of the sweet sorghum stalks in a glass vessel for freeze drying, pre-freezing for 18h at the temperature of minus 80 ℃, and freeze drying for 36h at the temperature of minus 60 ℃ to obtain the polyphenol of the sweet sorghum stalks.
As shown in fig. 1, the polyphenol of sweet sorghum stem is analyzed according to the first 20% substances detected by non-target metabolome, and the phenolic acids in the polyphenol of sweet sorghum stem specifically include gallic acid, gentisic acid, p-hydroxybenzoic acid, ferulic acid, caffeic acid, chlorogenic acid and o-coumaric acid.
Comparative examples 1,
1. Collecting freshly harvested sweet sorghum stems from a sweet sorghum planting base, juicing and crushing, taking 1000g of the sweet sorghum stem mixture, adding 5L of 60% acidic ethanol solution, and adjusting the pH value to 5.0-6.0 by using a buffer solution. Then placing the mixture in a water bath at 30 ℃ for heating, and stirring and leaching for 4 hours under 600W ultrasonic waves. After the leaching is finished, the hot solution is filtered, and filter residues are washed for 2 times in the same way.
2. Taking 2.5L of diluted solution of polyphenol crude extract, adding 125g AlCl at room temperature3Precipitating polyphenol with precipitant (mass volume ratio of sweet sorghum stalk is 1:20), and precipitating with 1mol/L NaHCO3Adjusting pH to 6.0, standing for 30min, precipitating completely, centrifuging at 6000r/min for 15min, and discarding supernatant to obtain complex of polyphenol and precipitant.
3. Taking 100g of complex of polyphenol and precipitator, adding 500mL of 60% acidic ethanol for washing, fully mixing washing solutions, immediately centrifuging for 15min under the condition of 6000r/min, discarding supernatant, and repeatedly washing for 3 times; to the washed precipitate was added 500mL of 1mol/L HCl solution to completely dissolve it. Then, ethyl acetate of the same volume was added to the solution to extract the mixture for 3 times.
4. Taking 1L of extract, performing rotary evaporation at 40 ℃ to obtain polyphenol concentrated solution, and recovering ethyl acetate; pouring the polyphenol concentrated solution into a glass dish, standing at room temperature for drying, and adding a small amount of deionized water for redissolving to obtain the polyphenol purified concentrated solution.
5. And placing the obtained purified concentrated solution of the polyphenol of the sweet sorghum stems in a glass vessel for freeze drying, pre-freezing for 18h at the temperature of minus 80 ℃, and freeze drying for 36h at the temperature of minus 60 ℃ to obtain the polyphenol purified product of the sweet sorghum stems.
Comparative examples 2,
1. Collecting freshly harvested sweet sorghum stems from a sweet sorghum planting base, juicing and crushing, taking 750g of the mixture of the sweet sorghum stems, adding 3.75L of 60% acidic ethanol solution, and adjusting the pH value to 5.0-6.0 by using a buffer solution. Then placing the mixture in a water bath at 30 ℃ for heating, and stirring and leaching for 4 hours under 600W ultrasonic waves. After the leaching is finished, the hot solution is filtered, and filter residues are washed for 2 times in the same way.
2. Collecting 1L of the crude extractive solution, performing rotary evaporation at 60 deg.C under reduced pressure to obtain polyphenol concentrated solution, and recovering ethanol.
3. And (3) placing the obtained sweet sorghum stem polyphenol concentrated solution in a glass vessel for freeze drying, pre-freezing for 18h at-80 ℃, and freeze drying for 36h at-60 ℃ to obtain the sweet sorghum stem polyphenol purified product.
The physiological and biochemical indexes of the polyphenol of the sweet sorghum stalks obtained in the above examples are measured as follows:
1) determination of polyphenol content
The polyphenol content is measured by referring to a method of a total polyphenol detection kit of plants of Beijing Soilebao Biotech limited. The results are expressed as gallic acid equivalent (mg GAE/g) contained in 1g of the extract.
TABLE 1 polyphenol content of sweet sorghum stalks in different examples
| Sweet sorghum stem purified product | Extract containing polyphenol mg GAE/g |
| Example 1 | 276.8±17.31 |
| Comparative example 1 | 225.5±7.84 |
| Comparative example 2 | 152.3±12.42 |
In the embodiment 1 of the invention, the purified polyphenol of the sweet sorghum stalks obtained by the composite ion precipitation method is 276.8mg of GAE/g extract, 51.3mg of GAE/g extract is added to the polyphenol of the sweet sorghum stalks obtained by the single ion precipitation method in the comparative example 1, and compared with the traditional method in the comparative example 2, the content of the polyphenol of the sweet sorghum stalks obtained by the method in the invention is 124.5mg of GAE/g extract, and the effect is obvious. At present, the prior art mostly adopts the steps of crushing and drying raw materials and then adding a solvent for extraction, so that the production cost is increased, and the polyphenol substances are damaged to a certain extent by high temperature, so that the polyphenol content of the obtained sweet sorghum stalks has no practical application value. The invention uses the fresh sweet sorghum stalks which are not dried and directly crushed and extracted, adopts ultrasonic wave to assist ethanol extraction to destroy the tissue structure of the sweet sorghum stalks, releases polyphenol substances as much as possible, then uses cheap and easily obtained composite metal ions as a precipitator to complex polyphenol with metal ions under acidic or neutral conditions, and the metal ions can remove a large amount of impurities such as polysaccharide and the like by precipitation, thereby greatly weakening the degradation effect of the impurities on the polyphenol substances. The polyphenol from sweet sorghum stems produced by the method has the characteristics of good safety, high yield, good product quality, low cost and the like; the method has the advantages of energy conservation, labor saving, time saving, no pollution to the environment and the like, is an application technology which can be advocated greatly, and can create higher economic benefit.
2) Determination of DPPH (dehydroepiandrosterone) free radical scavenging capacity of polyphenol of sorgo stems
The extracts in the embodiment 1 and the comparative examples 1-2 of the invention are respectively diluted into sample solutions of 50 mug/mL, each group of experiments are repeated for 3 times, and the detection is carried out according to the DPPH detection kit method of Beijing Sorbao Biotech Co.
As shown in fig. 2, the DPPH-radical clearance of the sweet sorghum stalk polyphenol of the present invention is significantly increased in a measurement-dependent manner, and at 50 μ g/mL, the DPPH-radical clearance of the sweet sorghum stalk polyphenol extracted by the ultrasonic ion precipitation composite method of the present invention reaches 82.08%, whereas the DPPH-radical clearance of the single ion precipitation method in comparative example 1 is 59.87%, and the DPPH-radical clearance of the traditional acidic ethanol extraction method in comparative example 2 is only 46.28%.
3) Determination of polyphenol hydroxyl radical scavenging capacity of sweet sorghum stems
The extracts of example 1 and comparative examples 1-2 were diluted to 50 μ g/mL sample solutions, and each test was repeated 3 times, and the test was performed according to the method of the kit for detecting scavenging ability of hydroxyl radicals of Beijing Sorbox Biotech, Inc.
As shown in figure 2, the hydroxyl radical clearance of the sweet sorghum stalk polyphenol is obviously increased in a metering-dependent mode, at 50 mug/mL, the clearance of the hydroxyl radical of the sweet sorghum stalk polyphenol extracted by the ultrasonic ion precipitation composite method reaches 61.46%, while the clearance of the hydroxyl radical of the sweet sorghum stalk polyphenol extracted by the ultrasonic ion precipitation composite method in comparative example 1 is 49.87%, and the clearance of the hydroxyl radical of the sweet sorghum stalk polyphenol extracted by the traditional acid leaching method in comparative example 2 is only 41.45%.
4) Determination of total antioxidant capacity of polyphenol of sweet sorghum stems
The extracts of example 1 and comparative examples 1-2 were diluted to 50. mu.g/mL of sample solutions. Each set of experiments was repeated 3 times. The specific method is to determine the total antioxidant capacity according to a determination kit of Suzhou Keming Biotechnology Co. And calculating the equivalent amount of mu mol of Trolox standard substance of the sample according to a standard curve formula to obtain the total antioxidant capacity of the sample, wherein the total antioxidant capacity can be calculated as mu mol of Trolox/mL.
As shown in figure 2, the total antioxidant capacity of the polyphenol from sweet sorghum stalks is obviously increased in a metering-dependent manner, when the total antioxidant capacity is 50 mu g/mL, the total antioxidant capacity of the polyphenol from the sweet sorghum stalks extracted by the ultrasonic ion precipitation composite method reaches 9.36 mu mol of Trolox/mL, while the phase of the single ion precipitation method in comparative example 1 is 7.51 mu mol of Trolox/mL, and the traditional acid ethanol leaching method in comparative example 2 is only 5.98 mu mol of Trolox/mL.
The high-activity sweet sorghum stem polyphenol obtained in the embodiment 1 is used in the research field of anti-food-borne microorganisms:
1) killing effect of sweet sorghum polyphenol on food-borne microorganisms
Inoculating 100 μ L of frozen bacteria liquid of Escherichia coli and Staphylococcus aureus into solid culture medium, coating, and culturing in 37 deg.C incubator for 16 hr. 2mL of liquid medium was aspirated and placed on a plate, and the cells were washed off with an inoculating loop, inoculated into the liquid medium, and cultured at 37 ℃ for 8 hours with a shaker at 200 rpm. Adjusting the concentration OD of the bacterial liquid600The value is equal to about 1. 100 mu L of the bacterial liquid is added into a sterilized 1.5mL centrifuge tube, and centrifuged for 15min at room temperature and 14000 r/min. The supernatant was aspirated, the cells were retained, and then 0, 5, 10, 20, 50g/L of the sweet sorghum polyphenol extract of example 1 of the present invention was added thereto. Vortex evenly, and stand for 10 min. Add 900. mu.L of sterile deionized water to the centrifuge tube and vortex well. According to the ratio 10-8～10-9After dilution, 100. mu.L of the suspension was spread evenly on a plate, and cultured in an incubator at 37 ℃ for 14 hours to count as shown in FIG. 3. As can be seen from figure 3, the sweet sorghum polyphenol can obviously kill food-borne microorganisms, and when the concentration of the sweet sorghum polyphenol is 20g/L, the survival rates of escherichia coli and staphylococcus aureus are respectively 26.58% and 17.36%.
2) Protein and nucleic acid leakage of food-borne microorganisms caused by sweet sorghum polyphenol
2.1 protein leakage
Inoculating 100 μ L of frozen bacteria liquid of Escherichia coli and Staphylococcus aureus into solid culture medium, coating, and culturing in 37 deg.C incubator for 16 hr. 2mL of liquid medium was aspirated and placed on a plate, and the cells were washed off with an inoculating loop, inoculated into the liquid medium, and cultured at 37 ℃ for 8 hours with a shaker at 200 rpm. Adjusting the concentration OD of the bacterial liquid600The value is equal to about 1. 100 mu L of the bacterial liquid is added into a sterilized 1.5mL centrifuge tube, and centrifuged for 15min at room temperature and 14000 r/min. Sucking supernatant, retaining thallus, and adding sweet sorghum polyphenol extract of 0, 5, 10, 20, 50 g/L. Vortex uniformly and stand stillAfter 10min, the mixture is centrifuged at 14000r/min for 15min at room temperature, and the supernatant is retained. 20 μ L of the supernatant was added to a 96-well plate, and 200 μ L of Coomassie brilliant blue G-250 was added and incubated at room temperature for 5min, and absorbance was measured at 595nm with a microplate reader. Protein content calculations were performed by substituting the BSA standard curve. y is 0.0021x +0.5431, R20.9936 (as shown in fig. 4 (a)). As can be seen from FIG. 4(A), the sweet sorghum polyphenol of the invention can significantly induce the protein leakage of food-borne microorganisms, and when the concentration of the sweet sorghum polyphenol is 20g/L, the protein leakage of Escherichia coli and Staphylococcus aureus is 15.87 and 18.52. mu.g/mL respectively.
2.2 nucleic acid leakage
Inoculating 100 μ L of frozen bacteria liquid of Escherichia coli and Staphylococcus aureus into solid culture medium, coating, and culturing in 37 deg.C incubator for 16 hr. 2mL of liquid medium was aspirated and placed on a plate, and the cells were washed off with an inoculating loop, inoculated into the liquid medium, and cultured at 37 ℃ for 8 hours with a shaker at 200 rpm. Adjusting the concentration OD of the bacterial liquid600The value is equal to about 1. 100 mu L of the bacterial liquid is added into a sterilized 1.5mL centrifuge tube, and centrifuged for 15min at room temperature and 14000 r/min. Sucking supernatant, retaining thallus, and adding sweet sorghum polyphenol extract of 0, 5, 10, 20, 50 g/L. Vortex evenly, after standing for 10min, room temperature, 14000r/min, centrifuge for 15min, and leave the supernatant. The supernatant was filtered using a 0.22 μm filter and diluted 100-fold. And (3) placing 200 mu L of liquid in an ultraviolet fluorescence 96-well plate, and measuring the absorbance at 260nm by using an enzyme-linked immunosorbent assay. The absorbance value is the amount of nucleic acid leakage (as shown in FIG. 4). As can be seen from FIG. 4(B), the sweet sorghum polyphenol of the present invention can significantly induce the leakage of nucleic acid from food-borne microorganisms, and when the concentration of the sweet sorghum polyphenol is 20g/L, the leakage of nucleic acid from Escherichia coli and Staphylococcus aureus reaches OD260＝0.25。
From the results, the high-activity sweet sorghum polyphenol extract obtained by the invention can obviously kill food-borne microorganisms in the research of resisting the food-borne microorganisms, so that the cell membranes of the food-borne microorganisms are damaged, and the leakage of protein and nucleic acid is caused.
Claims (10)
1. The preparation method of the high-activity sweet sorghum polyphenol comprises the following steps: 1) cleaning sweet sorghum stalks, squeezing to obtain juice, mixing the juice and the residues, adding an acidic ethanol solution, mixing, adjusting the pH of a system, leaching, and performing suction filtration after leaching to obtain a polyphenol crude extract;
2) mixing the polyphenol coarse extract with AlCl3+ZnCl2Mixing the composite precipitant, adjusting pH, standing for precipitation, and separating to obtain complex of polyphenol and precipitant;
3) washing the complex by using the acidic ethanol, centrifuging the obtained washing liquid, removing supernatant, mixing the washed precipitate with HCl solution, extracting by using ethyl acetate, and collecting the extract liquor of an organic layer;
4) concentrating the extract, standing at room temperature for drying, and then adding water for dissolving to obtain a purified polyphenol concentrated solution;
5) and (3) freeze-drying the purified concentrated solution of the polyphenol to obtain the powdery polyphenol of the sweet sorghum stalks.
2. The method of claim 1, wherein: the acidic ethanol is prepared by adding 1.6mol/L HCl and absolute ethanol into deionized water, wherein the volume percentage of the 1.6mol/L HCl is 2%, and the volume percentage of the absolute ethanol is 60%;
the mass-to-volume ratio of the juice and residue mixture to the acidic ethanol is 1g:5 mL;
in the step 1), a hydrochloric acid buffer solution is adopted to adjust the pH value of the system to 5.0-6.0;
in the step 1), the leaching conditions are as follows: heating in water bath at 30 deg.C; the ultrasonic treatment is carried out under the ultrasonic condition, and the ultrasonic power is 600W; the leaching time was 4 h.
3. The method according to claim 1 or 2, characterized in that: the step 1) also comprises the following steps, and the filter residue is repeatedly processed for 3 times by the following processes: adding an acidic ethanol solution into the filter residue, mixing, adjusting the pH of the system, leaching, and performing suction filtration after leaching to obtain filter residue and polyphenol crude extraction diluent; and then combining the polyphenol crude extract diluent with the polyphenol crude extract for the next operation.
4. The method according to any one of claims 1-3, wherein: the AlCl3+ZnCl2The mass-volume ratio of the composite precipitator to the coarse polyphenol extracting solution from the sweet sorghum stalks is 1: 20;
the AlCl3+ZnCl2AlCl in composite precipitator3And ZnCl2The mass ratio of (A) to (B) is 1: 2.
5. The method according to any one of claims 1-4, wherein: in the step 2), adjusting the pH value to 6.0; with 1mol/L NaHCO3Adjusting the pH value of the solution;
the standing time is 15-30 min;
the separation is carried out by centrifugation, wherein the centrifugation speed is 6000r/min, the temperature is 20 ℃, and the centrifugation time is 15 min.
6. The method according to any one of claims 1-5, wherein: the mass-volume ratio of the complex of the polyphenol and the precipitator to the acidic ethanol in the step 3) is 1g:5 mL;
the centrifugation speed is 6000r/min, and the temperature is 20 ℃;
the mass-to-volume ratio of the washed precipitate to the HCl solution is 1g:5 mL;
the concentration of the HCl solution is 1 mol/L;
the number of extractions was 3.
7. The method according to any one of claims 1-6, wherein: vacuum distillation is adopted for the concentration in the step 4); the temperature of the reduced pressure distillation is 40 ℃;
the drying temperature is 40 ℃, and the drying time is 6 h.
8. The method according to any one of claims 1-7, wherein: in step 5), the freeze-drying process is as follows: pre-freezing at-80 deg.C for 18h, and freeze-drying at-60 deg.C under vacuum degree of 2Pa for 36 h.
9. Sweet sorghum stalk polyphenol prepared by the method of any one of claims 1-8.
10. The use of the polyphenol derived from sweet sorghum stalks of claim 9 in the field of research on resistance to food-borne microorganisms.
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