阅读说明：本技术 一种睡莲茎细胞水的提取方法和得到的睡莲茎细胞水的应用 (Extraction method of water lily stem cell water and application of obtained water lily stem cell water ) 是由 张文环 蔡少纯 李佩晶 耿林 艾艳 艾勇 于 2020-06-03 设计创作，主要内容包括：本发明公开了一种睡莲茎细胞水的制备方法,采用低温真空提取技术与酶解技术相结合,不需要添加任何溶剂,在较低的温度下能得到纯天然的睡莲茎细胞水。通过本发明方法得到的睡莲茎细胞水品质高,澄清透明、含有50多种易挥发活性成分,具有清爽怡人的气味,具有抗氧化、保湿、抗炎的效果,可作为绿色天然的原料应用于食品、保健品、药品及化妆品等领域。(The invention discloses a preparation method of water lily stem cell water, which combines a low-temperature vacuum extraction technology with an enzymolysis technology, does not need to add any solvent, and can obtain pure natural water lily stem cell water at a lower temperature. The water lily stem cell water obtained by the method has high quality, is clear and transparent, contains more than 50 volatile active ingredients, has fresh and pleasant smell, has the effects of oxidation resistance, moisture retention and inflammation resistance, and can be used as a green and natural raw material to be applied to the fields of food, health care products, medicines, cosmetics and the like.)

1. A preparation method of water lily stem cell water is characterized by comprising the following steps: extracting the water lily stems primarily at 30-60 ℃ and under the pressure of-60 kPa to-101 kPa without adding a solvent, forming vapor in water of the water lily stem cells, condensing and collecting liquid, and extracting for 1-2.5 hours to obtain primary extracted cell water and primary extracted water lily stem residues; adding 0.2-0.4% of cellulase and 0-0.1% of pectinase into the primary water lily stem water, adding the primary water lily stem water into the primary water lily stem residue, extracting again at 35-55 ℃ under the pressure of-70 kPa to-101 kPa for 3-6 hours, and collecting the water lily stem water.

2. The method for preparing water lily stem cell water according to claim 1, wherein the temperature in the initial extraction stage is 40-50 ℃ and the pressure is-75 kPa-101 kPa.

3. The method for preparing water lily stem cell water according to claim 1, wherein the re-extraction conditions are 40-50 ℃ and-80 kPa-101 kPa.

4. The method for preparing water lily stem cell water according to claim 1, wherein 0.25-0.35% of cellulase and 0-0.06% of pectinase based on the total mass of the initial water lily stem is added to the initial water lily stem cell water.

5. The method for preparing water lily stem cell water according to claim 1, wherein stirring is performed during the extraction process, and the stirring speed is 1-150 rpm.

6. The method for preparing water lily stem cell water according to claim 1, wherein the water lily stem cell water is condensed during the collection process, and the temperature is-10 to 8 ℃.

7. The method for preparing water lily stem cell water according to claim 1, wherein the water lily stem is cut into a sheet with a thickness of 1-10 mm.

8. The use of the water lily stem cell water of any one of claims 1 to 7, which is used for skin care products, food products, health products and the like.

Technical Field

The invention relates to the technical field of agricultural product treatment, in particular to a preparation method of water lily stem cell water and application of the water lily stem cell water.

Background

Water lily, perennial aquatic herbs, thick rhizomes, distributed from northeast to Yunnan, west to Xinjiang, korean, japan, india, russia, north america, and the like. Grow in still water bodies such as ponds, lakes and the like. The water body in many parks is cultivated as an ornamental plant, and the rhizome is eaten or brewed with wine and is used as a medicine, so that the infantile chronic infantile convulsion can be treated; the whole grass can be used as green manure. Experimental research shows that the water lily has the function of adsorbing heavy metals. The water lily leachate has a certain inhibition effect on the growth of microcystis aeruginosa and shows an obvious phenomenon of low promotion and high inhibition. According to the analysis of the nutritional components of the water lily, the result shows that the water lily is rich in 17 amino acids, and the water lily protein belongs to high-quality protein. The analysis result also shows that the water lily contains rich VC, flavonoid glycoside and trace element zinc, and the combination of the two has strong lead-removing function. Animal acute toxicity experiments, micronucleus experiments and sperm aberration experiments show that the water lily is a safe and reliable substance without any toxic and side effects. The water lily pollen is rich in nutrition, has the characteristics of completeness, balance, concentration and the like, and is a natural nutrient source with development and utilization prospects.

Chinese patent application 2019102522047 discloses an extraction process of a water lily rhizome extract. The betulinic acid is extracted mainly by a solvent method. However, this method uses ethanol as an extractant, and ethanol cannot be completely removed, which makes it unusable for some people allergic to ethanol.

Enzymatic methods are also commonly used in the extraction of plant cell sap. The Chinese patent application CN109730948A discloses a method for preparing peony flower cell water by combining an ultrasonic low-temperature rotary steaming method and an enzyme method, which comprises the following steps: firstly, squeezing to obtain juice and residue 1, then carrying out rotary evaporation on the residue 1 to obtain cell water 1 and residue 2, and finally carrying out enzymolysis on the residue 2 and then carrying out rotary evaporation to obtain cell water 3. And mixing the juice and the cell water 1/2 to obtain the peony cell water with high yield. The method has high extraction efficiency, but has the following defects: (1) the squeezing method is adopted and then mixed with the liquid obtained by the vacuum extraction method, so that polysaccharide, pigment and pungent smell are brought in, and the problems of corrosion prevention and decoloration are caused; (2) the later stage of the process is matched with other plants for distillation, so that the problem of corrosion resistance is solved, but the original water components and smell of the peony cells are easily changed! The quality of the product cannot be controlled in the later period.

Disclosure of Invention

The invention aims to provide an extraction method of water lily stems, which can obtain pure natural water lily stem cell water, has more than 50 volatile active ingredients, has the effects of oxidation resistance, moisture retention and inflammation resistance, and can be used as a green and natural raw material to be applied to the fields of food, health care products, medicines, cosmetics and the like.

The invention is realized by the following technical scheme:

a preparation method of water lily stem cell water comprises the following steps: adding no solvent, primarily extracting the water lily stems at 30-60 ℃ and under the pressure of-60 kPa to-101 kPa, forming water lily stem cells into steam, condensing and collecting liquid, and extracting for 1-2.5 hours to obtain primary extracted cell water and primary extracted water lily stem residues; adding 0.2-0.4% of cellulase and 0-0.1% of pectinase based on the total mass of the initial water lily stem into the initial water lily stem, adding the initial water lily stem into the initial water lily stem residue, extracting again at 35-55 ℃ under the pressure of-70 kPa to-101 kPa, finishing the extraction for 3-6 hours, and collecting the water lily stem cell water.

The primary extraction time is one of key parameters, if the time is too short, the extracted water of the water lily stem cells is too little, and the water of the cells after the enzyme is added is difficult to wet the surface of the water lily stem, so that the enzymolysis can not be normally carried out. If the primary extraction time is too long, cell water flows out too much, so that the subsequent extraction efficiency is reduced, and the risk of excessive enzymolysis caused by enzymolysis time is increased. The method has the following advantages that a certain amount of enzyme is added into the primary cell extraction water, and the water lily stem residue is put into the container again for extraction. Firstly, the surface tension of primary extracted cell water is low, and the permeability is good; secondly, the pH of the primary cell extracting water is 3-7, and the pH does not need to be additionally adjusted, so that the enzyme activity is favorably improved; thirdly, the enzymolysis can accelerate the wall breaking; fourth, low temperature vacuum technique. Through the synergy of the four effects, the enzymolysis speed can be controlled at a lower temperature (35-55 ℃) to accelerate the outflow speed of cell sap. About the first 1 hour in the re-extraction step, the primary cell water poured back into the container can be steamed off, the enzymolysis speed is accelerated, the enzymolysis time is shortened (at the moment, the amount of the primary liquid is very important, the enzymolysis time is prolonged, and the enzymolysis time is shortened), and the problems that the traditional enzymolysis method needs to add a large amount of water to dilute the cell liquid and the pungent smell caused by excessive enzymolysis is avoided.

Regarding the permeability of the initial extract, it was found through experiments that about 70% more flavonoids and polysaccharides were extracted when the water lily stem residue was extracted using water lily stem cell sap as a solvent in the solvent method, compared to using pure water as a solvent.

Preferably, the temperature of the initial extraction stage is 40-50 ℃, and the pressure is-75 kPa-101 kPa. The preferable initial extraction conditions can achieve the balance of more extracted initial extraction cell fluid and active ingredient retention.

Preferably, the re-extraction conditions are 40 ℃ to 50 ℃ and the pressure is-80 kPa to-101 kPa. Through the optimized re-extraction step, the existence time of the primary extracted cell sap can be ensured to be within the range of 45 minutes to 1.5 hours, and the proper enzymolysis time is ensured.

Preferably, 0.25-0.35% of cellulase and 0-0.06% of pectinase based on the total mass of the initial water lily stem are added into the initial cell extracting water. The addition of enzyme is related to the enzymolysis speed, too much enzymolysis can result in excessive enzymolysis, too low enzymolysis can also result in insufficient enzymolysis, and the extraction rate of cell water is reduced.

Stirring is carried out during the extraction process, and the stirring speed is 1-150 revolutions per minute. Through stirring, the heat transfer effect can be ensured, the probability of exposing the water lily stems under the vacuum condition can be increased, and the extraction time is shortened.

Condensing in the collection process, wherein the temperature is-10-8 ℃.

The water lily stems are cut into sheets with the thickness of 1-10 mm. A reciprocating cutter may be used for cutting.

The water lily stem cell water has the effects of oxidation resistance, moisture retention and inflammation resistance, and is used for skin care products, foods, health care products and the like.

Compared with the prior art, the invention has the following beneficial effects:

the water lily stem cell water obtained by a single low-temperature-vacuum extraction technology has less volatile active ingredients, relatively light smell and relatively long extraction time. The invention does not add any solvent, and can accelerate the extraction speed of the cell sap by utilizing the low-temperature vacuum extraction technology and the enzymolysis technology and simultaneously can inhibit the defect that the excessive enzymolysis of the enzymolysis method can cause the precipitation of colored substances and heavy peculiar smell substances. The water lily stem cell water obtained by the method has more than 50 volatile active ingredients, is aromatic in flavor, clear and transparent in liquid, does not contain substances which are easy to mildew such as polysaccharide, flavone and the like, and has the effects of resisting oxidation, preserving moisture and resisting inflammation.

Drawings

FIG. 1: water lily stem cell water safety test result chart.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

The water lily stems used in the examples and the comparative examples are not mildewed, and the water is drained after cleaning to carry out the following extraction experiments of the examples and the comparative examples.

Example 1: cutting 50kg of water lily stem into 2-6mm thick pieces by a reciprocating cutter without adding any solvent, primarily extracting at 45 deg.C under-85 kPa, condensing water lily stem cell water to form vapor, collecting liquid, extracting for 2.5 hr to obtain primary extracted cell liquid and water lily stem primary extracted residue, adding 100g cellulase and 25g pectinase, adding the primary extracted cell liquid into the primary extracted residue, extracting again at 45 deg.C under-90 kPa for 8 hr, and collecting water lily stem cell water. The condensation temperature in the whole extraction process is-5 ℃, and the stirring is carried out at 45 r/min. The obtained water lily stem cell water is clear and transparent, and has aromatic flavor of 40.1 kg.

Example 2: example 2 differs from example 1 in that the temperature in the preliminary extraction stage is 35 ℃ and the pressure is-65 kPa. 37.9kg of water lily stem cell water is obtained, and the water lily stem cell water is clear and transparent and has aromatic flavor.

Example 3: example 3 differs from example 1 in that in the preliminary extraction stage, the temperature is 55 ℃ and the pressure is-100 kPa. 39.2kg of water lily stem cell water is obtained, and the water lily stem cell water is clear and transparent and has aromatic flavor.

Example 4: example 4 differs from example 1 in that in the re-extraction stage, the temperature is 35 ℃ and the pressure is-70 kPa. 37.5kg of water lily stem cell water is obtained, and the water lily stem cell water is clear and transparent and has aromatic flavor.

Example 5: example 5 differs from example 1 in that in the re-extraction stage, 55 ℃ at a pressure of-100 kPa. 38.1kg of water lily stem cell water is obtained, and the water lily stem cell water is clear and transparent and has aromatic flavor.

Example 6: example 6 differs from example 1 in that 200g of cellulase and 50g of pectinase were added and the extraction time was 6 hours again. 40.3kg of water lily stem cell water is obtained, is clear and transparent, has strong fragrance, but has a single-thread mixed taste.

Comparative example 1: without adding any solvent, cutting 50kg of flos Nymphaeae stem into 2-6mm thick pieces with reciprocating cutter, extracting at 45 deg.C under-100 kPa, condensing water to form vapor, collecting liquid (condensation temperature is-5 deg.C), extracting for 8 hr, stirring for 45 r/min to obtain clear and transparent water lily stem cell water, 34.3kg, and fragrance is not strong enough.

Comparative example 2: cutting 50kg of water lily stem into strips with the thickness of 2-5mm by a reciprocating cutting machine, mixing with 5kg of water, 200g of cellulase and 50g of pectinase, stirring at 50 ℃ for 45 minutes, extracting at 60 ℃ and under the pressure of-100 kPa, condensing water of water lily stem cells after forming steam, collecting liquid (the condensing temperature is-5 ℃), extracting for 8 hours, stirring for 45 revolutions per minute in the whole process, and obtaining 39.8kg of clear and transparent water lily stem cells (containing 5kg of water) with insufficient smell.

Comparative example 3: cutting 50kg of water lily stems into strips with the thickness of 2-5mm by using a reciprocating cutting machine, mixing the water lily stems with 5kg of water, 100g of cellulase and 25g of pectinase, stirring for 6 hours at 50-60 ℃, stirring for 45 revolutions per minute in the whole process, and filtering (double filtering, firstly adopting a centrifuge to separate solid from liquid, and then adopting a 0.22um filter membrane to carry out fine filtering) to obtain 41.4kg of water lily stem cell water (containing 5kg of added water), wherein the liquid contains tiny suspended matters and is heavy in abnormal taste.

Comparative example 4: the difference from example 1 is that the initial extraction time is 30 minutes, and 32.5kg of water lily stem cell water is obtained, which is clear and transparent and has light smell.

Comparative example 5: the difference from example 1 is that the initial extraction time is 4 hours, and clear and transparent 38.5kg of water lily stem cell water with obvious peculiar smell is obtained.

Table 1: test results of the Stem cell fluid of Water lily extracted in examples and comparative examples (the following contents are accurate to one decimal place)

Continuing with Table 1:

examples and comparative examples experimental data evaluation:

as can be seen from comparative example 1, the cell sap obtained by extraction by the single low-temperature-vacuum extraction method was acceptable in preservation performance, but had a small amount of active ingredients and a light odor.

As shown in comparative example 2, although 5kg of water was additionally added to the mixture of cellulase and pectinase, the entire surface of the plant could not be wetted due to the insufficient amount of water, and the complete evaporation was completed in half an hour, resulting in insufficient enzymatic hydrolysis. And the extra water will dilute the cell sap, reducing the quality.

As can be seen from comparative example 3, the use of pure enzymatic hydrolysis resulted in excessive enzymatic hydrolysis, a strong off-flavor and some fine visible suspensions in the liquid even with double filtration.

From comparative example 4/5, the initial extraction time also greatly affected the whole extraction process: if the initial extraction time is too short, the plant surface can not be covered, and the enzymolysis is insufficient after the evaporation is finished in less than half an hour. If the initial extraction time is too long, the enzymolysis time is too long in the process of re-extraction after adding enzyme, so that obvious peculiar smell is generated, and meanwhile, the surface of the initial extraction residue is too dry, so that the enzymolysis is difficult to enter the residue, and the yield of cell water is not high.

The test methods are as follows:

(1) water lily stem cell water activity component analysis: the headspace gas quality was checked at 80 ℃ injection temperature.

1. Instrument information:

Agilent 7980A GC；

MS:5975C；

50/30 μm CAR/PDMS/DVB extraction fiber head, SUPELCO USA.

GC-MS conditions:

the chromatographic column is HP-INNOWAX capillary column (30m × 0.25mm × 0.25 μm); the carrier gas is He, the flow rate is 1mL/min, and the separation ratio is 5: 1; the sample injection temperature is 250 ℃; the temperature raising procedure is that the initial temperature is 40 ℃, the temperature is kept for 5min, the temperature is raised to 250 ℃ at the speed of 8 ℃/min, and the temperature is kept for 5 min.

Mass spectrum conditions: EI ionization source, energy 70 eV; the ion source temperature is 230 ℃, the quadrupole rod temperature is 150 ℃, the interface temperature is 250 ℃, and the scanning range is 30-400 m/z.

3. Sample pretreatment:

5mL of the sample and 1g of NaCl were placed in a 20mL headspace bottle, and the cap was screwed down. After 5min of equilibrium at 80 ℃ in stirring mode, extracting for 5min at 80 ℃ with a solid phase micro-extraction needle, and then resolving for 5min at the sample inlet.

Table 2: example 1 Water lily stem cell water head air test results (only high content of components retained)

(2) Water lily stem cell water safety test

The HaCaT cell is a human immortal epidermal cell line, has cytotoxicity to the HaCaT cell, and can be used as reference data for safety of skin. The normal cells are in vigorous metabolism, succinate dehydrogenase in mitochondria can reduce tetrazolium salt substances into colored crystalline substances and deposit the crystalline substances around the cells, OD values can be read by an enzyme labeling instrument according to the change, and the relative growth condition of the cells can be known by comparing the OD values with a blank control group.

The water lily stem cell water basically has no toxicity to human body skin cells and is a very natural green raw material according to the specification and the attached figure 1 (example 1 water lily stem cell water safety test chart).

(3) Water lily stem cell water antioxidant property

By DPPH free radical scavenging method, the alcoholic solution is dark purple and has strong absorption near 517nm according to the stable nitrogen free radical existing in DPPH molecules. When the free radical scavenger exists, absorption of single electrons in molecules is gradually disappeared due to pairing, the fading degree and the number of the electrons received form a quantitative relation, and therefore, the antioxidant effect of the free radical scavenging capacity determination sample can be quantitatively analyzed by determining the reduction of the absorbance at the maximum absorption wavelength of 517 nm. Example 1 water lily stem cell water experimental results are shown in the table below.

Water lily stem cell Water concentration (%)	DPPH clearance/%)
		20	10
40	16
		50	23
60	30
		80	37
100	50

As can be seen from the data in the table above, the water lily stem cell water has good antioxidant effect.

(4) Water lily stem cell water anti-inflammatory effect

The water lily stem cells have more active ingredients in water, wherein the water lily stem cells have more obvious antibacterial and anti-inflammatory ingredients, such as phenethyl alcohol and the like, so the water lily stem cells are tested for anti-inflammatory efficacy.

Lipopolysaccharide is the main component of gram-negative bacteria cell wall, and can activate macrophage to release various inflammatory cytokines, so Lipopolysaccharide (LPS) is utilized to stimulate mouse mononuclear-macrophage to establish an in vitro inflammatory reaction model, samples are intervened, dexamethasone is used as a positive control, and enzyme-linked immunosorbent assay (ELISA) is adopted to determine the level change of inflammatory factors IL-6 and TNF-alpha, thereby discussing the in vitro anti-inflammatory action of the samples.

The water lily stem cell water stock solution of example 1 is prepared into different concentrations for testing, and the test results are shown in the following table.

Water concentration of water lily stem cell	IL-6(pg/mL)	TNF-α(pg/mL)
			100％	35	151
80％	37	162
			40％	36	189
15％	39	205
			10％	45	216
5％	48	230
			Blank space	25	150
LPS stimulation	50	260
			Dexamethasone	45	206

From the above table, it can be known that the water lily stem cell water with the concentration of 15% has the anti-inflammatory effect, and the IL-6 expression factor is obviously reduced after the skin is stimulated by LPS; and 100% concentration of water lily stem cell water can reduce the TNF-alpha expression factor of skin stimulated by LPS to be in a normal range.

(5) Water lily stem cell water preservation condition

The water lily stem cells have more active ingredients in water, and the storage is very important for preventing the quality of products from being reduced due to fungus reproduction.

The table below shows the physicochemical values and the fungus proliferation of the water lily stem cell water of example 1 at room temperature and 4 ℃ under two storage conditions, which are determined in one month.

1) No preservative is added at room temperature

2) No antiseptic at 4 deg.C

In the following table, physicochemical values and fungus reproduction conditions of the water lily stem cell water of the comparative example 3 at room temperature and 4 ℃ under two storage conditions were tested for one month.

3) No preservative is added at room temperature

4) No antiseptic at 4 deg.C