CN107279286A - A kind of preparation method of promotion wound healing Yoghourt - Google Patents
A kind of preparation method of promotion wound healing Yoghourt Download PDFInfo
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- CN107279286A CN107279286A CN201710402793.3A CN201710402793A CN107279286A CN 107279286 A CN107279286 A CN 107279286A CN 201710402793 A CN201710402793 A CN 201710402793A CN 107279286 A CN107279286 A CN 107279286A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
- A23C9/133—Fruit or vegetables
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1238—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt using specific L. bulgaricus or S. thermophilus microorganisms; using entrapped or encapsulated yoghurt bacteria; Physical or chemical treatment of L. bulgaricus or S. thermophilus cultures; Fermentation only with L. bulgaricus or only with S. thermophilus
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Abstract
The invention discloses a kind of preparation method of promotion wound healing Yoghourt, feature is included milk powder and warm water in mass ratio 15：The step of sterilizing obtains reconstituted milk after 100 ratio mixing；The step of Tomato juice, spinach juice, folium artemisiae argyi juice, black soya bean juice and hydromel are mixed by equal proportion；By reconstituted milk with mixing juice by volume 3：1 ratio is sufficiently mixed uniform homogeneous sterilization；In mass ratio 0.1%~0.11% inoculum concentration adds ferment agent for sour milk bacterium powder in mixed fermentation raw material, then the inoculum concentration for adding Lactobacillus casei XS1 and mass volume ratio 1~2% by the inoculum concentration of mass volume ratio 1~2% adds Lactobacillus plantarum RUB1 leavenings, stir after 5 min, it is fitted into the bottle after high pressure steam sterilization and seals fermentation, advantage is to improve immunity of organism to avoid wound infection, it is anti-oxidant, promote hematopoiesis, promote wound cells merisis, extension the Shelf-Life of Yogurt.
Description
Technical field
The present invention relates to a kind of Yoghourt, more particularly, to a kind of preparation method of promotion wound healing Yoghourt.
Background technology
Wound healing refers to that body is acted on by external force, and the tissue such as skin occurs more answering process after detachment or defect, is
Regeneration and granulation tissue hyperplasia including various tissues, the complex combination of cicatricial tissue formation, show the collaboration of various processes
Effect.General minor cut or wound can be easy to healing, but larger while situations such as with losing blood more for these wounds, patient or wound
Person's passive protective physical fitness restoring force, which declines, causes wound to be difficult to healing or even infected.So quickly and easily supplementing some nutrients
It is particularly important come the resistance recovery capability that improves body.Patient is typically very weak after surgery is difficult to mend by normal diet
Corresponding nutriment is filled, a kind of convenient easy absorption is at this moment accomplished by and is rich in the fluid food of required nutrition to promote patient's
Wound recovers.
The characteristics of being difficult to healing for postoperative patient wound, protein, carbohydrate, lipid, vitamin can be supplemented（C、A、B2
Deng）, trace element（Zinc, iron, calcium etc.）To promote hematopoiesis to improve immunity, promote cell division growth, prevent wound infection most
The purpose for promoting wound healing is reached eventually.Protein substance, carbohydrate, lipid be all cell growth metabolism fission process in can not
The nutriment lacked.Vitamin, trace element are reconciling body metabolism immunology with good effect.Certain zinc, iron,
The trace element such as calcium is also the important composition material of cell, with the function of promoting cell growth.These nutrients are also body
Produce blood indispensable, and body immunity and blood have very big contact.These content of material are added in Yoghourt
High raw material can undoubtedly accelerate the merisis of wound circumference cell, and then promote wound healing.
Tomato energy relieving heat and thirst, Yin and cool blood, promotes the production of body fluid to quench thirst.Eating something rare can replenishing vitamins C.Eaten after being cooked, it is contained
Lycopene and other oxidation preventive contents can significantly rise, and play a part of the cardiopathic risk of reduction and anti-aging.It is edible
With part 97%.Energy content 79kJ, moisture 94.4g, protein 0.9g, fat 0.2g, dietary fiber 0.5g, carbon water in per l00g
Compound 3.5g, the μ g of carrotene 550, the μ g of retinol equivalent 92, thiamine 0.03mg, riboflavin 0.03mg, niacin 0.6mg；
Vitamin 19mg, vitamin E 0.57mg；Potassium 163mg, sodium 5mg, calcium 10mg, magnesium 9mg, iron 0.4mg, manganese 0.08mg, zinc
0.13mg, copper 0.06mg, phosphorus 2mg；The μ g of selenium 0.15.And containing adenine, choline, tomatidine and anti-inflammatory, diuresis, decompression into
Point.
Spinach is the whole herb with root of amaranthaceous plant spinach, and spinach has functions that moistening lung laxation, clearing heat and relieving fidgetness.Spinach nutrition into
It enriches split pole, the leaf of spinach (dry weight) containing 56~68mg/Kg of zinc, folic acid 1.22ug/g, amino acid and lutein, beta carotene,
Neo-3-CaroteneB, new-beta carotene.The carotenoid such as spinacin U, also contain hitodesterol, cholesterine and sterol ester
Disappear with sterol glycoside, patuletin, spinacetin, and a kind of livid purple color fluorescent substance 2- acetyl group -3- para hydroxybenzene acryloyl groups
Revolve tartaric acid.Spinach moisture content, carbohydrate, protein, fat, dietary fiber, carrotene, vitamin B1, vitamin
The nutrients such as B2, vitamin C, potassium, sodium, calcium, phosphorus, milling, magnesium.Spinach can also promote health, and prevent aging, and preventing and treating lacks
Iron anaemia;Vitamin C and folate content in spinach enrich, and can strengthen absorbability of the puerpera to ferro element, be iron-deficients
Dietary fiber in the preferable food of anaemia, spinach can play good defecating feces excretion.Spinach, which has, promotes culture cell propagation
Effect.Contained carrotene, is transformed into vitamin A in human body in spinach, can safeguard the strong of twenty-twenty vision and epithelial cell
Health, increases the ability kept off infection.
Research is found, in every 100 grams of black soya beans：Moisture 9.9g, ash content：4 .6g, vitamin E (T) 1736mg, zinc 4.8mg,
Heat 381KJ, dietary fiber 10.2g, calcium：224mg, selenium 6.79ug, energy 1594KJ, carrotene 30mg, phosphorus 500mg, copper
156mg, protein 36g, thiamine ug, potassium 1377mg, the .83mg of manganese 2, fat 15.9g, the mg of riboflavin 0.33, sodium 3mg, iron
7mg, carbohydrate 33.6mg, niacin 2mg, magnesium 243mg, the mg of vitamin A 5, more than 12g, detection hair are up to containing crude fat
Now wherein containing at least 19 kinds aliphatic acid, and unsaturated fatty acid content is up to 80% unexpectedly, and wherein linoleic acid content just account for
About 55. 08%.Linoleic acid, as one kind of unrighted acid, is highly important necessary aliphatic acid in human body, to cholesterol
Metabolism has vital adjustment effect, only could in vivo transport and carry out normal when cholesterol is combined with linoleic acid
Metabolism.And when linoleic acid lacks, cholesterol will be combined with saturated fatty acid and in people's internal deposition, cause artery sclerosis
Occur.Therefore, linoleic acid has the good reputation of " blood vessel scavenger " again.
Chinese mugwort leaf Main Ingredients and Appearance is ethyl acetate, 1.8 ~ cineole, camphene hydrate, camphor, 2 ~ terpinol, Eucarvone etc.
Chemicals, the oil yield Chinese mugwort of its essential oil（1.06%）For general Chinese mugwort（0.54%）Twice.Meanwhile, Chinese mugwort contains absinthol
（15.6%）And isothujone（2.7%）, and general Ai Ze has no this composition.Chinese mugwork oil have significantly relieving asthma, antibechic, eliminating the phlegm and clear
Inflammation effect.Modern experimental research has shown that folium artemisiae argyi has antibacterial and antivirus action, and wormwood also has dispelling cold and removing dampness, regulating menstruation, hemostasis
Effect, to female irregular menstruation, dysmenorrhoea stomachache have certain curative effect.
Contain fructose, glucose, enzyme, protein, vitamin and several mineral materials in honey.Often eat can prevent and treat anaemia,
Heart disease, enterogastritis etc., and body immunity can be improved.Monose：Glucose and fructose can be directly digested.Enzyme
Class：Various enzymes can promote human body to digest and assimilate many kinds of substance.Acetylcholine：Dispelling fatigue, excitor nerve, raising memory
Power.Flavones：It is anti-oxidant, improve immunity of organisms.Honey is the natural nutrient food being widely recognized.Experiment shows, in honey
Containing the various inorganic salts similar to human serum concentration, such as iron, calcium, copper, manganese, potassium, phosphorus, also multivitamin and organic
Acid, therefore honey is referred to as " king of serum ".In addition, honey also has cosmetology, anti-inflammation, promotion organization regeneration, rush
Enter it is long-lived, improve sleep, hepatoprotective effect, it is antifatigue, promote upgrowth and development of children, protection is cardiovascular, moisten the lung and relieve the cough etc. acts on.Mesh
Before, there is not been reported for the functional yoghourt with promotion wound healing effect.
The content of the invention
The technical problems to be solved by the invention, which are to provide one kind and can improve immunity of organism, avoids wound infection, anti-oxidant,
Promote hematopoiesis, promote the Yoghourt of promotion wound healing and preparation method thereof of wound cells merisis, extension the Shelf-Life of Yogurt.
The present invention solve the technical scheme that is used of above-mentioned technical problem for：A kind of preparation side of promotion wound healing Yoghourt
Method, comprises the following steps：
（1）The making of reconstituted milk：By milk powder and 60~70 DEG C of warm water in mass ratio 15：After 100 ratio mixing, it is sufficiently stirred for
Dissolving, carries out pasteurization, obtains reconstituted milk；
（2）Mix the preparation of juice：By Tomato juice, spinach juice, folium artemisiae argyi juice, black soya bean juice and hydromel in mass ratio 1：1：1：
1：1 is mixed to form mixing juice, stirs；
（3）It is prepared by mixed liquor：By reconstituted milk with mixing juice by volume 3：After 1 ratio is sufficiently mixed uniformly, recovery is added
The white granulated sugar of breast matter amount 6~7%, is uniformly mixing to obtain mixed liquor；
（4）Homogeneous is sterilized：Mixed liquor is added in high pressure homogenizer, it is 65-75 DEG C to control mixeding liquid temperature, homogenization pressure is
15-25Mpa；The mixed liquor that homogeneous is crossed keeps 5min, obtains mixed fermentation raw material in 90~95 DEG C of high temperature sterilizations；
（5）The preparation of functional starter cultures：Respectively by the Lactobacillus casei XS1 and Lactobacillus plantarum that are preserved in -40 DEG C of refrigerators
RUB1 strains, are put in thaw at RT, add in the MRS fluid nutrient mediums after sterilizing, are placed in by the inoculum concentration of mass volume ratio 1%
The h of quiescent culture 8, obtains middle leavening in 37 DEG C of incubators；Middle leavening is drawn again with the inoculum concentration of mass volume ratio 2%
Add in the MRS fluid nutrient mediums after sterilizing, be placed in the h of quiescent culture 8 in 37 DEG C of incubators, that is, respectively obtain Lactobacillus casei
XS1 leavenings and Lactobacillus plantarum RUB1 leavenings；
（6）Inoculation：In step（4）In mass ratio 0.1%~0.11% inoculum concentration adds Yoghourt in obtained mixed fermentation raw material
Leavening bacterium powder, then adds Lactobacillus casei XS1 and mass volume ratio 1~2% by the inoculum concentration of mass volume ratio 1~2%
Inoculum concentration add after Lactobacillus plantarum RUB1 leavenings, 5 min of stirring, is fitted into the bottle after high pressure steam sterilization sealing；
（7）Fermentation：4~5h is left to ferment in 42 DEG C of constant incubator, is stopped after fermentation, in being refrigerated at 0~5 DEG C, is produced
To the Yoghourt for promoting wound healing.The purpose of after-ripening promotes the generation of aromatic substance to prevent peracid.
Step（2）The preparation process of described Tomato juice cleans stripping and slicing to choose new fresh tomato, squeezes the juice 3 with juice extractor
Minute, obtain Tomato juice after 8 layers of filtered through gauze；To choose, fresh spinach is clean to be cut the preparation process of described spinach juice
It is broken, squeezed the juice with juice extractor 3 minutes, spinach juice is obtained after 8 layers of filtered through gauze；The preparation process of described folium artemisiae argyi juice is new to choose
Fresh folium artemisiae argyi is cleaned, and is squeezed the juice with juice extractor 3 minutes, and folium artemisiae argyi juice is obtained through 8 layers of filtered through gauze；The preparation process of described black soya bean slurry
To select the black soya bean of full grains and free from admixture, with concentration 0.5wt% NaHCO3, will be black after 20 DEG C of 10~12 h of immersion of solution
Beans are 80 DEG C with temperature, the NaHCO that concentration is 0.5wt%3Solution blanching 10min, rapid cooling, sloughs kind of a skin, by black soya bean with
The mass ratio of water is 1：3 ratio, defibrination obtains black soya bean slurry, then black soya bean slurry is boiled 5 minutes, finally starches black soya bean with 8 layers of gauze
Filtering；The preparation process of described hydromel is to take 50mL plus 150mL warm water to melt the good honey of quality.
Step（6）Described in ferment agent for sour milk bacterium powder by lactobacillus bulgaricus and streptococcus thermophilus in mass ratio 1：1
Ratio mix.
Compared with prior art, the advantage of the invention is that：A kind of preparation method of promotion wound healing Yoghourt of the present invention,
It the sour environment of folium artemisiae argyi, the antibacterial action that folium artemisiae argyi has, and Yoghourt is with the addition of in Yoghourt first can improve in folium artemisiae argyi and wave
The antibacterial action of hair oil, while under the antibacterial double action of functional starter cultures, makes the extended shelf-life of Yoghourt to 40-
50d；Because honey is rich in organic carbohydrate, its addition promotes the dissolving and protection of Flavonoid substances in black soya bean so that this acid
Milk also has good antioxidation while wound healing is promoted.About contain 2.5 ~ 3.0mg's in per 100mL Yoghourts
Fe elements.The Yoghourt promotes cell division to improve 20% ~ 25%.This Yoghourt can reconcile the macrophages secrete TNF-α of culture（It is swollen
Tumor necrosis factor α）With IL-1 β（Interleukin-1 beta）Amount（P<0.05）.With constipation is improved, prevent and treat anaemia, increase
Strong immune, antibacterial anti-inflammatory, promotes effect of wound healing.
In summary, the present invention promotes the preparation method of wound healing Yoghourt, with tomato, spinach, folium artemisiae argyi, black soya bean, honeybee
These sweet food are raw material, are made an addition in milk, and production promotes the Yoghourt of wound healing, simplifies production technology, to greatest extent
Reservation raw material nutritive value, reduce between cost, each nutriment and mutually promote the battalion for absorbing and improving product
Support value and economic benefit.The Yoghourt color of resulting promotion wound healing is yellowish, homogeneous, beans perfume, the vegetable for having black soya bean
The delicate fragrance of dish folium artemisiae argyi, it is edible when can feel the texture of sweetened bean paste, not coarse, not puckery mouth；Sweet mouthfeel is suitable, and aftertaste has honey
Sweetness, can improve immunity of organism rich in nutritional ingredient, vitamin, trace element and avoid wound infection, anti-oxidant, promote hematopoiesis,
Promote wound cells merisis, the Yoghourt of extended shelf-life, contribute to wound healing, reduce operation or the pain of injured patient
It is bitter.
Classification And Nomenclature be Lactobacillus plantarum (Lactobacillus plantarum), bacterial strain is RUB1, and preserving number is
CGMCC No.8211, were deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms on 09 17th, 2013
The heart, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
Classification And Nomenclature be Lactobacillus casei (Lactobacillus casei), bacterial strain is XS1, and preserving number is CGMCC
No.8213, China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation were deposited on 09 17th, 2013
Address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
Above-mentioned Lactobacillus plantarum RUB1（Deposit number is CGMCC No.8211）With Lactobacillus casei XS1（Deposit number is
CGMCC No.8213）With good bacteriostasis, to salmonella, Listeria monocytogenes, staphylococcus aureus, large intestine
The pathogenic bacteria such as bacillus and bacillus cereus have good inhibiting effect.
Embodiment
The present invention is described in further detail with reference to embodiments.
First, experimental method
It is often accompanied by losing blood after injury, and Fe elements play important role during hematopoiesis.The process of the healing of wound
The middle merisis for needing epithelial cell is realized.Caused by general bacterium infection inflammation be suppress wound healing it is important because
Element.So this experiment promotes merisis and the culture of mouse enterocyte by determining the contents of Fe elements in Yoghourt
The inflammatory factor TNF-α of macrophages secrete and IL-1 β amount the function of the Yoghourt is determined as examination criteria.
1st, in Yoghourt Fe constituent contents measure
（1）Instrument and material：Atomic absorption spectrophotometer（Shimadzu Corporation of AA-6800 Japan）, Muffle furnace, ferro element it is hollow
Cathode modulation etc..The Yoghourt of this patent, common sour milk, hydrogen peroxide, the concentrated sulfuric acid, nitric acid, ultra-pure water, standard working solution of ferro element etc.
（2）Experiment detection：4mL samples are pipetted with pipette at room temperature with dry pot, adding 4mL concentrated sulfuric acid 2mL hydrogen peroxide, it is quiet
About 10min is put, untill the white cigarette that is heated to emerging then is placed on electric furnace.Treat that it is cooled to 50 ~ 60 DEG C.Added again into crucible
The 2mL concentrated sulfuric acids, cover crucible cover.The crucible that will be equipped with pretreatment sample is sequentially placed into Muffle furnace and is ashed completely.Temperature adjustment is to 600
~ 650 DEG C, take out after about 2h, whether the sample in observation crucible is into lily ash content, if it is ashing completely.
After cooling, ash content is fully dissolved with dust technology, and is moved into 100mL volumetric flasks, ultra-pure water constant volume is used, shakes up standby.Do simultaneously
Sample blank.
The standard liquid series of table 1
The instrument condition of work of table 2
Note：Air mass flow is 8.0L/min.
The rate of recovery and precision test：3 parts of same sample is removed, a certain amount of standard liquid is separately added into, with other standards sample
Product same method digestion process, carries out the quasi- recovery test of sample mark-on to examine the reliability of measurement result.Sample is taken to repeat
Determine 3 times, complete respectively plus determining 3 times with each sample treatment of the precision of the method for inspection, it is testing result to take its average value.
The content of Fe elements in sample：According to the range of linearity of Instrument measuring, appropriate dilution or concentrating sample solution survey its suction
Luminosity.Regression equation is drawn through regression analysis, by the absorbance measured for people's regression equation, Fe members in the sample are calculated
The content of element, the content of Fe elements in sample is represented with the average value of 5 parallel determinations.
2nd, detection of the Yoghourt to promotion enterocyte merisis
（1）Cell culture：Trained using containing 200mL/L hyclones, the dual anti-liquid of Pen .- Strep, pH value for 7.2 DMEM
Nutrient solution cultivates mouse enterocyte under the conditions of 37 DEG C, 50mL/LCO2, and liquid is changed daily once；
（2）Effect of the Yoghourt to enterocyte merisis is detected using cell counting：When cell growth reaches plocoid
1. number during state, had digestive transfer culture is counted, and uniform 3 bottles of passage is put into after 37 DEG C of 50mL/LCO2 incubator culture 24h, cell attachment,
The appropriate Yoghourt is added in bottle, common sour milk is added in 2. number bottle, 3. a number bottle does blank control.Respectively the 1st, 2,3,4 days
Digestion counts cell.
3rd, the TNF-α of the macrophage strain of culture and the detection of IL-1 β secretory volumes
This programme is by setting up lipopolysaccharides（LPS）The external inflammatory model of stimulating expression of macrophage strain RAW246.7 activation determines acid
The antiinflammatory action of milk.LPS induction, can cause relevant inflammatory factors such as TNF-α, the change of IL-1 β amounts, thin by determining macrophage
Judge whether Yoghourt has before and after the secretory volume modeling of various inflammatory factors, to the change before and after Yoghourt extract in born of the same parents' supernatant
There is anti-infectious function.
（1）Main solution and preparation
1. 1640 culture medium：13.4g DMEM dry-type culture mediums are dissolved with 90ml ultra-pure waters, magnetic agitation makes dry powder completely molten
Solution, plus 2.23g NaHCO3 and 3.08g Hepes, adjustment pH value add water to 7.2 ~ 7.4 and are settled to 1000ml, 0.22 μm of filter membrane
Dispensed after filtration sterilization, -20 DEG C of preservations；
2. 0.25% trypsase：Trypsase 0.5g is weighed, is added in 200ml PBS, magnetic agitation is completely dissolved it,
Dispensed after 0.22 μm of membrane filtration is degerming, -20 DEG C of preservations；
3. Yoghourt extract：100ml Yoghourts are taken to carry out refrigerated centrifuge（10000r/min, 10min）, with twice of 0.2 μm of filter membrane,
Take supernatant；
4. instrument：Sorvall ST16R refrigerated centrifuges（Thermo Fishr Scientific companies of the U.S.）；Mouse TNF-
α ELISA kits（R ＆ D companies of the U.S., lot number：201207）；Mouse IL-1 β ELISA kits（R ＆ D companies of the U.S., batch
Number：201207.
（2）Cell culture
1. source property macrophage strain RAW264.7 is incubated in RPMI1640 culture mediums（Containing 10% NBCS, 100 μ g/ml chains
Mycin, 100 μ/ml penicillin）.Condition of culture is 37 DEG C, and gas concentration lwevel is 5%, continuous culture；
2. cell is added cold culture medium after Microscopic observation is rounded and stops digestion, blown repeatedly with 0.25% Trypsin Induced
Centrifuging and taking precipitation is resuspended with culture medium after beating, and is inoculated in culture dish, 2 × 105/ml of initial density is inoculated with, after culture to 80% fusion
For testing, serum-free processing in 12 hours makes cell synchronization before experiment；
3. Yoghourt extract is added by various concentrations, 2h is incubated in advance, solvent control is DMSO, the rear LPS for adding 200ng/ml
（Final concentration）Different time is stimulated, supernatant culture is taken respectively, cell is used for different Indexs measures.
（3）MTT colorimetric determinations
Principle：The entitled tetrazolium bromide of MTT business, is faint yellow, it can be reduced into by the succinate dehydrogenase in living cells mitochondria
The bluish violet crystal violet thing first a ceremonial jade-ladle, used in libation of slightly solubility is simultaneously deposited in cell body, but MTT can not be reduced to bluish violet knot in dead cell
Brilliant thing.The crystallization can be by dimethyl sulfoxide (DMSO)（DMSO）Or have one wider at 560-610nm after being acidified the dissolving such as isopropanol
Maximum absworption peak, the light absorbs at 570nm can be quantitative determined with ELIASA.Due to the growing amount and mitochondrial succinate of first a ceremonial jade-ladle, used in libation
Dehydrogenase activity is proportionate, and can reflect dehydrogenase activity with the size of absorbance, so that reflect the quantity of living cells indirectly,
Living cells is more, and purple product is more, then light absorbs（OD values）It is higher.
Method：With 96 orifice plate culture cells, the Yoghourt extract of various concentrations is added according to experiment packet（0.8,4,20,
40,80μM）, 18h is incubated in 37 DEG C, the incubator containing 5%CO2, final concentration of 0.5mg/ml MTT is added, is incubated after 4h,
Supernatant is abandoned in suction, and dimethyl sulfoxide (DMSO) is added per hole（DMSO）, shaken at room temperature 10min treats that purple crystal thing fully dissolves, with it is complete from
Dynamic ELIASA reads OD value, every group of hole of repetition 5 at 570nm.Calculate cell survival rate.Cell survival rate=（The OD of detection hole
The OD values of value/control wells）×100%.
（4）The Yoghourt measures the detection of influence on RAW264.7 cell TNF secretions-α
Yoghourt extract is added by various concentrations（0.8,4,20μM）, 2h is incubated in advance, and other groups of addition solvents are compareed, and change liquid
Remove the LPS that 200ng/ml is added after Yoghourt（Final concentration）18h is stimulated, supernatant nutrient solution is taken respectively, -20 DEG C is placed in and preserves to inspection
Survey.Each sample does multiple holes measure.Determination step is as follows：
1. sample 0.1ml is added per hole, ELISA Plate is plus lid, and 37 DEG C are reacted 90 minutes；
2. with the liquid got rid of in ELISA Plate after reacting, then against blotting paper clap it is several under, do not wash；
3. ready biotin anti-TNF-α antibody working solution is sequentially added by every hole 0.1ml.37 DEG C are reacted 60 minutes；
4. add 0.01M PBS 0.35ml per hole to wash 3 times, every time immersion 1 minute or so；
5. ready ABC working solutions are sequentially added by every hole 0.1ml,（Except TMB blank colour developing hole）.37 DEG C are reacted 30 points
Clock；
6. 0.01 M PBS 0.35ml are added per hole to wash 5 times, are soaked 1 ~ 2 minute or so every time；
7. sequentially added by every μ l of hole 90 in 37 DEG C of balances TMB nitrite ions of 30 minutes, 37 DEG C of lucifuges are reacted 15 minutes；
8. TMB terminate liquids are sequentially added by every hole 0.1ml, now blueness turns yellow.With ELIASA OD values are determined in 50nm.According to
Standard curve calculates corresponding concentration.
（5）Cell supernatant IL-1 β are detected
Using mouse IL-1 β ELISA kits, operated in strict accordance with kit specification, step is as follows：
1. the dilution of standard items and sample-adding：Set on enzyme mark coating plate in the hole of standard sample wells 10, sub 1,2 holes and add standard items respectively
100 μ l, then add the μ l of standard dilutions 50 in the 1st, 2 holes, mix；Then 100 μ l are respectively taken to be added separately to from the 1st, 2 holes
3rd, 4 holes, add the μ l of standard dilutions 50 respectively in the 3rd, 4 holes, mix；Then 50 μ l are respectively taken to discard in the 3rd, 4 holes,
Respectively take 50 to be added separately in the 5th, 6 holes again, add the μ l of standard dilutions 50 respectively in the 5th, 6 holes, mix；From the 5th, 6 holes
Respectively take 50 to be added separately in 7,8 holes, add the μ l of standard dilutions 50 respectively in 7,8 holes, mix；Respectively taken from the 7th, 8 holes
50 μ l are added separately in the 9th, 10 holes, add the μ l of standard dilutions 50 to mix respectively in the 9th, 10 holes, then the 9th, 10
50 μ l are respectively taken to discard in hole.Each hole adds sample size to be 50 μ l after dilution, concentration be respectively 90ng/L, 60ng/L, 30ng μ g/L,
15ng/L、7.5ng/L；
2. it is loaded：White hole is set respectively（Sample and enzyme marking reagent are not added with, remaining each step operation is identical）, testing sample hole.
Respectively add the μ l of sample diluting liquid 40 in testing sample hole, then add the μ l of testing sample 10 respectively again（The final dilution factor of sample is 5 times）.
Sample is added on ELIASA plate hole bottom by sample-adding, is not touched hole wall as far as possible, is gently rocked mixing；
3. incubate：Incubated 30 minutes with being placed in 37 °C of electro-heating standing-temperature cultivator after shrouding film shrouding；
4. liquid is matched somebody with somebody：By 30（20 times of 48T）Times concentrated cleaning solution distilled water 30（20 times of 48T）It is standby after diluting again；
5. board-washing：ELISA Plate is taken out, shrouding film is carefully thrown off, discards liquid, is dried, board-washing liquid is filled it up with per hole, after standing 30 seconds
Discard, be so repeated 5 times, pat dry；
6. it is enzyme-added：Added per hole except the μ l of enzyme marking reagent 50, blank well；
7. incubate：Incubated 30 minutes with being placed in 37 °C of electro-heating standing-temperature cultivator after shrouding film shrouding；
8. board-washing：ELISA Plate is taken out, shrouding film is carefully thrown off, discards liquid, is dried, board-washing liquid is filled it up with per hole, after standing 30 seconds
Discard, be so repeated 5 times, pat dry；
9. develop the color：50 μ l developer A are first added per hole, 50 μ l developer B are added, gently concussion is mixed, 37 °C of lucifuge colour developings
15 minutes；
10. terminate：The μ l color development stoppings of terminate liquid 50 are added to react per hole.Returned to zero with blank well, 450nm wavelength sequentially determines each hole and inhaled
Luminosity.Measure should be carried out within after adding terminate liquid 15 minutes.
Calculate：According to the concentration of standard items and corresponding OD values, the linear regression equation of standard curve is calculated, in basis
The OD values of sample, calculate corresponding sample concentration on regression equation, and ultimate density is that actual concentrations are multiplied by extension rate.
2nd, specific embodiment
Embodiment 1
A kind of preparation method of promotion wound healing Yoghourt, comprises the following steps：
（1）The making of reconstituted milk：By milk powder and 65 DEG C of warm water in mass ratio 15：After 100 ratio mixing, it is sufficiently stirred for molten
Solution, carries out pasteurization, obtains reconstituted milk；
（2）Mix the preparation of juice：By Tomato juice, spinach juice, folium artemisiae argyi juice, black soya bean juice and hydromel in mass ratio 1：1：1：
1：1 is mixed to form mixing juice, stirs；Wherein
The preparation process of Tomato juice cleans stripping and slicing to choose new fresh tomato, is squeezed the juice with juice extractor 3 minutes, through 8 layers of gauze mistake
Tomato juice is obtained after filter；The preparation process of spinach juice is washed and chopped to choose fresh spinach, is squeezed the juice with juice extractor 3 minutes,
Spinach juice is obtained after 8 layers of filtered through gauze；The preparation process of folium artemisiae argyi juice is cleaned to choose fresh folium artemisiae argyi, squeezes the juice 3 with juice extractor
Minute, obtain folium artemisiae argyi juice through 8 layers of filtered through gauze；The preparation process of black soya bean slurry is selection full grains and the black soya bean of free from admixture, is used
Concentration 0.5wt% NaHCO3After 20 DEG C of solution immersion, 10~12 h, by black soya bean with temperature be 80 DEG C, concentration be 0.5wt%'s
NaHCO3Solution blanching 10min, rapid cooling, sloughs kind of a skin, is 1 by the mass ratio of black soya bean and water：3 ratio, defibrination obtains black
Soya-bean milk（So that the nutriment such as the rupture of black soya bean histocyte, protein is soluble in water, black soya bean fiber precipitation）, then by black soya bean slurry boil
Boiling 5 minutes, finally starches black soya bean with 8 layers of filtered through gauze；The preparation process of hydromel is to take 50mL to add the good honey of quality
150mL warm water is melted；
（3）It is prepared by mixed liquor：By reconstituted milk with mixing juice by volume 3：After 1 ratio is sufficiently mixed uniformly, recovery is added
The white granulated sugar of breast matter amount 6~7%, is uniformly mixing to obtain mixed liquor；
（4）Homogeneous is sterilized：Mixed liquor is added in high pressure homogenizer, it is 70 DEG C to control mixeding liquid temperature, homogenization pressure is
20Mpa；The mixed liquor that homogeneous is crossed keeps 5min, obtains mixed fermentation raw material in 92 DEG C of high temperature sterilizations；
（5）The preparation of functional starter cultures：Respectively by the Lactobacillus casei XS1 and Lactobacillus plantarum that are preserved in -40 DEG C of refrigerators
RUB1 strains, are put in thaw at RT, add in the MRS fluid nutrient mediums after sterilizing, are placed in by the inoculum concentration of mass volume ratio 1%
The h of quiescent culture 8, obtains middle leavening in 37 DEG C of incubators；Middle leavening is drawn again with the inoculum concentration of mass volume ratio 2%
Add in the MRS fluid nutrient mediums after sterilizing, be placed in the h of quiescent culture 8 in 37 DEG C of incubators, that is, respectively obtain Lactobacillus casei
XS1 leavenings and Lactobacillus plantarum RUB1 leavenings；
（6）Inoculation：In step（4）In mass ratio 0.105% inoculum concentration adds ferment agent for sour milk in obtained mixed fermentation raw material
Bacterium powder（By lactobacillus bulgaricus and streptococcus thermophilus in mass ratio 1：1 ratio is mixed）, then by mass volume ratio
1.5% inoculum concentration adds Lactobacillus casei XS1 and the inoculum concentration of mass volume ratio 1.5% adds Lactobacillus plantarum RUB1 fermentations
After agent, 5 min of stirring, it is fitted into the bottle after high pressure steam sterilization and seals；
（7）Fermentation：4.5h is left to ferment in 42 DEG C of constant incubator, is stopped after fermentation, in being refrigerated at 0~5 DEG C, is produced
To the Yoghourt for promoting wound healing.The purpose of after-ripening promotes the generation of aromatic substance to prevent peracid.
Embodiment 2
With above-described embodiment 1, its difference is：
Step（1）It is middle milk powder is mixed with 60 DEG C of warm water after；
Step（4）Middle to control mixeding liquid temperature to be 65 DEG C, homogenization pressure is 15Mpa；The mixed liquor that homogeneous is crossed is high in 9095 DEG C
Temperature sterilization, keeps 5min；
Step（6）In mass ratio 0.1%% inoculum concentration adds ferment agent for sour milk bacterium powder in middle mixed fermentation raw material, then by quality
The inoculum concentration of volume ratio 1% adds Lactobacillus casei XS1 and the inoculum concentration of mass volume ratio 1% adds Lactobacillus plantarum RUB1 hairs
Ferment agent；
Step（7）In：4h is left to ferment in constant incubator.
Embodiment 3
With above-described embodiment 1, its difference is：
Step（1）It is middle milk powder is mixed with 70 DEG C of warm water after；
Step（4）Middle to control mixeding liquid temperature to be 75 DEG C, homogenization pressure is 25Mpa；The mixed liquor that homogeneous is crossed is in 95 DEG C of high temperature
Sterilization, keeps 5min；
Step（6）In mass ratio 0.11% inoculum concentration adds ferment agent for sour milk bacterium powder in middle mixed fermentation raw material, then by quality
The inoculum concentration of volume ratio 2% adds Lactobacillus casei XS1 and the inoculum concentration of mass volume ratio 2% adds Lactobacillus plantarum RUB1 hairs
Ferment agent；
Step（7）In 5h is left to ferment in constant incubator.
Certainly, it is not limitation of the present invention to state above, and the present invention is also not limited to the example above.The art
Technical staff is in the essential scope of the present invention, the variations, modifications, additions or substitutions made, and should also belong to the protection of the present invention
Scope.
Claims (3)
1. a kind of preparation method of promotion wound healing Yoghourt, it is characterised in that comprise the following steps：
（1）The making of reconstituted milk：By milk powder and 60~70 DEG C of warm water in mass ratio 15：After 100 ratio mixing, it is sufficiently stirred for
Dissolving, carries out pasteurization, obtains reconstituted milk；
（2）Mix the preparation of juice：By Tomato juice, spinach juice, folium artemisiae argyi juice, black soya bean juice and hydromel in mass ratio 1：1：1：
1：1 is mixed to form mixing juice, stirs；
（3）It is prepared by mixed liquor：By reconstituted milk with mixing juice by volume 3：After 1 ratio is sufficiently mixed uniformly, recovery is added
The white granulated sugar of breast matter amount 6~7%, is uniformly mixing to obtain mixed liquor；
（4）Homogeneous is sterilized：Mixed liquor is added in high pressure homogenizer, it is 65-75 DEG C to control mixeding liquid temperature, homogenization pressure is
15-25Mpa；The mixed liquor that homogeneous is crossed keeps 5min, obtains mixed fermentation raw material in 90~95 DEG C of high temperature sterilizations；
（5）The preparation of functional starter cultures：Respectively by the Lactobacillus casei XS1 and Lactobacillus plantarum that are preserved in -40 DEG C of refrigerators
RUB1 strains, are put in thaw at RT, add in the MRS fluid nutrient mediums after sterilizing, are placed in by the inoculum concentration of mass volume ratio 1%
The h of quiescent culture 8, obtains middle leavening in 37 DEG C of incubators；Middle leavening is drawn again with the inoculum concentration of mass volume ratio 2%
Add in the MRS fluid nutrient mediums after sterilizing, be placed in the h of quiescent culture 8 in 37 DEG C of incubators, that is, respectively obtain Lactobacillus casei
XS1 leavenings and Lactobacillus plantarum RUB1 leavenings；
（6）Inoculation：In step（4）In mass ratio 0.1%~0.11% inoculum concentration adds Yoghourt in obtained mixed fermentation raw material
Leavening bacterium powder, then adds Lactobacillus casei XS1 and mass volume ratio 1~2% by the inoculum concentration of mass volume ratio 1~2%
Inoculum concentration add after Lactobacillus plantarum RUB1 leavenings, 5 min of stirring, is fitted into the bottle after high pressure steam sterilization sealing；
（7）Fermentation：4~5h is left to ferment in 42 DEG C of constant incubator, is stopped after fermentation, in being refrigerated at 0~5 DEG C, is produced
To the Yoghourt for promoting wound healing.
2. a kind of preparation method of promotion wound healing Yoghourt according to right 1, it is characterised in that：Step（2）Described
The preparation process of Tomato juice cleans stripping and slicing to choose new fresh tomato, is squeezed the juice with juice extractor 3 minutes, after 8 layers of filtered through gauze
Obtain Tomato juice；The preparation process of described spinach juice is washed and chopped to choose fresh spinach, is squeezed the juice with juice extractor 3 points
Clock, spinach juice is obtained after 8 layers of filtered through gauze；The preparation process of described folium artemisiae argyi juice is cleaned to choose fresh folium artemisiae argyi, with squeezing
Juice machine is squeezed the juice 3 minutes, and folium artemisiae argyi juice is obtained through 8 layers of filtered through gauze；The preparation process of described black soya bean slurry for selection full grains and
The black soya bean of free from admixture, with concentration 0.5wt% NaHCO320 DEG C of solution is soaked after 10~12 h, be 80 DEG C by black soya bean temperature,
Concentration is 0.5wt% NaHCO3Solution blanching 10min, rapid cooling, sloughs kind of a skin, is 1 by the mass ratio of black soya bean and water：3
Ratio, defibrination obtains black soya bean slurry, then black soya bean slurry boiled into 5 minutes, finally by black soya bean slurry with 8 layers of filtered through gauze；Described honey
The preparation process of water is to take 50mL plus 150mL warm water to melt the good honey of quality.
3. a kind of preparation method of promotion wound healing Yoghourt according to right 1, it is characterised in that：Step（6）Described in
Ferment agent for sour milk bacterium powder by lactobacillus bulgaricus and streptococcus thermophilus in mass ratio 1：1 ratio is mixed.
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| CN110651864A (en) * | 2019-10-29 | 2020-01-07 | 陕西仙喜辣木茯茶有限公司 | Functional beverage for promoting wound healing and preparation method and application thereof |
| CN110934188A (en) * | 2019-11-20 | 2020-03-31 | 黄冈师范学院 | Purple perilla and Chinese mugwort yoghourt and preparation method thereof |
| CN113679655A (en) * | 2021-08-26 | 2021-11-23 | 浙江辰海生命科学有限公司 | Honey fermentation product, skin external preparation containing honey fermentation product, and preparation method and application of honey fermentation product |
| CN114470007A (en) * | 2020-11-13 | 2022-05-13 | 葡萄王生技股份有限公司 | Wound external composition containing lactic acid bacteria fermentation product and use thereof |
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Application publication date: 20171024 |