CN108850924A - A kind of fructus lycii probiotics ferment and preparation method thereof - Google Patents
A kind of fructus lycii probiotics ferment and preparation method thereof Download PDFInfo
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- CN108850924A CN108850924A CN201810805214.4A CN201810805214A CN108850924A CN 108850924 A CN108850924 A CN 108850924A CN 201810805214 A CN201810805214 A CN 201810805214A CN 108850924 A CN108850924 A CN 108850924A
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- fermentation
- fructus lycii
- probiotics
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- ferment
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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
- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
- A23L19/09—Mashed or comminuted products, e.g. pulp, purée, sauce, or products made therefrom, e.g. snacks
-
- 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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- 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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/14—Yeasts or derivatives thereof
-
- 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
-
- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/113—Acidophilus
-
- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/123—Bulgaricus
-
- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/143—Fermentum
-
- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/249—Thermophilus
-
- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/51—Bifidobacterium
Abstract
The invention belongs to ferment production technical fields, and in particular to a kind of fructus lycii probiotics ferment and preparation method thereof.The fructus lycii probiotics ferment is using fructus lycii, pineapple, strawberry, tomato, brown granulated sugar as primary raw material, by fermenting three times.The nutritional ingredient of raw material is more abundant in scientific and reasonable collocation, under the fermentation of probiotics, the type, dosage of strict control probiotics and fermentation duration simultaneously, guarantee that the fermentation of raw material is more abundant, finally enable the nutriment in raw material, active material, antioxidant to release completely, finally obtains a kind of nutritional ingredient and be easy to absorption, good mouthfeel, the fructus lycii probiotics ferment for being able to ascend immunity.
Description
Technical field
The invention belongs to ferment production technical fields, and in particular to a kind of fructus lycii probiotics ferment and preparation method thereof.
Background technique
Ferment, which refers to, ferments to fruit and vegetable by microorganism, a kind of liquid containing bioactive ingredients of extraction.Above-mentioned life
Object active constituent includes enzyme but is not limited to enzyme, including active antioxidants, biomass element etc., participates in bacterium from fermentation
With the food materials for fermentation.
The ferment rationally made, the active substance of plant extracted are from food materials, and participation bacterium of fermenting is even more people
The component part of body microecosystem.Therefore, the side effect of ferment is almost nil.Powerful effect, minimum side effect, closely
It is always in explosive increase trend over year.
Containing the substances such as glycine betaine, atropine, hyoscyamine, polysaccharides are contained in fructus lycii, now to the further of fructus lycii
Exploitation is also carrying out always, has at present and fructus lycii is made into Chinese wolfberry fruit wine (CN104845858A), is made into Chinese wolfberry fruit drink
(CN103892389A), it is made into juice from Chinese wolfberry (CN104432311A) etc., also has and fructus lycii is made into fructus lycii ferment
(CN105167072B), fructus lycii composite enzyme (CN107125735A), but these products all not too much fill the fermentation of raw material
Point.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of fructus lycii probiotics ferment and preparation method thereof, fructus lycii benefit
The rich in nutrition content of raw bacterium ferment.
The technical scheme adopted by the invention is as follows：
A kind of preparation method of fructus lycii probiotics ferment, includes the following steps：
S1 is by fructus lycii 500-650kg, pineapple 150-350kg, strawberry 100-400kg, tomato 100-200kg, brown granulated sugar 5-
25kg is added in Zymolysis Equipment；
S2 continuously adds saccharomycete 1-5g and lactobacillus fermenti 10-15g into Zymolysis Equipment and carries out just fermentation, when fermentation
Between be 7-9 months；Lactobacillus acidophilus 10-15g, lactobacillus bulgaricus 1-5g are just added after fermentation and carries out second of hair
Ferment, fermentation time 7-9 months；Bifidobacterium lactis 5-10g, streptococcus thermophilus 1-5g, acetic acid bacteria is added after fermentation for the second time
1-5g continues third time fermentation 7-9 months；
After fermentation, detection pH stablizes between 2.6-3.5 S3, and when alcoholic strength < 0.5%, fermentation is completed；
The fermentation liquid that fermentation is completed is filtered by S4；
S5 stands purifying：By filtered fermentation liquid stand purify, when alcoholic strength < 0.5%, lactic acid bacteria sum >=1 ×
106It purifies and completes when CFU/ml, obtain fructus lycii probiotics enzyme stoste.
When just fermentation, second of fermentation and third time are fermented in the S2, isolation air carries out anaerobic fermentation.
Zymolysis Equipment is fermenter.
Raw material is in the S1：Fructus lycii 500-650kg, pineapple 200-300kg, strawberry 200-300kg, tomato 100-
150kg, brown granulated sugar 5-10kg.
Raw material is in the S1：Fructus lycii 550-650kg, pineapple 150-200kg, strawberry 200-400kg, tomato 100-
200kg, brown granulated sugar 10-15kg.
Raw material is in the S1：Fructus lycii 500-650kg, pineapple 300-350kg, strawberry 100-200kg, tomato 100-
150kg, brown granulated sugar 15-25kg.
Raw material is in the S1：Fructus lycii 500kg, pineapple 200kg, strawberry 200kg, tomato 100kg, brown granulated sugar 5kg.
Using fructus lycii probiotics ferment made from method made above.
The progress of fructus lycii probiotics enzyme stoste is filling to obtain finished product.
Filling condition：Control Cleanliness reaches 100,000 grades of prescribed requirements, and shop worker must wash before entering workshop
Hand changes one's clothes, sterilizes, and is strictly operated by the operating instruction of bottle placer and Cover whirling Machine, filling check at any time in filling progress
Amount, the links such as seal quality, it is ensured that product complies with standard technical requirements.Equipment and pipeline are carried out afterwards before filling the container thoroughly clear
It washes and sterilizes；Filling is bottled, bottle and bottle cap cleaning：Bottle first uses pure water reverse-flush, then is blown with clean compressed air
Dry, lid is carried out disinfection processing with disinfection cabinet, guarantees to reach sterility requirements.
It is preferred that following technical scheme：In S2, fermenting bucket is 850, and each 1200 liters of fermentation ladle capacity is (every time
Raw material total amount 1000kg or so is added), electronic scale 2,1 ton of every fermenter charge, fermentation temperature is room temperature, and workshop seals,
Keep clean, normal temperature fermentation, fermentation time is 24 months.
In S5, standing purifying tank is 50, and each standing purifying tankage size is 30 tons, the use of material is food-grade modeling
Expect material.
When filling, the model TD-8 of bottle placer, the quantity of bottle placer is 2 sets；The model TD-SP1 of Cover whirling Machine, spiral cover
The quantity of machine is 2 sets, and filling speed is 2000 bottles/hour, and bottle device is 30ml vial.
When bottle and bottle cap clean, bottle device 2 are managed；Full-automatic inverting bottle cleaning machine 2, the model of full-automatic inverting bottle cleaning machine
For CP-15；Disinfection cabinet 1, the model ZTP-390 of disinfection cabinet.
Compared with prior art, the method have the benefit that：
The present invention using fructus lycii, pineapple, strawberry, tomato, brown granulated sugar as primary raw material, close in science by the nutritional ingredient of raw material
It is more abundant in the collocation of reason, under the fermentation of probiotics, while the type of strict control probiotics, dosage and hair
Ferment duration guarantees that the fermentation of raw material is more abundant, and fermentation process is divided into three phases and is added respectively according to the characteristic of different phase
Enter different strains, fermentation is made to have level more reasonable；Especially secondary fermentation stage, spontaneous fermentation are not further added by sugar
Source can consume in original fermentation liquor remaining sugar, sufficiently consumption carbohydrate in this way, finally make nutriment in raw material, activity
Substance, antioxidant can release completely, finally obtain a kind of nutritional ingredient and are easy to absorption, good mouthfeel, Neng Gouti
The fructus lycii probiotics ferment of immunity is risen, which has effects that promote immunity, increases the functionality and valence of product
Value.
Fructus lycii probiotics ferment produced by the present invention is free of any food additives, safe and healthy, has no toxic side effect, can
It trusts and enjoys, be suitable for various people.
A kind of fructus lycii probiotics ferment provided by the invention and its preparation process improve existing ferment using raw material
Add ferment fermentation efficiency caused by the production method of water by fermentation or fruit juice fermentation low, active material, nutrient composition content are low,
And the technical problems such as health-care effect is not significant.
Specific embodiment
Illustrate a specific embodiment of the invention below with reference to embodiment, but following embodiment is used only to specifically
The bright present invention, does not limit the scope of the invention in any way.
Raw material is added before fermenter through over cleaning, drying, ultraviolet disinfection, crushing in following embodiment.
Embodiment 1：A kind of preparation method of fructus lycii probiotics ferment, includes the following steps：
Fructus lycii 500kg, pineapple 200kg, strawberry 200kg, tomato 100kg, brown granulated sugar 5kg are added in fermenter S1；
S2 continuously adds saccharomycete 5g and lactobacillus fermenti 15g into fermenter and carries out just fermentation, fermentation process
Fermentation time is 7-9 months；Lactobacillus acidophilus 15g, lactobacillus bulgaricus 5g are just added after fermentation to carry out
Second of fermentation, fermentation time 8 months；Bifidobacterium lactis 10g, streptococcus thermophilus 5g, acetic acid is added after fermentation for the second time
Bacterium 5g continues third time fermentation 8 months；
After fermentation, detection pH stablizes at 3.0, alcoholic strength < 0.5% S3, and fermentation is completed；
The fermentation liquid that fermentation is completed is filtered by S4；
S5 stands purifying：By filtered fermentation liquid stand purify, when alcoholic strength < 0.5%, lactic acid bacteria sum >=1 ×
106It purifies and completes when CFU/ml, obtain fructus lycii probiotics enzyme stoste.
Embodiment 2：A kind of preparation method of fructus lycii probiotics ferment, includes the following steps：
Fructus lycii 550kg, pineapple 150kg, strawberry 150kg, tomato 150kg, brown granulated sugar 15kg are added in fermenter S1；
S2 continuously adds saccharomycete 3g and lactobacillus fermenti 13g into fermenter and carries out just fermentation, fermentation process
Fermentation time is 7 months；Lactobacillus acidophilus 13g, lactobacillus bulgaricus 3g are just added after fermentation and carries out the
Secondary fermentation, fermentation time 9 months；Bifidobacterium lactis 8g, streptococcus thermophilus 3g, acetic acid bacteria is added after fermentation for the second time
3g continues third time fermentation 7 months；
After fermentation, detection pH stablizes at 2.6, alcoholic strength < 0.5% S3, and fermentation is completed；
The fermentation liquid that fermentation is completed is filtered by S4；
S5 stands purifying：By filtered fermentation liquid stand purify, when alcoholic strength < 0.5%, lactic acid bacteria sum >=1 ×
106It purifies and completes when CFU/ml, obtain fructus lycii probiotics enzyme stoste.
Embodiment 3：A kind of preparation method of fructus lycii probiotics ferment, includes the following steps：
Fructus lycii 650kg, pineapple 150kg, strawberry 100kg, tomato 100kg, brown granulated sugar 10kg are added in fermenter S1；
S2 continuously adds saccharomycete 1g and lactobacillus fermenti 15g into fermenter and carries out just fermentation, fermentation process
Fermentation time is 9 months；Lactobacillus acidophilus 10g, lactobacillus bulgaricus 5g are just added after fermentation and carries out the
Secondary fermentation, fermentation time 7 months；Bifidobacterium lactis 5g, streptococcus thermophilus 5g, acetic acid bacteria is added after fermentation for the second time
1g continues third time fermentation 9 months；
After fermentation, detection pH stablizes at 3.5, alcoholic strength < 0.5% S3, and fermentation is completed；
The fermentation liquid that fermentation is completed is filtered by S4；
S5 stands purifying：By filtered fermentation liquid stand purify, when alcoholic strength < 0.5%, lactic acid bacteria sum >=1 ×
106It purifies and completes when CFU/ml, obtain fructus lycii probiotics enzyme stoste.
Embodiment 4：A kind of preparation method of fructus lycii probiotics ferment, includes the following steps：
Fructus lycii 500kg, pineapple 150kg, strawberry 250kg, tomato 100kg, brown granulated sugar 25kg are added in fermenter S1；
S2 continuously adds saccharomycete 5g and lactobacillus fermenti 1g into fermenter and carries out just fermentation, fermentation process
Fermentation time is 9 months；Lactobacillus acidophilus 10g, lactobacillus bulgaricus 5g are just added after fermentation and carries out the
Secondary fermentation, fermentation time 7 months；Bifidobacterium lactis 10g, streptococcus thermophilus 1g, acetic acid bacteria is added after fermentation for the second time
5g continues third time fermentation 7 months；
After fermentation, detection pH stablizes at 3.2, alcoholic strength < 0.5% S3, and fermentation is completed；
The fermentation liquid that fermentation is completed is filtered by S4；
S5 stands purifying：By filtered fermentation liquid stand purify, when alcoholic strength < 0.5%, lactic acid bacteria sum >=1 ×
106It purifies and completes when CFU/ml, obtain fructus lycii probiotics enzyme stoste.
After embodiment 1-4 obtains fructus lycii probiotics enzyme stoste, microbiological indicator, again need to be detected by national standard GB/T31121
Metal indicator is up to standard, other indexs reach professional standard T/CBFIA08003-2016, and then fructus lycii probiotics enzyme stoste passes through
Pipe special is delivered to racking room, filling to obtain fructus lycii probiotics ferment finished product.
Effect experiment：Fructus lycii probiotics ferment promotes immunity verifying
Stomach-filling in 30 days is carried out to mouse using fructus lycii probiotics ferment made of embodiment 1-4, carries out the change of mouse delayed
State reacts (DTH), and ConA inducing mouse Splenic vein hemodynamics test (mtt assay), serum hemolysin measurement, and antibody tormation is thin
Born of the same parents' experiment, the experiment of mouse carbonic clearance and Turnover of Mouse Peritoneal Macrophages phagocytosis chicken red blood cell experiment.Wherein the indices of mouse are all
It makes moderate progress, it was confirmed that fructus lycii probiotics ferment can change in terms of humoral immunity, cellular immunity and mononuclear phagocyte system
Kind immunity of organisms.
Experimental principle：
The immunity function of body includes cellular immune function, humoral immune function, mononuclear macrophage function etc.
Whether aspect has the function of strengthen immunity come judgement sample by the variation of immune system after detection zoopery.
Experimental method and step
1, sample treatment and mode is given
(1) mouse is given in stomach-filling；
2, zoopery
(1) animal selects：With inbred mouse, 25g ± 2g, single gender, every group 20.
(2) dosage is grouped：Experiment sets four experimental groups and a blank control group, control group using physiological saline stomach-filling,
Experimental group 1-4 uses fructus lycii probiotics ferment stomach-filling obtained in embodiment 1-4 respectively, and stomach-filling amount, mode are consistent.
(3) sample gives the time：The edible full nutrition mixed feed of all experimental animals, animal freely ingest, take the photograph
Water.The stomach-filling volume for giving tested material is 20ml/ (kgBw).Given the test agent gives the time 30 days.
3, Indexs measure
(1) the mouse spleen lymphocyte transformation experiment (mtt assay) of ConA induction
1. preparation of reagents
A, complete culture solution：RPMI1640 culture solution filtration sterilization, with 10% calf serum of preceding addition, 1% glutamine
(200mmol/L), penicillin (100U/ml), streptomysin (100 μ g/L) and 5 × 10-52 mercaptoethanols of mol/L, with sterile
The NaOH tune pH to 7.0~7.2 of the HCl or 1mol/L of 1mol/L, i.e. complete culture solution.
B, ConA liquid：The solution of 100 μ g/ml is configured to distilled water, filtration sterilization is protected in low temperature refrigerator (- 20 DEG C)
It deposits.
C, sterile Hank ' s liquid：With preceding with 3.5% sterile NaHCO3 tune pH7.2~7.4.
D, MTT liquid：5mg MTT is dissolved in the PBS of 1ml pH7.2, it is ready-to-use.
E, acid isopropyl alcoholic solution：The HCl of 4ml 1mol/L, prepared before use are added in 96ml isopropanol.
2. prepared by splenocyte suspension：It is sterile to take spleen, it is placed in and fills in appropriate sterile Hank ' s liquid plate, gently with tweezers
Spleen is ground, individual cells suspension is made.Spleen is ground through 200 mesh net filtrations, or with 4 layers of gauze, washes 2 with Hank ' s liquid
It is secondary, it is centrifuged 10min (1000r/min) every time.Then cell is suspended in 1ml complete culture solution, with platform phenol indigo plant dyeing counting
Viable count (should be 95% or more), adjustment cell concentration are 3 × 106A/ml.
3. lymphproliferation response：It is divided to two holes to be added in 24 well culture plates cell suspension, every hole 1ml, a hole adds 75
μ l ConA liquid (is equivalent to 7.5 μ g/ml), and another hole sets 5%CO2, cultivate 72h in 37 DEG C of CO2 incubators as control.Culture
4h before terminating, every hole gently suck supernatant 0.7ml, the RPMI1640 culture solution that 0.7ml is free of calf serum are added, simultaneously
50 hole μ l/ MTT (5mg/ml) is added, continues to cultivate 4h.After culture, 1ml acid isopropyl alcohol is added in every hole, and piping and druming mixes,
It is completely dissolved purple crystal.Then it is dispensed into 96 well culture plates, each hole dispenses 3~6 holes as Duplicate Samples, and use is enzyme-linked
Immune detector measures OD value with 570nm wavelength.
Influence of the fructus lycii probiotics ferment to the ConA mouse spleen lymphocyte conversion capability induced
Note：Compared with the control group,*P < 0.05
(2) dinitrofluorobenzene inducing mouse delayed allergy (DTH)
1. the preparation of DNFB solution：DNFB solution answers Fresh, weighs DNFB 50mg, sets in the dry bottle of cleaning,
By the 5ml acetone sesame oil solution prepared in advance, (acetone: sesame oil=1: 1) pouring into bottle, covers the sealing of bottle stopper blend compounds cloth.It is mixed
After even, taken with 250 μ l syringes by bottle cap.
2. sensitization：Every mouse skin of abdomen is lost hair or feathers with barium sulphide, range about 3cm × 3cm, is uniformly applied with 50 μ l of DNFB solution
Smear sensitization.
3. the generation and measurement of DTH：After 5 days, mouse right ear (two sides) is uniformly applied to 10 μ l of DNFB solution and is attacked
It hits.Cervical dislocation puts to death mouse for 24 hours after attack, cuts left and right auricular concha.The auricle of diameter 8mm is removed with punch, is weighed.
Influence of the fructus lycii probiotics ferment to mouse delayed allergy (DTH)
Note：Compared with the control group,*P < 0.05
(3) antibody-producting cell detection (Jerne improves slide method)
①SRBC：Sheep blood is put into the sterilizing conical flask of bead, shakes in one direction by sheep taking blood from jugular vein
It is dynamic, it to take off fiber, is put into 4 DEG C of refrigerators and saves backup, can be reserved for 2 weeks.
2. preparing complement：Guinea pig blood is acquired, serum (pooled serum of at least 5 cavys) is isolated, by 1ml hematocrit
SRBC is added in 5ml guinea pig serum, and 4 DEG C of refrigerators place 30min, often vibrates, centrifuging and taking supernatant, packing, -70 DEG C of preservations.With
When with SA liquid by 1: 8~15 dilution.
3. slide film：Painting a thin layer agarose on cleaning slide, (0.5g agarose adds distilled water to 100ml, adds
Heat of solution), after dry film releasing box can long-term preservation it is spare.
4. immune animal：Sheep blood is taken, with brine 3 times, is centrifuged (2000r/min) 10min every time, is counted thin
Born of the same parents, every mouse is through abdominal cavity or intravenous injection SRBC 5 × 107~2 × 108It is a.Hematocrit SRBC can also be made into physiological saline
0.2ml is injected intraperitoneally in the cell suspension of 2% (V/V), every mouse.
5. prepared by splenocyte suspension：Mouse cervical dislocation after SRBC is immunized 4~5 days is put to death, and is taken out spleen, is placed on
In the small plate for filling Hank ' s liquid, spleen is gently torn up, cell suspension is made, through 200 mesh net filtrations, or with 4 layers of gauze
Spleen is ground, (1000r/min) 10min is centrifuged, is washed 2 times with Hank ' s liquid, cell is finally suspended in 5ml RPMI1640 training
In nutrient solution, cell is counted, and cell concentration is adjusted to 5 × 106A/ml.Cell can also be suspended in 8ml Hank ' s liquid,
Measure spleen plaque test number.
6. the measurement of plaque：After surface layer culture medium (1g agarose adds distilled water to 100ml) is dissolved by heating, 45 DEG C are put
Water-bath heat preservation, mix with Hank ' the s liquid of equivalent pH7.2~7.4,2 times of concentration, packing small test tube, every pipe 0.5ml, then to pipe
Interior plus 50 μ l 10%SRBC (V/V is prepared with SA liquid), 20 μ l splenocyte suspensions (5 × 106A/ml) or 25 μ l splenocytes it is outstanding
Liquid mixes rapidly, is poured on the slide of brush agarose thin layer, does parallel plate, after agar solidification, slide level is buckled
It is placed on horse, is put into 1~1.5h of incubation in carbon dioxide incubator, is then added to the diluted complement of SA buffer (1: 8)
In glass frame groove, after continuing 1~1.5h of incubation, hemolysis plaque number is counted.
Influence of the fructus lycii probiotics ferment to mouse antibodies cellulation number
Note：Compared with the control group,**P < 0.001
(4) measurement (Hemagglutination Method) of serum hemolysin
①SRBC：Blood is put into the sterilizing conical flask of bead, shakes in one direction by sheep taking blood from jugular vein,
To take off fiber, it is put into 4 DEG C of refrigerators and saves backup.
2. immune animal and serum separation：Sheep blood is taken, with brine 3 times, is centrifuged (2000r/min) every time
10min.Hematocrit SRBC is made into the cell suspension of 2% (V/V) with physiological saline, every mouse intraperitoneal injection 0.2ml is exempted from
Epidemic disease.After 4~5 days, extracts eyeball and take blood in centrifuge tube, place about 1h, solidification blood and tube wall are removed, analyse serum sufficiently
Out, 2000r/min is centrifuged 10min, collects serum.
3. agglutinating reaction：With physiological saline by serum doubling dilution, the serum of different dilutions is respectively placed in Trace Blood
In solidifying experimental plate, every 100 μ l of hole adds the SRBC suspension of 100 μ l 0.5% (V/V), mixes, is packed into wet square position
It covers, in 37 DEG C of incubation 3h, observes hemagglutination degree.
Serum agglutination degree is generally divided into 5 grades (0~IV) records, and antibody product is calculated as follows, and test sample group resists
Volume number is significantly higher than the antibody level of control group, can determine that this experimental result positive.
Antibody level=(S1+2S2+3S3 ...+nSn)
1,2,3 ... n represent the index of two-fold dilution in formula, and S represents the rank of agglutination degree, and antibody product is bigger, indicate
Serum antibody is higher.
0 grade：Red blood cell all sinks, and concentrates on hole bottom and forms fine and close round point shape, surrounding liquid clear.
I grade：Red blood cell is largely deposited on bottom hole into round point shape, and surrounding has the red blood cell being aggregated on a small quantity.
II grade：The red blood cell of agglutination forms thin layer in bottom hole, and center can obviously see a loose red dot.
III grade：The uniform shakedown of the red blood cell of agglutination is dispersed in bottom hole into a thin layer, and may be seen indistinctly a small red dot at center.
IV grade：The uniform shakedown of the red blood cell of agglutination is dispersed in bottom hole into a thin layer, and grumeleuse is sometimes at convolution shape.
Influence of the fructus lycii probiotics ferment to mice serum hemolysin
Note：Compared with the control group,**P < 0.001
5) mouse carbonic clearance is tested
1. solution is prepared
A, injection prepared Chinese ink：By india ink stoste with 3~4 times of normal saline dilution.
B, Na2CO3 solution takes 0.1g Na2CO3, adds distilled water to 100ml.
2. injecting prepared Chinese ink：It weighs, diluted india ink is injected from mouse tail vein, based on every 10g weight 0.1ml
It calculates.It is injected to prepared Chinese ink, timing immediately.
3. measuring：2,10min after injection prepared Chinese ink, take 20 μ l of blood from angular vein clump respectively, exist side by side and be added into 2ml
In 0.1%Na2CO3 solution.With 721 type spectrophotometers at 600nm wavelength densitometric value (OD), with Na2CO3 solution
Make blank control.Mouse is put to death, liver and spleen are taken, blots organ surface blood stains with filter paper, is weighed.Based on following equation
Calculate phagocytic indexIn formula：
Fructus lycii probiotics ferment is to the influence of mouse monokaryon-macrophage carbonic clearance
(6) Turnover of Mouse Peritoneal Macrophages phagocytosis chicken red blood cell experiment
1. prepared by chicken erythrocyte suspension：It takes chicken blood to be placed in the conical flask of bead, sufficiently shakes in one direction,
To take off fiber.With brine 2~3 times, it is centrifuged (2000r/min, 10min), removes supernatant, be made into physiological saline
The chicken erythrocyte suspension of 20% (V/V).
2. detection of phagocytic function：20% chicken erythrocyte suspension 1ml is injected intraperitoneally in every mouse.It is spaced 30min, at cervical dislocation
Dead animal is faced upward position and is fixed on mouse plate, and abdominal skin is cut off in center, through Intraperitoneal injection physiological saline 2ml, rotates mouse plate
1min.Then abdominal cavity washing lotion 1ml is sucked out, average mark is dripped on 2 glass slides, is put into the enamel box for being lined with wet gauze, dislocation
37 DEG C of incubators incubate 30min.It incubates complete, is rinsed in physiological saline, to remove non-patch cell.It dries, with 1: 1 acetone methanol
Solution is fixed, and 4% (V/V) Giemsa phosphate buffer dyes 3min, then is dried with distilled water rinsing, is seen under every piece oil mirror
100 macrophages are examined, phagocytic percentage and phagocytic index is calculated as follows：Phagocytic percentage (%)=phagocytosis chicken red blood cell
Macrophage counting number 100 macrophage × 100, the 100 of the total counting number of phagocytic index=phagocytosis chicken red blood cell
A macrophage：When counting, the degree that chicken red blood cell is digested should be observed simultaneously.So as to determining macrophage phagocytosis and disappearing
Change function, is generally divided into 4 grades：
I grade：It does not digest.The chicken red blood cell swallowed is complete, and cytoplasm is pale red or pale yellow band green, karyon lilac.
II grade：Slight digestion.Cytoplasm chartreuse, nuclear pyknosis are in hyacinthine.
III grade：Severe digestion.The light dye of cytoplasm, the light light gray of karyon.
IV grade：Digestion completely.Vacuole of rarely seen form similar to chicken red blood cell size, neat in edge, karyon in macrophage
It mays be seen indistinctly.
Influence of the fructus lycii probiotics ferment to Turnover of Mouse Peritoneal Macrophages phagocytosis chicken red blood cell ability
Note：Compared with the control group,*P < 0.05
(7) NK cytoactive detection (determination of lactate dehydrogenase method)
1. the preparation of lactate dehydrogenase L DH matrix liquid
Lithium lactate 5 × 10-2mol/L
Nitro tetrazolium chloride (INT) 6.6 × 10-4mol/L
Phenazine Dimethyl Sulfate (PMS) 2.8 × 10-4mol/L
Oxidized coenzyme I (NAD) 1.3 × 10-3mol/L
Mentioned reagent is dissolved in the TrisHCl buffer of 0.2mol/L (pH8.2)
2. the passage (YAC1 cell) of target cell：Target cell is subjected to secondary culture for 24 hours before experiment.Using preceding with Hank '
S liquid is washed 3 times, is 4 × 10 with RPMI1640 complete culture solution adjustment cell concentration5A/ml.
3. the preparation (effector cell) of splenocyte suspension：It is sterile to take spleen, it is placed in and fills the small flat of appropriate sterile Hank ' s liquid
In ware, gently spleen is ground with tweezers, single cell suspension is made.Spleen is ground through 200 mesh net filtrations, or with 4 layers of gauze,
Or washed 2 times with Hank ' s liquid, it is centrifuged 10min (1000r/min) every time.It abandons supernatant to bounce cytoplasm, 0.5ml sterilizing is added
Water 20s, adds 2 times of Hank ' s liquid of 0.5ml and 8ml Hank ' s liquid after splitting erythrocyte, 1000r/min, 10min centrifugation,
RPMI1640 complete culture solution with 1ml containing 10% calf serum is resuspended, and with counting after the dilution of 1% glacial acetic acid, (viable count is answered
95% or more), with platform phenol orchid dyeing counting viable count (should be 95% or more), finally use RPMI1640 complete culture solution
Adjusting cell concentration is 2 × 107A/ml.
4. NK cytoactive detection：Target cell and each 100 μ l of effector cell (effect target is than 50: 1) are taken, the training of 96 hole of U type is added
It supports in plate；Target cell Spontaneous release hole adds target cell and each 100 μ l of culture solution, and target cell maximum relief hole adds target cell and 1%
Each 100 μ l of NP40 or 2.5%Triton；Above-mentioned items are all provided with three multiple holes, cultivate 4h in 37 DEG C, 5%CO2 incubator, so
96 well culture plates are centrifuged 5min with 1500r/min afterwards, every hole is drawn in 100 μ l horizontalization bottom of supernatant, 96 well culture plate, is added simultaneously
Enter 100 μ l of LDH matrix liquid, react 3min, the 30 μ l of HCl of 1mol/L is added in every hole, and optical density is measured at microplate reader 490nm
It is worth (OD).NK cell activity is calculated as follows：
Fructus lycii probiotics ferment is on the active influence of NK cells in mice
This experimental result surface, the fructus lycii probiotics ferment of the embodiment 1-4 through stomach-filling 20ml/ (kgBw) dosage, energy
Spleen lymphocyte proliferation, the transformation for promoting mouse, promote the delayed allergy of mouse, promote the antibody of mouse raw
At cell Proliferation, the serum hemolysin of mouse is improved, the weight of mouse is increased, the phagocytosis function of monocytes/macrophages
Energy and NK cell activity have no significant effect, it was demonstrated that fructus lycii probiotics ferment made from embodiment 1-4 has the function of strengthen immunity
Effect, wherein all data shows that fructus lycii probiotics ferment strengthen immunity obtained effect is most strong in embodiment 1.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, this field is common
Other modifications or equivalent replacement that technical staff makes technical solution of the present invention, without departing from the technology of the present invention side
The spirit and scope of case, are intended to be within the scope of the claims of the invention.
Claims (8)
1. a kind of preparation method of fructus lycii probiotics ferment, which is characterized in that include the following steps：
S1 is by fructus lycii 500-650kg, pineapple 150-350kg, strawberry 100-400kg, tomato 100-200kg, brown granulated sugar 5-25 kg
It is added in Zymolysis Equipment；
S2 continuously adds saccharomycete 1-5g and lactobacillus fermenti 10-15g into Zymolysis Equipment and carries out just fermentation, fermentation time 7-
9 months；Lactobacillus acidophilus 10-15g, lactobacillus bulgaricus 1-5g are just added after fermentation and carries out second of fermentation, fermentation
Time 7-9 month；Bifidobacterium lactis 5-10g, streptococcus thermophilus 1-5g, acetic acid bacteria 1-5g is added after fermentation for the second time to continue
Third time fermentation 7-9 months；
S3 after fermentation, obtains fructus lycii probiotics ferment；PH is detected to stablize between 2.6-3.5, when alcoholic strength < 0.5%, hair
Ferment is completed；
The fermentation liquid that fermentation is completed is filtered by S4；
S5 stands purifying：Filtered fermentation liquid is stood and is purified, as alcoholic strength < 0.5%, lactic acid bacteria sum >=1 × 106CFU/
It purifies and completes when ml, obtain fructus lycii probiotics enzyme stoste.
2. the preparation method of fructus lycii probiotics ferment according to claim 1, it is characterised in that：Just ferment in the S2,
When second of fermentation and third time are fermented, isolation air carries out anaerobic fermentation.
3. the preparation method of fructus lycii probiotics ferment according to claim 1, it is characterised in that：Zymolysis Equipment is fermentation
Bucket.
4. the preparation method of fructus lycii probiotics ferment according to claim 1, it is characterised in that：Raw material is in the S1：
Fructus lycii 500-650 kg, pineapple 200-300kg, strawberry 200-300 kg, tomato 100-150 kg, brown granulated sugar 5-10 kg.
5. the preparation method of fructus lycii probiotics ferment according to claim 1, it is characterised in that：Raw material is in the S1：
Fructus lycii 550-650 kg, pineapple 150-200 kg, strawberry 200-400 kg, tomato 100-200 kg, brown granulated sugar 10-15 kg.
6. the preparation method of fructus lycii probiotics ferment according to claim 1, it is characterised in that：Raw material is in the S1：
Fructus lycii 500-650 kg, pineapple 300-350 kg, strawberry 100-200 kg, tomato 100-150 kg, brown granulated sugar 15-25 kg.
7. the preparation method of fructus lycii probiotics ferment according to claim 1, it is characterised in that：Raw material is in the S1：
500 kg of fructus lycii, 200 kg of pineapple, 200 kg of strawberry, 100 kg of tomato, 5 kg of brown granulated sugar.
8. using fructus lycii probiotics ferment made from any one of claim 1-7 preparation method.
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| CN112106909A (en) * | 2019-06-21 | 2020-12-22 | 百岳特生物技术（上海）有限公司 | Ginseng and medlar fermentation liquor and preparation method thereof |
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| CN106578794A (en) * | 2016-12-01 | 2017-04-26 | 李恒 | Fermented fruit and vegetable juice beverage preparation process |
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| CN106578794A (en) * | 2016-12-01 | 2017-04-26 | 李恒 | Fermented fruit and vegetable juice beverage preparation process |
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Application publication date: 20181123 |