CN110464003A - A kind of anti-oxidation function food of probiotics solid state fermentation and preparation method thereof - Google Patents

A kind of anti-oxidation function food of probiotics solid state fermentation and preparation method thereof Download PDF

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CN110464003A
CN110464003A CN201910879857.8A CN201910879857A CN110464003A CN 110464003 A CN110464003 A CN 110464003A CN 201910879857 A CN201910879857 A CN 201910879857A CN 110464003 A CN110464003 A CN 110464003A
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fermentation
solid
probiotics
lactobacillus
state fermentation
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韩雪梅
李树森
王飞
王海宽
梁武
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Tianjin Inncorigin Biological Technology Co ltd
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Tianjin Inncorigin Biological Technology Co ltd
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    • 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
    • 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
    • A23V2400/117—Animalis
    • 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
    • A23V2400/165—Paracasei
    • 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
    • A23V2400/169—Plantarum
    • 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
    • A23V2400/173—Reuteri
    • 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
    • A23V2400/41—Pediococcus
    • A23V2400/413—Acidilactici
    • 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
    • A23V2400/513—Adolescentes
    • 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
    • A23V2400/533—Longum

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  • Life Sciences & Earth Sciences (AREA)
  • Mycology (AREA)
  • Health & Medical Sciences (AREA)
  • Nutrition Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The present invention provides a kind of anti-oxidation function food of probiotics solid state fermentation, be using sea-buckthorn, jujube, lens, soybean mixture as fermentation substrate, solid-state stepwise fermentation is carried out using lactobacillus reuteri, bifidobacterium longum, Pediococcus acidilactici, lactobacillus plantarum, animal bifidobacteria, lactobacillus paracasei, bifidobacterium adolescentis, bacillus coagulans and bacillus subtilis and is prepared.The functional food has anti-aging, toxin-expelling and face nourishing, freckle removing and whitening and other effects.

Description

A kind of anti-oxidation function food of probiotics solid state fermentation and preparation method thereof
Technical field
The invention belongs to microbial fermentation application field more particularly to a kind of anti-oxidation functions of probiotics solid state fermentation Food and preparation method thereof.
Background technique
Aging is that most biologies increase with the age and slowly occur, generally occur, irreversible degenerative process, is Body moves towards the inevitable step of natural death, is old pathogenetic common risk factors.With scientific and technological progress and living condition Improve, countries in the world aging of population constantly aggravates, it has also become important common problem.The precise mechanism that aging generates, it is related Causes of senescence successively has as many as tens of kinds, such as free radical theory, mitochondrial DNA damage theory, tolemerase, cross linkage theory, life Object membrane damage theory, genetic program theory, chromosome mutation theory, immunological theory, endocrine theory, Apoptosis theory etc.. At present to Aging mechanism most study and compare receiving is free radical theory, mitochondrial DNA damage theory and tolemerase.
Free radical theory is thought: organism metabolism constantly generates free radicals, while the Free-radical ring opening polymerization of internal body In constantly cleaning free radical；With the aging of people, intracorporal removing system cannot remove free radical in time, and excessive free radicals can produce Raw peroxidating cell membrane and nucleic acid, protein and other, cause peroxidatic reaction of lipid, and a series of metabolism of generation produce Object will affect the activity of the transmitting expression and enzyme of DNA, so as to cause cell ageing apoptosis.Mitochondrial DNA damage theory thinks: leading Another reason for causing cell ageing, denaturation, leakage and the rupture of mitochondria have with cell ageing and death directly to be contacted, and is reduced The damage process of mitochondrial DNA, may extend cell survival, to extend the service life of body.Tolemerase is thought, eukaryon is located at The telomere of biological stain body end can be gradually shortened with the increase of frequency dividing cell, after division reaches a certain level, Telomere, which will start related mechanism, leads to cell ageing apoptosis.
The reason of aging and mechanism are complicated, can explain all aging phenomenons there are no a kind of theory so far, also not have The reason of a kind of theory can be fully contemplated by and illustrate aging and mechanism.Gene function disorder, albumen homeostasis and nutrition generation It thanks to the Multiple factors such as signal changes, injury of mitochondria and stem cell are exhausted, cell ageing and inflammatory aging to participate in and act on jointly, Various mechanism interact, and jointly promote the aging of body, and skin is as the maximum organ of human body, undertake protection, feeling, The effects of secretion, excretion, body heat regulation, immunological regulation, increases, the also gradually aging as other organs with the age.Skin Aging major influence factors are divided into internal cause (such as age, gene mutation, cell metabolism, hormonal milieu) and external cause (such as chemistry Substance, toxin, environmental pollution, ultraviolet light and ion irradiation).Wherein, it is because of solar radiation that external cause, which causes skin aging mostly,.Skin Skin aging show as wrinkle and pigementation increases, cutis laxa is coarse, nonelastic.Ageing skin it is exposed, become human body The external presentation of aging is easiest to discover during body aging, and the most obvious with face, and the psychology for aggravating people is negative Load.So how effectively to remove the free radical of body metabolism generation, vivotoxin is excluded, reasonable supplement nutriment delays skin Skin aging, appearance of remaining youthful forever are the topics of all trades and professions of all times all extensive concerns, and anti-aging product has wide city , instantly with anti-senescence function food patent mainly there are several types of.
A kind of anti-senescence function food of the disclosure of the invention of publication number 106722434A, which is characterized in that comprising as follows Active component: European blueberry extract, water-soluble haematococcus pluvialis, olive fruit powder and acerola concentrate concentrate, the Europe are blue Anthocyanin containing weight fraction 35% in certain kind of berries extract, it is described water solubility haematococcus pluvialis in the shrimp containing weight fraction 2% Blueness is plain, the hydroxytyrosol containing weight fraction 1.5% in the olive fruit powder, contains weight point in the acerola concentrate concentrate The natural vitamin C of number 17%.
A kind of Amti-sanility health-care food of the disclosure of the invention of publication number 107927738A, which is characterized in that it includes following The active principle of parts by weight: 10~25 parts of Cordyceps militaris powder, 3~10 parts of astaxanthin, 15~30 parts of ginseng, 5~20 parts of lycopene, 30~60 parts of procyanidine, 20~50 parts of maca powder.
The disclosure of the invention of the publication number 108294155A preparation method of anti-aging blueberry dried fruit, which is characterized in that including Following steps: S1 sugar system；S2 elution；S3 draining；S4 is put；S5 drying；S6 selects shaping；S7 secondary drying；S8 selects high-quality Dried fruit；S9 sorted and packaged；S10 storage.
The disclosed above-mentioned prior art is mostly various nature foods and each nutrient compounding for antisenility cistanche food It forms, the generally simple processing mixing of preparation method prepares such functional food using probiotics solid state fermentation and has not been reported.
Summary of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide a kind of husky using probiotics solid-state stepwise fermentation The raw materials such as spine, jujube, lens, soybean production functional food, the functional food have anti-aging, toxin-expelling and face nourishing, Freckle removing and whitening and other effects.
To achieve the above object, technical scheme is as follows:
A kind of anti-oxidation function food of probiotics solid state fermentation, is with the mixing of sea-buckthorn, jujube, lens, soybean Object is as fermentation substrate, using lactobacillus reuteri, bifidobacterium longum, Pediococcus acidilactici, lactobacillus plantarum, animal bifid bar Bacterium, lactobacillus paracasei, bifidobacterium adolescentis, bacillus coagulans and bacillus subtilis carry out vacuum after solid-state stepwise fermentation Freeze-drying is prepared.
The preparation method of the anti-oxidation function food of the probiotics solid state fermentation, includes the following steps:
(1) good lens, soybean, sea-buckthorn, jujube is chosen to be milled sieve with 100 mesh sieve respectively；
(2) it prepares activation medium: adding 10g soy meal in every 100mL water；It sterilizes to activation medium, condition For, 121 DEG C, 20min；
(3) it prepares fermentation medium: lens powder, soy meal, seabuckthorn fruit powder, jujube powder being uniformly mixed, mixture and water 1:1.5-2.0 in mass ratio prepare solid-state fermentation culture medium, preparation be placed in solid-state fermenter sterilize it is spare；Sterilising conditions: 121 DEG C, 20min；
(4) actication of culture: lactobacillus reuteri, bifidobacterium longum, Pediococcus acidilactici, lactobacillus plantarum, animal bifid bar Bacterium, lactobacillus paracasei, bifidobacterium adolescentis, bacillus coagulans and bacillus subtilis glycerol tube press 1% (v/v) respectively It is inoculated in activation medium to be activated, pH is naturally, controlled at 37 DEG C；After activating for 24 hours respectively, bacterium is connect by 5% (v/v) Switching activation medium carries out re-activation to amount again, and reactivation obtains secondary seed solution afterwards for 24 hours；
(5) probiotics solid-state stepwise fermentation:
1. maintaining temperature 37 in the secondary seed solution access solid-state fermenter of bacillus subtilis, bacillus coagulans DEG C, ventilatory capacity 60L/min, ferment 10-18h；
2. 1. walked after fermentation to the, Pediococcus acidilactici, lactobacillus plantarum secondary seed solution access solid-state fermenter In, 37 DEG C of temperature are maintained, sealed fermenting 12-24h；
3. to 2. walk after fermentation, by animal bifidobacteria, bifidobacterium adolescentis, long Bifidobacterium Bifidum secondary seed Liquid accesses in solid-state fermenter, maintains 37 DEG C of temperature, sealed fermenting 18-36h；
4. 3. walking after fermentation to the, the secondary seed solution of lactobacillus paracasei, lactobacillus reuteri is accessed into solid-state In fermentor, 37 DEG C of temperature are maintained, sealed fermenting 24-48h；
(6) after fermentation ends through vacuum freeze drying to get probiotics fermention functional food.
The lens, soybean, sea-buckthorn, jujube are commercially available.
Preferably, the seabuckthorn fruit powder: jujube powder: lens powder: soy meal mass ratio is 2:3:5:6.
Preferably, the mass ratio of the mixture and water is 1:1.4 when preparing solid-state fermentation culture medium.
Preferably, the bacillus subtilis, bacillus coagulans, Pediococcus acidilactici, lactobacillus plantarum, animal bifid bar Bacterium, bifidobacterium adolescentis, long Bifidobacterium Bifidum, lactobacillus paracasei, lactobacillus reuteri inoculum concentration be respectively 107-108CFU/g Solid-state fermentation culture medium.
It is furthermore preferred that the bacterium amount that connects of above-mentioned each bacterium is respectively 5 × 107CFU/g solid-state fermentation culture medium；
Preferably, the condition of the vacuum freeze drying are as follows: vacuum freeze drying for 24 hours, until water content be not more than 12%.
Preferably, the lactobacillus plantarum is IOB602, has been preserved in Chinese microorganism strain guarantor on June 29th, 2018 Hide administration committee's common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microorganism Research institute, postcode 100101, classification naming: Lactobacillus plantarum, deposit number are CGMCC No.16021. This lactobacillus plantarum has the ability of preferable anti-gastric acid, anti-cholate, the 2h survival rate 86.05% in pH 3,0.3% cholate 4h survival rate 78.04%, and there is extensive antifungal activity, it does not need to add additional preservative, beverage can also be extended With the food preservation time.
Preferably, the animal bifidobacteria is IOB402, is preserved in Chinese microorganism strain on June 29th, 2018 Preservation administration committee common micro-organisms center, address: the micro- life of the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences Object research institute, postcode 100101, classification naming: animal bifidobacteria Bifidobacterium animalis, deposit number are CGMCC No.16028。
Preferably, the lactobacillus paracasei is IOB413, is preserved in Chinese microorganism strain on June 29th, 2018 Preservation administration committee common micro-organisms center, address: the micro- life of the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences Object research institute, postcode 100101, classification naming: lactobacillus paracasei Lactobacillus paracasei, deposit number are CGMCC No.16022。
Preferably, the bifidobacterium adolescentis is IOB506, is preserved in Chinese microorganism strain on June 29th, 2018 Preservation administration committee common micro-organisms center, address: the micro- life of the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences Object research institute, postcode 100101, classification naming: bifidobacterium adolescentis Bifidobacterium adolescentis, preservation are compiled Number be CGMCC No.16026.
Preferably, the bacillus coagulans are I0B502, are preserved in Chinese microorganism strain on June 29th, 2018 Preservation administration committee common micro-organisms center, address are as follows: the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences is micro- Biological study institute, postcode 100101, classification naming: bacillus coagulans Bacillus coagulans, deposit number are as follows: CGMCC No.16025。
Preferably, the bacillus subtilis is IOB430, is preserved in Chinese microorganism strain on June 29th, 2018 Preservation administration committee common micro-organisms center, address are as follows: the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences is micro- Biological study institute, postcode 100101, classification naming: bacillus subtilis Bacillus subtilis, deposit number CGMCC No.16024。
Preferably, the lactobacillus reuteri is IOB423, is preserved in Chinese microorganism strain on June 29th, 2018 Preservation administration committee common micro-organisms center, address are as follows: the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences is micro- Biological study institute, postcode 100101, classification naming: lactobacillus reuteri Lactobacillus reuteri, deposit number are CGMCC No.16023。
Preferably, the bifidobacterium longum is IOB515, has been preserved in Chinese microorganism strain guarantor on June 29th, 2018 Hide administration committee's common micro-organisms center, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the micro- life of the Chinese Academy of Sciences Object research institute, postcode 100101, classification naming: bifidobacterium longum Bifidobacterium longum, deposit number CGMCC No.16027。
Preferably, the Pediococcus acidilactici is IOB701, has been preserved in Chinese microorganism strain guarantor on July 10th, 2018 Hide administration committee's common micro-organisms center, address are as follows: the micro- life of the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences Object research institute, postcode 100101, classification naming: Pediococcus acidilactici pediococcus acidilactici, deposit number are CGMCC No.16077。
The utility model has the advantages that
Sea-buckthorn is warm-natured, sour-puckery flavor, has the effect of relieving cough and reducing sputum, stomach strengthening and digestion promoting, promoting blood circulation to remove blood stasis.Modern pharmacology research table Bright, sea-buckthorn, which has, removes human free radical, improves immunity of organism, anti-aging, antitumor, anticancer, allevating angina pectoris, improves heart function Can, relieving cough and asthma, sharp lung resolving sputum, relieving dyspepsia, anti-inflammatory myogenic, the effect for promoting regeneration etc..Every 100g fructus hippophae Contain 360~798mg of flavone compound in reality；VC content is minimum to contain 800mg, after probiotics is segmented solid state fermentation, Victoria C Content has reached 1646mg, and the VC amount is far more than the amount in common fruit and vegetables.
Jujube pharmacological activity is extensive, and there is anti-oxidant, antitumor, adjusting to be immunized, repair hepatic injury, fatigue-resisting function, Also there is improvement result to hematopoiesis function and intestinal movement etc..After probiotics is segmented solid state fermentation, date polysaccharide is converted into Bacterial polysaccharides, antioxidant effect significantly improve.
Lens protein rich in and vitamin B, in addition iron-content is also 2 times of other beans.Food fiber It is also very abundant with folate content, have and eliminate constipation, prevents heart disease, cancer, anti-aging function.Pigment in dark hyacinth bean There is antioxidant, it can be with anti-aging, prevention heart disease and cancer.After probiotics is segmented solid state fermentation, in lens Procyanidin content increase.
Soybean protein rich in, isoflavones, phytosterol, soyabean oligosaccharides etc., can enhance immunity of organism, It prevents vascular sclerosis, have the effects that hypoglycemic, anti-oxidant, the Flavonoid substances in soybean are segmented solid state fermentation through probiotics Afterwards, the aglycon for being easier to be absorbed by the body is changed by plant glycosidic bond, it is possible to reduce ascorbic loss is assisted with vitamin C Same synergy.
A kind of anti-aging of probiotics solid-state stepwise fermentation of the present invention, toxin-expelling and face nourishing, freckle removing and whitening functions food, Not only the homologous original nutritive value of natural medicine-food, but also the nutrition health-care functions with probiotics fermention product had been saved, it is unique in taste. The food had both made different characteristics probiotics coordinated with each other by probiotics solid-state stepwise fermentation, common to breed, and reached Tiny ecosystem system The standard of agent, and the macromolecular substances contained in natural material can be degraded to the small molecule object for being easier to absorb by probiotics Matter.Importantly, the food preparation process energy conservation and environmental protection, generating without waste water and gas waste material, it is easy to accomplish large-scale production.
Detailed description of the invention
Fig. 1: HE dyeing nude mice skin form (Normal group)；
Fig. 2: HE dyeing nude mice skin form (model control group)；
Fig. 3: HE dyeing nude mice skin form (non-fermented food group)；
Fig. 4: HE dyeing nude mice skin form (probiotics fermention food group)；
Fig. 5: HE dyeing nude mice skin form (positive controls).
Specific embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention Range, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this Under the premise of invention spirit and scope, to the various changes or change of material component and dosage progress in these embodiments It belongs to the scope of protection of the present invention.
Embodiment 1: anti-oxidation function food of probiotics solid state fermentation and preparation method thereof
1, the determination of raw material proportioning
(1) good lens, soybean, sea-buckthorn, jujube is chosen to be milled sieve with 100 mesh sieve respectively；
(2) it prepares activation medium: adding 10g soy meal in every 100mL water；Sterilising conditions: 121 DEG C, 20min；
(3) it prepares fermentation medium: seabuckthorn fruit powder, jujube powder, lens powder, soy meal is mixed by certain weight ratio Even, mixture and water 1:1.5 in mass ratio prepare solid-state fermentation culture medium, be placed in 6T solid-state fermenter sterilize it is spare；Sterilizing Condition: 121 DEG C, 20min；
(4) actication of culture: lactobacillus reuteri, bifidobacterium longum, Pediococcus acidilactici, lactobacillus plantarum, animal bifid bar Bacterium, lactobacillus paracasei, bifidobacterium adolescentis, bacillus coagulans and bacillus subtilis glycerol tube press 1% (v/v respectively The volume ratio of activation medium) it is inoculated in activation medium and is activated, pH is naturally, controlled at 37 DEG C；It activates respectively for 24 hours Afterwards, by 5% (v/v) connect bacterium amount transfer again activation medium carry out re-activation, reactivation for 24 hours afterwards secondary seed solution；
(5) probiotics solid-state stepwise fermentation:
1. the bacillus subtilis that activation is completed and bacillus coagulans access 6T solid-state fermenter by inoculation pipeline In, make the inoculum concentration 10 of bacillus subtilis in every gram of fermentation substrate7-108The inoculum concentration of CFU/g, bacillus coagulans is 107-108CFU/g maintains 37 DEG C of temperature, ventilatory capacity 60L/min, and ferment 10h；
2. 1. walking after fermentation to the, activated Pediococcus acidilactici and lactobacillus plantarum are passed through into inoculation pipeline access In 6T solid-state fermenter, make the inoculum concentration 10 of Pediococcus acidilactici in every gram of fermentation substrate7-108CFU/g, lactobacillus plantarum connect Kind amount is 107-108CFU/g maintains 37 DEG C of temperature, sealed fermenting 12h；
3. 2. being walked after fermentation to, activated animal bifidobacteria, bifidobacterium adolescentis, long Bifidobacterium Bifidum are led to It crosses in inoculation pipeline access 6T solid-state fermenter, makes the inoculum concentration 10 of B. animais in every gram of fermentation substrate7-108CFU/ G, the inoculum concentration of bifidobacterium adolescentis are 107-108CFU/g, the inoculum concentration of long Bifidobacterium Bifidum are 107-108CFU/g maintains temperature 37 DEG C, sealed fermenting 18h；
4. 3. walking after fermentation to the, activated lactobacillus reuteri, lactobacillus paracasei are passed through into inoculation pipeline It accesses in 6T solid-state fermenter, makes the inoculum concentration 10 of lactobacillus reuteri in every gram of fermentation substrate7-108CFU/g, secondary cheese cream The inoculum concentration of bacillus is 107-108CFU/g maintains 37 DEG C of temperature, and sealed fermenting is for 24 hours；
(6) after fermenting, the number of live bacteria of probiotics in fermented sample is detected respectively, the results are shown in Table 1；
The seabuckthorn fruit powder, jujube powder, lens powder, soy meal raw material proportioning label: group unification: 6:5:4:3, combination Two: 6:5:6:5, combination three: 3:6:6:3, combination four: 2:3:5:6, combination five: 3:5:5:4.
Influence of 1 raw material proportioning of table to number of live bacteria of probiotics
Group unification Combination two Combination three Combination four Combination five
Bacillus subtilis 3.3×108 4.5×108 2.1×108 5.8×108 4.1×108
Lactobacillus plantarum 1.7×109 1.6×109 2.3×109 3.6×109 3.1×109
Lactobacillus paracasei 1.8×109 2.4×109 3.3×109 5.1×109 4.2×109
Lactobacillus reuteri 3.3×108 3.5×108 4.9×108 5.1×108 4.7×108
Pediococcus acidilactici 2.2×109 1.4×109 1.8×109 2.6×109 2.1×109
Bifidobacterium longum 4.5×108 4.9×108 5.1×108 5.3×108 3.0×108
Bifidobacterium adolescentis 1.4×107 1.9×107 2.8×107 3.3×107 2.0×107
Animal bifidobacteria 3.2×107 1.4×107 2.5×107 6.9×107 1.3×107
Bacillus coagulans 1.6×108 2.4×108 3.8×108 8.5×108 6.2×108
Total viable count 3.2×108 5.5×108 1.9×109 2.7×109 9.8×108
As shown in Table 1, the optimal proportion of raw material is combination four, as seabuckthorn fruit powder: jujube powder: lens powder: soy meal Mass ratio is 2:3:5:6, and total viable count is 2.7 × 10 after fermentation9CFU/g。
2, the determination of inoculum concentration
(1) good lens, soybean, sea-buckthorn, jujube is chosen to be milled sieve with 100 mesh sieve respectively；
(2) it prepares activation medium: adding 10g soy meal in every 100mL water；Sterilising conditions: 121 DEG C, 20min；
(3) it prepares fermentation medium: seabuckthorn fruit powder, jujube powder, lens powder, soy meal 2:3:5:6 in mass ratio is mixed, Mixture and water prepare solid-state fermentation culture medium according to mass ratio 1:1.5, be placed in 6T solid-state fermenter sterilize it is spare；Sterilize item Part: 121 DEG C, 20min；
(4) actication of culture: lactobacillus reuteri, bifidobacterium longum, Pediococcus acidilactici, lactobacillus plantarum, animal bifid bar Bacterium, lactobacillus paracasei, bifidobacterium adolescentis, bacillus coagulans and bacillus subtilis glycerol tube press 1% (v/v) respectively It is inoculated in activation medium to be activated, pH is naturally, controlled at 37 DEG C；After activating for 24 hours respectively, bacterium is connect by 5% (v/v) Switching activation medium carries out re-activation to amount again, and reactivation obtains secondary seed solution afterwards for 24 hours；
(5) probiotics solid-state stepwise fermentation:
1. the bacillus subtilis that activation is completed and bacillus coagulans access 6T solid-state fermenter by inoculation pipeline In, 37 DEG C of temperature, ventilatory capacity 60L/min are maintained, ferment 10h；
2. 1. walking after fermentation to the, activated Pediococcus acidilactici and lactobacillus plantarum are passed through into inoculation pipeline access In 6T solid-state fermenter, 37 DEG C of temperature are maintained, sealed fermenting 12h；
3. 2. being walked after fermentation to, activated animal bifidobacteria, bifidobacterium adolescentis, long Bifidobacterium Bifidum are led to It crosses in inoculation pipeline access 6T solid-state fermenter, maintains 37 DEG C of temperature, sealed fermenting 18h；
4. 3. walking after fermentation to the, activated lactobacillus reuteri, lactobacillus paracasei are passed through into inoculation pipeline It accesses in 6T solid-state fermenter, maintains 37 DEG C of temperature, sealed fermenting is for 24 hours；
(6) after fermenting, the number of live bacteria of probiotics in fermented sample is detected respectively；
The inoculum concentration difference of each bacterium is equal when the probiotics solid-state stepwise fermentation are as follows: 1 × 107CFU/g；5×107CFU/g；1 ×108CFU/g；5×108Tetra- groups of CFU/g, it the results are shown in Table 2.
Influence (unit: CFU/g) of 2 inoculum concentration of table to number of live bacteria of probiotics
Each bacterium inoculum concentration 1×107 5×107 1×108 5×108
Bacillus subtilis 2.1×108 4.5×108 3.3×108 1.0×108
Lactobacillus plantarum 1.8×109 2.5×109 2.3×109 1.2×109
Lactobacillus paracasei 4.9×109 7.4×109 6.3×109 5.5×109
Lactobacillus reuteri 2.8×108 6.0×108 3.7×108 5.1×108
Pediococcus acidilactici 1.0×109 3.9×109 1.8×109 2.2×109
Bifidobacterium longum 5.3×108 8.7×108 6.2×108 4.1×108
Bifidobacterium adolescentis 1.5×107 2.9×107 2.0×107 2.5×107
Animal bifidobacteria 3.6×107 5.8×107 2.5×107 4.9×107
Bacillus coagulans 3.3×108 6.1×108 5.7×108 4.8×108
Total viable count 3.7×109 9.4×109 6.8×109 5.5×109
As shown in Table 2: optimum inoculation amount is bacillus subtilis, lactobacillus plantarum, lactobacillus paracasei, Luo Yishi cream bar Bacterium, Pediococcus acidilactici, bifidobacterium longum, bifidobacterium adolescentis, animal bifidobacteria, bacillus coagulans are 5 × 107CFU/ G, total viable count is 9.4 × 10 after fermentation9CFU/g。
3, the determination of material-water ratio
(1) good lens, soybean, sea-buckthorn, jujube is chosen to be milled sieve with 100 mesh sieve respectively；
(2) it prepares activation medium: adding 10g soy meal in every 100mL water；Sterilising conditions: 121 DEG C, 20min；
(3) it prepares fermentation medium: seabuckthorn fruit powder, jujube powder, lens powder, soy meal 2:3:5:6 in mass ratio is mixed, It is solid to be placed in 6T according to mass ratio 1:1.5-2.0 preparation solid-state fermentation culture medium (grouping of different ratio is shown in Table 3) for mixture and water It sterilizes in state fermentor spare；Sterilising conditions: 121 DEG C, 20min；
(4) actication of culture: lactobacillus reuteri, bifidobacterium longum, Pediococcus acidilactici, lactobacillus plantarum, animal bifid bar Bacterium, lactobacillus paracasei, bifidobacterium adolescentis, bacillus coagulans and bacillus subtilis glycerol tube press 1% (v/v) respectively It is inoculated in activation medium to be activated, pH is naturally, controlled at 37 DEG C；After activating for 24 hours respectively, bacterium is connect by 5% (v/v) Switching activation medium carries out re-activation to amount again, and reactivation obtains secondary seed solution afterwards for 24 hours；
(5) probiotics solid-state stepwise fermentation:
1. the bacillus subtilis that activation is completed and bacillus coagulans access 6T solid-state fermenter by inoculation pipeline In, 37 DEG C of temperature, ventilatory capacity 60L/min are maintained, ferment 10h；
2. 1. walking after fermentation to the, activated Pediococcus acidilactici and lactobacillus plantarum are passed through into inoculation pipeline access In 6T solid-state fermenter, 37 DEG C of temperature are maintained, sealed fermenting 12h；
3. 2. being walked after fermentation to, activated animal bifidobacteria, bifidobacterium adolescentis, long Bifidobacterium Bifidum are led to It crosses in inoculation pipeline access 6T solid-state fermenter, maintains 37 DEG C of temperature, sealed fermenting 18h；
4. 3. walking after fermentation to the, activated lactobacillus reuteri, lactobacillus paracasei are passed through into inoculation pipeline It accesses in 6T solid-state fermenter, maintains 37 DEG C of temperature, sealed fermenting is for 24 hours；
Above 1. -4. to walk each bacterium inoculum concentration be 5 × 10 in every gram of solid medium7CFU/g；
(6) after fermentation, the number of live bacteria of probiotics in fermented sample is detected respectively, the results are shown in Table 3.
Influence (unit: CFU/g) of 3 material-water ratio of table to number of live bacteria of probiotics
As shown in Table 3, the best material-water ratio for preparing solid-state fermentation culture medium is 1:1.4, at this time number of live bacteria of probiotics highest It is 6.4 × 109CFU/g。
4, the determination of fermentation time
(1) good lens, soybean, sea-buckthorn, jujube is chosen to be milled sieve with 100 mesh sieve respectively；
(2) it prepares activation medium: adding 10g soy meal in every 100mL water；Sterilising conditions: 121 DEG C, 20min；
(3) it prepares fermentation medium: seabuckthorn fruit powder, jujube powder, lens powder, soy meal 2:3:5:6 in mass ratio is mixed, Mixture and water are placed in 6T solid-state hair according to mass ratio 1:1.4 preparation solid-state fermentation culture medium (grouping of different ratio is shown in Table 3) It sterilizes in fermentation tank spare；Sterilising conditions: 121 DEG C, 20min；
(4) actication of culture: lactobacillus reuteri, bifidobacterium longum, Pediococcus acidilactici, lactobacillus plantarum, animal bifid bar Bacterium, lactobacillus paracasei, bifidobacterium adolescentis, bacillus coagulans and bacillus subtilis glycerol tube press 1% (v/v) respectively It is inoculated in activation medium to be activated, pH is naturally, controlled at 37 DEG C；After activating for 24 hours respectively, bacterium is connect by 5% (v/v) Switching activation medium carries out re-activation to amount again, and reactivation obtains secondary seed solution afterwards for 24 hours；
(5) probiotics solid-state stepwise fermentation:
1. the bacillus subtilis that activation is completed and bacillus coagulans access 6T solid-state fermenter by inoculation pipeline In, 37 DEG C of temperature, ventilatory capacity 60L/min are maintained, ferment 10-24h；
2. 1. walking after fermentation to the, activated Pediococcus acidilactici and lactobacillus plantarum are passed through into inoculation pipeline access In 6T solid-state fermenter, sealed fermenting 12-24h；
3. 2. being walked after fermentation to, activated animal bifidobacteria, bifidobacterium adolescentis, long Bifidobacterium Bifidum are led to It crosses in inoculation pipeline access 6T solid-state fermenter, maintains 37 DEG C of temperature, sealed fermenting 18-36h；
4. 3. walking after fermentation to the, activated lactobacillus reuteri, lactobacillus paracasei are passed through into inoculation pipeline It accesses in 6T solid-state fermenter, maintains 37 DEG C of temperature, sealed fermenting 24-48h；
Above 1. -4. to walk each bacterium inoculum concentration be 5 × 10 in every gram of solid medium7CFU/g；
(6) assembled scheme of specific fermentation time is shown in Table 4；After fermentation, living to the probiotics in fermented sample respectively Bacterium number is detected, and the results are shown in Table 5.
4 probiotics solid-state stepwise fermentation ageing of table
5 fermentation time of table combines the influence (unit: CFU/g) to number of live bacteria of probiotics
Group unification Combination two Combination three Combination four
Bacillus subtilis 2.5×108 5.3×108 4.3×108 3.9×108
Lactobacillus plantarum 2.8×109 5.5×109 3.1×109 4.2×109
Lactobacillus paracasei 6.1×109 7.7×109 5.4×109 6.4×109
Lactobacillus reuteri 4.8×108 7.1×108 4.1×108 6.9×108
Pediococcus acidilactici 2.2×109 4.8×109 3.3×109 1.1×109
Bifidobacterium longum 2.9×108 4.0×108 3.4×108 3.8×108
Bifidobacterium adolescentis 2.9×107 4.4×107 1.1×107 3.5×107
Animal bifidobacteria 6.3×107 8.1×107 7.2×107 5.5×107
Bacillus coagulans 6.9×108 9.4×108 8.0×108 7.1×108
Total viable count 8.2×109 9.7×109 7.1×109 8.9×109
As shown in Table 5, the optimal stepwise fermentation time is combination two, and viable count is 9.7 × 10 after fermentation9CFU/g is that is, withered Careless bacillus and bacillus coagulans fermentation 12h；Pediococcus acidilactici and lactobacillus plantarum sealed fermenting 48h；Animal bifid bar Bacterium, bifidobacterium adolescentis, long Bifidobacterium Bifidum sealed fermenting are for 24 hours；Lactobacillus reuteri, lactobacillus paracasei sealed fermenting 12h.
Embodiment 2: the anti-oxidation function biological food determination of bioactive constituent of probiotics solid state fermentation
It is different according to type, the sequence of access strain using the anti-oxidation function food of probiotics solid-state stepwise fermentation, Different metabolites is produced, the type of metabolite is more, activity is higher.
After 1. probiotics is segmented solid state fermentation access bafillus natto and bacillus coagulans, in 37 DEG C of environment, After aerobic fementation 12h, colorimetric method for determining carotenoid, procyanidin content are utilized.Referring to the method for the reports such as Ndolo, slightly It is quickly measured after adding change with 96 hole elisa Plates.In every hole of 96 orifice plates, the probiotics for being diluted to suitable multiple is sequentially added The water-soluble extracting solution of food under solid-state stepwise fermentation senile-resistant efficacy, measures extinction with UV-Vis multi-function microplate reader at 450 nm It spends (EL 340, Bio-Tek Instruments, Inc.Winooski, VT, USA), the results are shown in Table 6.It is surveyed using high-efficient liquid phase technique Determine Hyperoside, rutin, Isorhamnetin -3-O- glucoside, Quercetin, Kaempferol, each flavonoids of Isorhamnetin.Chromatographic column Hypersil-OD column (5 μm, 4.6 × 250mm)；Mobile phase: A phase is 0.1% phosphate aqueous solution, and B phase is methanol, gradient elution (0~35min, 32%B → 35%B；35~45min, 35%B → 58%B；45~60min, B phase maintain 58% ratio not Become)；Flow velocity: 1.0mL/min；Detection wavelength: 370nm；Column temperature: 30 DEG C；Sample volume: 20 μ L the results are shown in Table 6.
2. after accessing Pediococcus acidilactici and lactobacillus plantarum, in 37 DEG C of environment, after sealed fermenting 48h, using efficient Liquid phase method measures ascorbic acid, malic acid.Chromatographic column: Sepax GP-C18 column (5 μm, 4.6 × 250mm)；Mobile phase: A phase is 0.05mol/L KH2PO4Aqueous solution (pH 2.70), B phase are methanol, and (0~30min, 1%B → 0.5%B are linearly washed gradient elution It is de-)；Flow velocity: 0.5mL/min；Detection wavelength: 210nm；Column temperature: 30 DEG C；Sample volume: 2.0 μ L the results are shown in Table 6.
3. accessing bifidobacterium longum, bifidobacterium adolescentis, animal bifidobacteria, in 37 DEG C of environment, sealed fermenting For 24 hours, Syrups by HPLC ursolic acid, oleanolic acid are utilized.Chromatographic column Eclipse XDB-C18 column (5 μm, 4.6 × 250mm)；Mobile phase: isocratic elution, methanol-water-glacial acetic acid-triethylamine (90: 10: 0.03: 0.06, v: v: v: v)；Flow velocity: 0.5mL/min；Detection wavelength: 210nm；Column temperature: 30 DEG C；Sample volume: 10 μ L the results are shown in Table 6.
4. access lactobacillus reuteri and lactobacillus paracasei after sealed fermenting 12h, utilize fire in 37 DEG C of environment Flame aas determination mineral element.Referring to GB/T 5009.14-2017, with Varian FS-220AA atomic absorption spectrum Instrument measures iron content and total mineral constituent content, the results are shown in Table 6.
Using the content of one Sulphuric acid Colorimetry date polysaccharide of phenol, it the results are shown in Table 6.
The anti-oxidation function biological food determination of bioactive constituent of 6 probiotics solid state fermentation of table
At present about preventing the drug of skin aging to be based on Free radicals injury theory, which thinks skin aging more With body is anti-oxidant, to remove the ability of free radical closely related, increase in right amount antioxidant, remove internal excessive free radicals can With delay skin aging.As shown in Table 6, through probiotics solid-state stepwise fermentation, different strains is accessed in a different order, is pressed After different condition fermentations, the macromolecular substances in the raw materials such as sea-buckthorn, jujube, lens, soybean are by probiotics katabolism For small-molecule substance, especially Flavonoid substances, content is dramatically increased, and activity significantly improves, can be more effective, direct clear Except excessive free radicals in human body, cytotoxin is excluded.Meanwhile vitamin C, procyanidine, date polysaccharide etc., these substances are days Right, efficient antioxidant, and plant by the metabolism of microorganism is anti-oxidant can be directly absorbed by the body utilization, To improve the human body availability of the substance, the generation of cell degradation, aging and cancer is prevented, the effect got twice the result with half the effort is reached Fruit.Meanwhile probiotics can use organic acid synthesizing ester, and the food is made to have better flavor and taste.
Embodiment 3: the measurement of anti-oxidation function
10 monthly age of Healthy female mouse 72 are selected, is randomly divided into six groups, every group of 12 animals, wherein three blank controls Group, three non-fermented food experimental groups, three probiotics solid state fermentation food experiment groups.Non- fermented food experimental group and probiotics Solid state fermentation food experiment group is a three times a day 10g, and edible with the mode of canteen, blank control group, which is given, distills Water continuous 48 days.It is given in last and indices measurement is carried out to animal after by food 30 minutes, the results are shown in Table 7；
1, mouse red blood cell superoxide dismutase (SOD) determination of activity
SOD is catalyzed ultra-oxygen anion free radical (O2-) generate H2O2, then by such as glutathione peroxidating of other antioxidases Object enzyme and hydrogen peroxide enzyme effect generate water, can remove O in this way2-Toxic action to cell.SOD, GSH-Px are in animal Content in a little organs and human body erythrocyte has apparent increasing age variation, and the growth of enzymatic activity and biological age is inversely proportional. The ability for eliminating free radical is directly proportional to enzymatic activity.O2-The final product for aoxidizing hydroxyl is nitrite, and the latter is in p-aminophenyl Sulfonic acid and the lower presentation aubergine of alpha naphthylamine effect, have maximum absorption peak at wavelength 530nm, can be measured with spectrophotometry, when SOD eliminates O2-The nitrite formed afterwards is reduced.Corresponding SOD amount is one when with SOD inhibiting rate in 1ml reaction solution up to 50% A unit, sample are red blood cell extract, are as a result indicated with U/g Hb.
2, Mouse whole blood glutathione peroxidase (GSH-Px) determination of activity
Glutathione peroxidase (GSH-Px) is that a kind of existing selenium that contains removes free radical and inhibits free radical in vivo The system of reaction.To preventing interior free yl from causing membrane lipid peroxidatio especially important, vigor is to be catalyzed the reaction that GSH is aoxidized The speed and amount of GSH reduction indicates that GSH and 5,5- bis- sulphur paranitrobenzoic acid (DTNB) is reacted in GSH-Px in the unit time The thio 2- nitrobenzoyl acid anion of the 5- of producible yellow under catalysis, has maximum absorption band in 423nm wavelength, measures the ion Concentration can calculate the amount of GSH reduction, since GSH can be carried out non-enzyme reaction oxidation, so when finally calculating enzyme activity, it must GSH caused by non-enzyme reaction must be deducted to reduce.Mouse whole blood GSH-Px unit of activity deducts per minute, it is specified that every 1ml whole blood After the log [GSH] of non-enzyme reaction is reduced, reduces log [GSH] by 1 one enzyme activity units, as a result indicated with U/ml whole blood.
3 mouse liver lipid peroxide (LPO) assays
Lipid peroxidation can form malonaldehyde, ethane, conjugated diene, fluorescence-causing substance and can generate chemiluminescent object Matter.If content of these products in body fluid and tissue increases, show that body lipid peroxidization enhances.MDA (malondiadehycle) be Cell membrane lipids peroxidating one of final product, lipid peroxidation can be estimated indirectly by surveying its content Degree.Heat together, formation are pink in acid condition for 1 malonaldehyde (MDA) molecule and 2 thiobarbituricacidα- (TBA) molecules Color compound.The substance has maximum absorption peak in wavelength 532nm, can be measured with spectrophotometry, with mmol/mg Pr table Show.
The measurement of the anti-oxidation function food oxydating resistance ability of 7 probiotics solid state fermentation of table
As shown in Table 7, mouse red blood cell SOD activity and negative control group have extremely significant property (P < 0.01) than difference, with Non- fermented food control group has conspicuousness (P < 0.05) than difference, illustrates the anti-oxidation function food tool of probiotics solid state fermentation It is improved the active effect of mouse red blood cell SOD；Mouse whole blood GSH-PX activity is compared with negative control group and non-fermented food pair It is significantly improved according to group, difference has extremely significant (P < 0.01), illustrates that probiotics solid-state stepwise fermentation anti-senescence function is eaten Product, which have, improves the active effect of Mouse whole blood GSH-PX；Mouse liver LPO content reduces, and do not ferment with negative control group and food Product control group is compared, and difference has extremely significant property (P < 0.01), illustrates the anti-oxidation function food tool of probiotics solid state fermentation Play the role of reducing mouse liver LPO content.
Embodiment 4: the measurement of free radical scavenging ability
1、ABTS+The Scavenging activity of free radical
Using the green skies kit measurement of ABTS, 200 microlitres of ABTS working solutions are added to each detection hole of 96 orifice plates, it is empty In white control wells be added 10 microlitres of distilled water, standard curve detection hole be added 10 microlitres of various concentration Trolox (hydroxyl -2 6-, 5,7,8- tetramethyl benzodihydropyran -2- carboxylic acids) standard solution, 10 microlitres of various samples are added in sample detection hole, and (water mentions Object), it mixes gently；After incubation at room temperature 2-6 minutes, absorbance is measured at 734nm, the results are shown in Table 7.
2, the Scavenging activity of DPPH free radical
Measuring method is with reference to Zhang Fangang etc. and is suitably modified, and weighs 0.004g 1,1- diphenyl -2- picryl hydrazine is certainly By base (DDPH), 100mL is dissolved and be settled to dehydrated alcohol, is stored in brown bottle.Hydroxyl -2,5 0.025g 6- are weighed, 7,8- tetramethyl benzodihydropyran -2- carboxylic acids (Trolox) are dissolved with 20mL dehydrated alcohol, are configured to the mother liquor of 5mmol/L, Trolox mother liquor is diluted to 1000,500,250,125,62.5,31.25 μm of ol/L with dehydrated alcohol.Measuring hole is 20 prebiotic Bacterium fermented sample (water extract), 80 μ L DDPH；Control wells are the non-fermented sample of 20 μ L, 180 μ L DDPH；Blank control wells are 200 μ L dehydrated alcohols.Reaction solution is added in ELISA Plate, 30min is protected from light, absorbance is measured in microplate reader, is measured Wavelength is 517nm, the results are shown in Table 8.
3, the Scavenging activity of hydroxy radical
Using Feton system of determination.6mmo1/L ferrous sulfate, each 2.5mL of 6mmo1/L hydrogen peroxide are taken respectively, after mixing 6mmo1/L salicylic acid 7.5mL is added, after mixing well, system is placed in 37 DEG C of water-baths and reacts 15min, Yu Bochang 510nm Place's measurement absorbance, absorbance A0, 1mL probiotics fermention food (water extract) is added to system, is reacted in 37 DEG C of water-baths Absorbance, absorbance Ax are measured at 15min, Yu Bochang 510nm；Control experiment is done with non-fermented sample；Distilled water does blank Control, vitamin C is positive control, the results are shown in Table 7.
The measurement of the anti-oxidation function food free radical scavenging ability of 8 probiotics solid state fermentation of table
As shown in Table 8, using Trolox as positive control, the anti-oxidation function food of probiotics solid state fermentation is to ABTS+、 The Scavenging activity of DPPH free radical is remarkably reinforced compared with non-fermented sample, to ABTS+The IC of free radical50Value is followed successively by 1.14, 2.12mg/mL, to DPPH free radical IC50Value is followed successively by 2.32,6.07mg/mL, and all has significant difference (p < 0.05), and The IC of reference substance Trolox50Value is 0.44,0.38mg/mL, illustrates that the anti-oxidation function food of probiotics solid state fermentation has Remove ABTS+With the ability of DPPH free radical；Hydroxyl radical free radical has extremely strong electronic capability, that is, oxidability, and raw material at There are the electron donating groups such as a variety of sulfydryls, phenolic hydroxyl group in point, redox reaction can occur with hydroxyl radical free radical, to remove hydroxyl Base free radical.The ability that the anti-oxidation function food of probiotics solid state fermentation removes hydroxyl radical free radical is better than unfermentable original Powder, and to the IC of Hydroxyl radical-scavenging50Value is lower than vitamin C, illustrates the Hydroxyl radical-scavenging ability of the fermented food than single It is more effective when solely using vitamin C.
Embodiment 5: the measurement of freckle removing and whitening effect
Human body can all form a kind of pigmentation, referred to as lipofuscin in aging course in various tissues (liPofuscin), such as in cardiac muscle, brain tissue, kidney, dermis of skin all it is likely to form lipofuscin.In dermis of skin Lipofuscin pigmentation be commonly referred to as senile plaque.This is because cell-membrane lipid bilayer is generally polyunsaturated fatty acids (PUFA) it is formed.When interior free yl is excessive, these polyunsaturated fatty acids are in the Free Radical as initiator Under will do it peroxidation, and occur oxidation scission reaction and become polyunsaturated fatty acids various peroxide.When Pigmentation, i.e. lipofuscin (senile plaque) are just formed after the latter and protein cross.It is body that body, which respectively organizes lipofuscin increase, Interior free radical disequilibrium and performance on the high side, and it is generally acknowledged that the peroxidating of cell membrane bilayer is the symbol of aging.
80 senile plaque patients are screened, are grouped at random, share two groups, every group 40, respectively fermented food is tested Group and non-fermented food experimental group.Wherein fermented food experimental group eats the anti-oxidation function food of probiotics solid state fermentation, The edible non-fermentation and sterilization raw material freeze-drying mixture of non-fermented food experimental group is 3 times a day used with canteen, a 10g；Continuous food With 100 days, meanwhile, during the experiment, experimental subjects first is it is noted that sun-proof, and especially ultraviolet light irradiates；Second is to forbid Oral contraceptive is taken, avoids leading to hyperpigmentation because of oral contraceptive；Third is to be forbidden to use irritation article, is avoided The state of an illness is aggravated, to influence test result；Fourth is that be forbidden to use hormone drug；Fifth is that avoid fatigue, and experimental subjects exists In process of experimental, it is ensured that the regularity of work and rest avoids fatigue；Sixth is that facial-care simplification, to avoid pair The influence of test result；Seventh is that on diet based on light food.After experiment, by VISIA skinanalysis apparatus pair The patient for participating in experiment records, and the results are shown in Table 9.
The measurement (example, %) of the anti-oxidation function food freckle removing and whitening effect of 9 probiotics solid state fermentation of table
Group Number of cases Recovery from illness It is effective Effectively In vain Total effective rate
Fermented food group 40 24 9 4 3 92.5%
Non- fermented food group 40 15 6 10 9 77.5%
X2 8.248
P 0.041
As shown in Table 9, the anti-oxidation function food of probiotics solid state fermentation of the present invention can be effectively improved skin pigment Calmness lightens the stain, and provides scheme for market freckle removing and whitening product diversification.
Embodiment 6: skin aging Biochemical Indexes
Daily subcutaneous injection D- galactolipin 1000mgkg- 1·d- 1Combine 350~400nm UV prolonged exposure 40min, even Continuous 40d, induced subacute aging model.Male nude mouse is randomly divided into Normal group, model group, non-fermented food group, prebiotic Bacterium solid-state stepwise fermentation anti-senescence function food group (15g/d), positive controls (Nutrilite moistens beautiful drink 30ml/d), every group 10.In the 11st day difference feeding 30d of modeling.Detect hydroxyproline, total collagen, III collagen type, elasticity in serum The content of albumen, the results are shown in Table 10；HE dyeing observation skin molds change, the result is shown in Figure 1-5.
The anti-oxidation function food skin aging Biochemical Indexes of 10 probiotics solid state fermentation of table
D- galactolipin generates free radicals in the metabolic process under the action of galactolipin synzyme, influences the function of mitochondria Can, cause cell oxidative damage occur, hydroxyproline content is reduced, and skin corium is significantly thinning, and collagenous fibres are reduced, elastic fibers The skin agings phenomenons such as denaturation.In addition, UV irradiation is the important extrinsic factor of skin aging, matrix metalloproteinase can induce (MMPs) it over-expresses, the dermal extracellular matrix ingredients such as collagen of then degrading lead to skin photoage.By 10 result of table It has been shown that, compared with Normal group, hydroxyproline, total collagen, III collagen type, elastin laminin contain in model group serum Amount is remarkably decreased (P < 0.05 or P < 0.01), and skin molds change obvious, prompt modeling success.Collagen is a kind of Structural proteins are the important compositions in dermis of skin, and content is decreased obviously with age, make cutis laxa, elasticity drop It is low, therefore collagen is the important indicator of skin aging；Elastin peptide is the pyrolysis product of elastin laminin, with protein phase Than Elastin peptide shows better improvement to indexs such as moisture of skin, elasticity.As shown in Table 10, with model group pair Than probiotics segmentation solid state fermentation anti-senescence function food can dramatically increase skin hydroxyproline, total collagen, III type glue The content of former albumen and elastin laminin.By Fig. 1-5 it is found that Normal group nude mice skin structural integrity skin layer thickness is uniform, It has no and obviously thickens or thinning, basal cell arrangement is uniform sequential, and epidermis and skin corium distinct, fibr tissue are arranged with Sequence does not find inflammatory cell infiltration.Model group epidermis thickens, uneven thickness, and the cell number of plies is reduced, and basal cell arrangement is loose disorderly Disorderly, size is uneven, and epidermis and skin corium boundary are fuzzy, and dermis thickness is thinning, and fibr tissue arrangement is loose, it is seen that inflammatory cell Infiltration.Compared with model group, non-fermented food group, probiotics solid-state stepwise fermentation anti-senescence function food and positive controls Pathological change mitigated, especially probiotics solid-state stepwise fermentation anti-senescence function food group, epidermis is relatively thin, fiber group It is more neat to knit arrangement, it is prompted to have the function of improving skin molds, anti aging effect.
It is above-mentioned referring to embodiment to probiotics solid-state stepwise fermentation temperature through cold extermination functional food and preparation method thereof into Capable detailed description is illustrative without being restrictive, and can enumerate several embodiments according to limited range, therefore Change and modification in the case where not departing from present general inventive concept should belong within protection scope of the present invention.

Claims (8)

1. a kind of anti-oxidation function food of probiotics solid state fermentation, which is characterized in that the probiotics solid state fermentation resists Oxidative function food be using sea-buckthorn, jujube, lens, soybean mixture as fermentation substrate, using lactobacillus reuteri, Bifidobacterium longum, Pediococcus acidilactici, lactobacillus plantarum, animal bifidobacteria, lactobacillus paracasei, bifidobacterium adolescentis, condensation Vacuum freeze drying is prepared after bacillus and bacillus subtilis carry out solid-state stepwise fermentation.
2. the anti-oxidation function food of probiotics solid state fermentation as described in claim 1, which is characterized in that the plant cream bar The deposit number of bacterium is CGMCC No.16021；The deposit number of the animal bifidobacteria is CGMCC No.16028；It is described The deposit number of lactobacillus paracasei is CGMCC No.16022；The deposit number of the bifidobacterium adolescentis is CGMCC No.16026；The deposit number of the bacillus coagulans are as follows: CGMCC No.16025；The preservation of the bacillus subtilis is compiled Number be CGMCC No.16024；The deposit number of the lactobacillus reuteri is CGMCC No.16023；The bifidobacterium longum Deposit number be CGMCC No.16027；The deposit number of the Pediococcus acidilactici is CGMCC No.16077.
3. the anti-oxidation function food of probiotics solid state fermentation as claimed in claim 2, which is characterized in that the sea-buckthorn: big Jujube: lens: the mass ratio of soybean is 2:3:5:6.
4. the preparation method of the anti-oxidation function food of probiotics solid state fermentation described in claim 3, includes the following steps:
(1) good lens, soybean, sea-buckthorn, jujube is chosen to be milled sieve with 100 mesh sieve respectively；
(2) it prepares activation medium: adding 10g soy meal in every 100mL water；It sterilizing to activation medium, condition is, 121 DEG C, 20min；
(3) it prepares fermentation medium: lens powder, soy meal, seabuckthorn fruit powder, jujube powder is uniformly mixed, mixture and water press matter Amount than 1:1.5-2.0 prepare solid-state fermentation culture medium, preparation be placed in solid-state fermenter sterilize it is spare；Sterilising conditions: 121 DEG C, 20min；
(4) actication of culture: lactobacillus reuteri, bifidobacterium longum, Pediococcus acidilactici, lactobacillus plantarum, animal bifidobacteria, pair Lactobacillus casei, bifidobacterium adolescentis, bacillus coagulans and the glycerol tube of bacillus subtilis press 1% (v/v) inoculation respectively It is activated in activation medium, pH is naturally, controlled at 37 DEG C；After activating for 24 hours respectively, bacterium amount is connect again by 5% (v/v) Secondary switching activation medium carries out re-activation, and reactivation obtains secondary seed solution afterwards for 24 hours；
(5) probiotics solid-state stepwise fermentation:
1. maintaining 37 DEG C of temperature by the secondary seed solution access solid-state fermenter of bacillus subtilis, bacillus coagulans, leading to Tolerance 60L/min, ferment 10-18h；
2. 1. walked after fermentation to the, Pediococcus acidilactici, lactobacillus plantarum secondary seed solution access solid-state fermenter in, dimension 37 DEG C of temperature are held, sealed fermenting 12-24h；
3. 2. being walked after fermentation to, the secondary seed solution of animal bifidobacteria, bifidobacterium adolescentis, long Bifidobacterium Bifidum is connect Enter in solid-state fermenter, maintains 37 DEG C of temperature, sealed fermenting 18-36h；
4. 3. walking after fermentation to the, the secondary seed solution of lactobacillus paracasei, lactobacillus reuteri is accessed into solid state fermentation In tank, 37 DEG C of temperature are maintained, sealed fermenting 24-48h；
(6) after fermentation ends through vacuum freeze drying to get probiotics fermention functional food.
5. the preparation method of the anti-oxidation function food of probiotics solid state fermentation as claimed in claim 4, which is characterized in that match The mass ratio of the mixture and water is 1:1.4 when solid-state fermentation culture medium processed.
6. the preparation method of the anti-oxidation function food of probiotics solid state fermentation as claimed in claim 4, which is characterized in that institute State bacillus subtilis, bacillus coagulans, Pediococcus acidilactici, lactobacillus plantarum, animal bifidobacteria, bifidobacterium adolescentis, Long Bifidobacterium Bifidum, lactobacillus paracasei, lactobacillus reuteri inoculum concentration be respectively 107-108CFU/g solid-state fermentation culture medium.
7. the preparation method of the anti-oxidation function food of probiotics solid state fermentation as claimed in claim 6, which is characterized in that on The bacterium amount that connects for stating each bacterium is respectively 5 × 107CFU/g solid-state fermentation culture medium.
8. the preparation method of the anti-oxidation function food of probiotics solid state fermentation as claimed in claim 4, which is characterized in that institute State the condition of vacuum freeze drying are as follows: vacuum freeze drying for 24 hours, until water content be not more than 12%.
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Application publication date: 20191119