CN105831771B - A kind of dietary supplements with the probiotics and microelement of alleviating lead poisoning sexual function - Google Patents
A kind of dietary supplements with the probiotics and microelement of alleviating lead poisoning sexual function Download PDFInfo
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- CN105831771B CN105831771B CN201610169922.4A CN201610169922A CN105831771B CN 105831771 B CN105831771 B CN 105831771B CN 201610169922 A CN201610169922 A CN 201610169922A CN 105831771 B CN105831771 B CN 105831771B
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- 230000002485 urinary effect Effects 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- 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
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- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses a kind of dietary supplements with the probiotics and microelement of alleviating lead poisoning sexual function, belong to food technology field.The present invention is by this plant of bacterium to be freeze-dried the bacterium powder of acquisition as main ingredient, it is aided with antioxidant and microelement, the probiotics of tablet form and the dietary supplements of microelement are prepared with direct powder compression after addition proper auxiliary materials, it contains viable bacteria content greater than 109The lactobacillus plantarum CGMCC 5494 of CFU/g.The dietary supplements obtained in this way can reduce lead content in lead exposure Mice Body；Restore oxidativestress damage caused by lead exposure；Improve the activity of aminolevulinate dehydratase in lead exposure Mice Body；Reduce zinc protoporphyrin content in lead exposure Mice Body；Restore the intellectual damage of lead exposure mouse.It is with a wide range of applications.
Description
Technical field
The present invention relates to a kind of dietary supplements with the probiotics and microelement of alleviating lead poisoning sexual function, belong to food
Product technical field.
Background technique
Lead is a kind of silvery white heavy metal with blue, oxidizable in air, dimmed.Lead is widely used, both can be with
Effectively stop X-ray and radioactive ray, and the main material of manufacture battery.In addition, the compound of lead is many pigment
Raw material.Lead contamination refers to compound pollution caused by ambient enviroment of lead or lead, and concentration and chemical form endanger journey with it
It spends directly related.The strong feature of lead enriching makes it be difficult to degrade in the environment, cannot pass through the catharsis of environment itself
It reduces, exceeds normal range (NR), can not only be detrimental to health, also result in environmental quality deterioration.Lead content increases in environment
The main reason for being the generation of lead contamination.Lead content in China's atmospheric environment is up to fifty-five million ton.High lead content in air
It on the one hand is caused by natural cause, such as volcano eruption, on the other hand forest fire etc. is then due to industrial pollution and traffic
Exhaust emission.Furthermore also extremely serious the phenomenon that Lead Pollution in Soil.After sewage irrigation soil, heavy metals exceeding standard that soil contains
Directly contribute heavy metal pollution of soil.The cereal of this soil output and vegetables, content of beary metal can be significantly exceeded.National soil
Background value basic statistics amount the result shows that, China's Content of Pb in Soil reaches as high as 1143 μ g/g, minimum 0.68 μ g/g, average
It can reach 26 μ g/g.Food, most of lead contamination of drinking water is all extraneous contamination.Leaded food, drinking water after peptic digest,
Adult absorbs up to 11%, and children absorb and are up to 30%-75%.After people drinks this tap water, lead is lodged in vivo, directly
Health is damaged.
Lead can enter human body by multiple channel, and intracorporal lead content seldom also can cause to damage to human body.Even if controlling
Decline intracorporal lead level after treatment, but the damage that has resulted in of lead be also it is permanent, can not restore as former state.Current grinds
Study carefully discovery lead poisoning to be mainly reflected in the following aspects to the toxic action of human body: A influences nervous system, be mainly shown as
Damage to intellectual aspect；B damages hematological system, and main cash is anaemia；C influences urinary system, is mainly shown as to kidney
The damage of tubule；D damages cardiovascular system, is mainly shown as the diseases such as myocarditis.
The drug of traditional two kinds treatment lead poisonings is while being effectively discharged intracorporal lead in the market, bring side effect
Also extremely serious.EDTA metalloid chelating agent calcium disodium edetate (CaNa2EDTA), to mucous membrane, the upper respiratory tract, eyes, skin
Irritating effect, and there is serious renal toxicity, other the essential trace elements of the human bodys have also been discharged while driving lead.DMSA
Class competitiveness antidote dimercaptosuccinic acid (DMSA) is possible to that midfield as above is caused to go up painful after enteron aisle is absorbed into human body,
Certain side effects such as nausea.Furthermore there is serious hepatic disorder person that cannot use DMSA.
Therefore make great efforts to explore safer, more efficient while again economic method alleviation lead poisoning to be very necessary.
Especially for the children also in the growth stage, very serious toxic side effect can be had to children by chelating agent therapy, safely and effectively
Therapy is particularly important.By the synergy of probiotics and nutrient, can to avoid or reduce the dosage of chelating agent,
The side effect for reducing chelating agent is more beneficial to health more suitable for medication.
Lactic acid bacteria is one kind of probiotics, is one of intracorporal necessary flora of people, is widely present in human body intestinal canal.Currently
Domestic and international biologist is it was demonstrated that lactic acid bacteria inhibits to cause a disease there are many important physiological function, including adjusting human body microecological balance
Bacterium, prevents and treats lactose intolerance, and antitumor, adjusting autoimmunity intervenes oxidative stress, reduction cholesterol etc..Current multinomial research
Confirm that lactic acid bacteria adsorbs the ultrahigh in efficiency of heavy metal in vitro, it is very outstanding to alleviation effect of body oxidative stress.Calcium, zinc are
The essential trace elements of the human body.Calcium is many biochemical processes and physiology course necessary element for organism, and zinc is in human body
Growth and development maintains to play an important role in the physiology courses such as immune function, immune, endocrine.Current multinomial research
Calcium, zinc and lead are proved to intestinal absorption site, the action target spot etc. of enzyme has Competition from each other, therefore supplement calcium, zinc can
To mitigate lead poisoning burden.Vitamin C is also known as ascorbic acid, and Vc is a kind of antioxidant, strong reductant, its energy in vivo
Enough threat of the protection body from oxidant.Multiple studies have shown that Vc can prevent the combination of lead He other molecules, enteron aisle is prevented
Absorption to lead improves oxidation resistance to prevent lipid peroxidation caused by lead.
The continuous transformation of nutrient health theory, ill to cure the disease, the lactobacillus micro-ecological preparation product of no treating diseases and making health-care is increasingly
It is welcome.It is more common with liquid lactic acid bacterium probiotics preparation however in lactobacillus micro-ecological preparation, and the benefit of tablet form
Raw bacterium dietary supplements is not common.Therefore tablet form is developed, has and alleviate lead poisoning effect, while can also mends
Fill the probiotics of micro elements needed by human and the dietary supplements of microelement, be it is a kind of be suitable for children, it is in the pink of condition
Therapeutic scheme, have a vast market application prospect.
Summary of the invention
The present invention is to provide a kind of purposes of lactobacillus plantarum (Lactobacillus plantarum), is by this plant of bacterium
To be freeze-dried the bacterium powder obtained as main ingredient, it is aided with antioxidant and microelement, prepares piece dosage form after adding proper auxiliary materials
The probiotics of formula and the dietary supplements of microelement, it contains viable bacteria content greater than 109The lactobacillus plantarum CGMCC of CFU/g
5494.The bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 29th, 2011,
Preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, deposit number CGMCC
5494。
Related lactobacillus plantarum CGMCC 5494 has following biological characteristics:
(1) thallus feature: Gram's staining is positive, and cell is in rod-short, and thallus is about 0.5-1.0 μm wide, 2-4 μm
Long, Cheng Dan, pairs of or chaining do not form gemma, and both ends are round；
(2) colony characteristics: forming apparent bacterium colony on MRS culture medium, and diameter is round between 0.3-2.0mm, milky white
Color, translucent, surface wettability is smooth, neat in edge, not chromogenesis；
(3) growth characteristics: the bacterial strain minimum growth temperature is 20 DEG C, and maximum growth temperature is 40 DEG C, at 30-37 DEG C of temperature
Lower growth is best, and the highest and lowest initial growth pH being resistant to is 9.0 and 2.5, and the initial pH of the most suitable growth is 6.0；The present invention
The period of delay of lactobacillus plantarum strain is relatively short, and 4h or so initially enters logarithmic growth phase, and 12h just reaches stationary phase.
A kind of dietary supplements containing profitable probliotics and microelement simultaneously provided by the invention, is to be freeze-dried and obtain
Probiotics bacterial powder be main ingredient, be aided with antioxidant and microelement, prepared with powder through pressed disc method after adding proper auxiliary materials
It obtains；The probiotics is the lactobacillus plantarum (Lactobacillus plantarum) that deposit number is CGMCC 5494.
In one embodiment of the invention, the probiotics bacterial powder the preparation method is as follows: by lactobacillus plantarum
CGMCC 5494 expands culture, centrifugation, washing after activating, freeze drying protectant is then added, thallus is resuspended, reach cell concentration
1011CFU/mL or more, pre-freeze after mixing, is then freeze-dried.
In one embodiment of the invention, the freeze-drying is the trehalose or 100- with 100-150g/L
The degreasing milk solution of 250g/L is as freeze drying protectant.
In one embodiment of the invention, the freeze-drying is using the trehalose of 100g/L as frozen-dried protective
Agent.
In one embodiment of the invention, the probiotics bacterial powder preparation the following steps are included:
A. the preparation of culture medium: using by based on culture medium gross mass 87.7% water by 10% enzyme hydrolysis skimmed milk,
0.5% glucose, 1.5% tryptone and 0.3% yeast extract dissolve, and then adjusting its pH is 6.8, obtain culture medium；
B. the preparation of freezing drying protective agent: being mixed with using water and protective agent raw material, obtains the freeze-drying of respective concentration
Protective agent；
C. 5494 strain of lactobacillus plantarum CGMCC is inoculated into based on the quality of the culture medium with the inoculum concentration of 2-4%
At 110-120 DEG C of temperature in culture medium cooling after sterilizing 8-12min, 18h then is cultivated at 37 DEG C, with pH7.2 phosphate
Buffer solution for cleaning 2-4 times, the resuspension of the freezing drying protective agent described in step B make viable bacteria concentration up to 1011CFU/mL or more allows this
Suspension preculture 60min at 37 DEG C of temperature is freeze-dried to obtain the freeze-dried vaccine powder later.
In one embodiment of the invention, the dietary supplements, according to parts by weight containing the bacterium of 30-50%
Powder, the antioxidant of 1-2%, the substance that can be added in food of the offer microelement of 6-8%, the auxiliary material of 40-63%.
In one embodiment of the invention, the antioxidant is vitamin C；Microelement is calcium, zinc；Auxiliary material
It is one or more usually used fillers in tablets, adhesive, disintegrating agent, lubricant.
In one embodiment of the invention, the dietary supplements contains 35.9-56.7% according to parts by weight
Microcrystalline cellulose, the low-substituted hydroxypropyl cellulose of 4-6%, the superfine silica gel powder of 0.1-0.3%, the vitamin C of 1-2%,
The calcium carbonate of 5.7-7.5%, the zinc acetate of 0.3-0.5%, the bacterium powder of 30-50%.
In one embodiment of the invention, it is through tablet press machine with 2.4t pressure tabletting that the pressed disc method, which carries out tabletting,
Obtained tablet weight is 250mg ± 15mg.
In one embodiment of the invention, the dietary supplements, according to parts by weight containing 46.5% crystallite
Cellulose, 5% low-substituted hydroxypropyl cellulose, 0.1% superfine silica gel powder, 1.6% vitamin C, 6.4% calcium carbonate,
0.4% zinc acetate, 40% bacterium powder.Formula is as shown in table 1 below:
The dietary supplements of 1 probiotics of table and microelement composition and content
In one embodiment of the invention, the viable bacteria of lactobacillus plantarum CGMCC 5494 contains in the dietary supplements
Amount reaches 109CFU/g or more.
Tablet form described in one embodiment of the present invention, the dietary supplements system of probiotics and microelement
It is as shown in Fig. 1 to make method.
Beneficial effects of the present invention:
(1) meal supplement tablet while being prepared for the first time containing profitable probliotics and microelement；
(2) dietary supplements active component content of the invention is high, shelf life is long；Prebiotic bacterial content can reach 8.3 ×
109Cfu/g, viable count is still greater than 10 after storing 6 months at 4 DEG C6CFU/g；Ascorbic acid content is up to 15.45mg/g, at 20 DEG C
Storage still had 90% or more retention rate after 6 months；
(3) dietary supplements of the invention can reduce the lead content and blood of the internal organs such as liver in Mice Body, kidney, brain
Lead content, alleviate the lead poisoning of Mice Body internal cause caused by oxidativestress damage, aminolevulinate dehydratase activity reduce, zinc
Protoporphyrin content rises, and intelligence caused by alleviating because of lead poisoning reduces, and has very much application prospect.
Biomaterial preservation
It is micro- to be preserved in China on November 29th, 2011 for a kind of lactobacillus plantarum (Lactobacillus plantarum)
Biological inoculum preservation administration committee common micro-organisms center, preservation address are in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Institute of microbiology, the academy of sciences, state, deposit number are CGMCC 5494.
Detailed description of the invention
Fig. 1 is the production method process of dietary supplements；
Fig. 2 is that type ascorbic acid measurement standard curve is restored in dietary supplements.
Specific embodiment
Embodiment 1: influence of the different freezing drying protective agents to 5494 survival rate of lactobacillus plantarum CGMCC
After lactobacillus plantarum CGMCC 5494 was activated for two generations in MRS fluid nutrient medium, 1L is accessed by 2% inoculum concentration
In MRS fluid nutrient medium, it is placed in constant temperature at 37 DEG C and expands culture 18h.5000g is centrifuged 15min at 4 DEG C, discards supernatant and collects bacterium
Body, thallus are washed twice with sterile phosphate buffer (PBS, 60mmol/L, pH6.5), collect thallus.Above-mentioned experiment repeats 5
It is secondary, 5 parts of thallus are collected, the sterile PBS solution of 25mL, 150g/L sucrose solution, 100g/L aqueous trehalose, 50g/ are separately added into
L sorbitol solution, 100g/L skimmed milk power solution are mixed as freezing drying protective agent and are resuspended.Protectant bacteria suspension will be contained
1mL is divided in sterile cillin bottle.Pre-freeze 3h at -80 DEG C.It is freeze-dried later, time 48h, vacuum degree 4Pa, cold-trap
- 50 DEG C of temperature.Freeze-drying front and back viable bacteria content is detected with bacterium colony colony counting method, number of viable is denoted as before being freeze-dried
M0；Number of viable is denoted as M after freeze-drying, obtains survival rate=M/M0× 100%.The results are shown in Table 2:
Influence of the different freezing drying protective agents of table 2 to 5494 survival rate of lactobacillus plantarum CGMCC
Note: different letters have significant difference (P < 0.05) between representing group
By table 2 it is found that the difference on effect of different freezing drying protective agents is obvious.10% trehalose, 10% degreasing
Milk solution effect is preferable, and survival rate is respectively 40.67% and 37.87% after freeze-drying.And sterile PBS solution protecting effect is most
Difference, thallus survival rate is only 0.07%, almost without effect.The result shows that being freezed in 5494 thallus of lactobacillus plantarum CGMCC
In drying process, trehalose, skimmed milk reduce somatic cells damage.Trehalose is selected, skimmed milk is as lactobacillus plantarum
The freezing drying protective agent of CGMCC 5494 is than convenient.
The present invention considers the non-functional component for simplifying the dietary supplements of probiotics and microelement as far as possible, not to more
The combination of kind freeze drying protectant is tested, and is only compared to the protecting effect of single kind freezing drying protective agent.
Embodiment 2: influence of the trehalose concentration to 5494 thallus freeze drying activity of lactobacillus plantarum CGMCC
The protective agent for preparing the various concentration gradient that trehalose concentration is 100g/L, 150g/L, 200g/L, 250g/L is molten
Liquid carries out expansion culture, centrifugation, washing and the freeze-drying of lactobacillus plantarum CGMCC 5494 according to the process of embodiment 1, living
Bacterium counts the concentration for investigating trehalose to the active influence of 5494 thallus of CGMCC freeze-drying.The results are shown in Table 3:
Protecting effect of the trehalose of 3 various concentration gradient of table for CGMCC 5494 in freezing dry process
Note: different letters have significant difference (P < 0.05) between representing group
By table 3 it is found that with trehalose concentration raising, protecting effect is obviously improved.But experiment discovery, trehalose
When concentration is greater than 150g/L, the thallus viscosity after freeze-drying is larger, can not grind bacterium powder, this may be the water suction work of trehalose
Caused by, thus controlling trehalose concentration is that 100g/L is appropriate.
Embodiment 3: influence of the skimmed milk concentration to 5494 thallus freeze drying activity of lactobacillus plantarum CGMCC
The freeze-drying for preparing the various concentration gradient that skimmed milk concentration is 100g/L, 150g/L, 200g/L, 250g/L is protected
Agent solution is protected, expansion culture, centrifugation, washing and the freezing of lactobacillus plantarum CGMCC 5494 are carried out according to the process of embodiment 1
Dry, the concentration that count plate investigates skimmed milk is freeze-dried active influence to 5494 thallus of lactobacillus plantarum CGMCC.Knot
Fruit is as shown in table 4:
Influence of the skimmed milk power of 4 various concentration of table to 5494 survival rate of lactobacillus plantarum CGMCC
Note: different letters have significant difference (P < 0.05) between representing group
By table 4 it is found that with skimmed milk concentration raising, protecting effect is obviously improved.But skimmed milk concentration is more than
After 150g/L, influence of the concentration to protecting effect be not significant, 150g/L, 200g/L, the guarantor of tri- concentration skimmed milks of 250g/L
It is almost the same to protect effect, no significant difference (P > 0.05).After using each concentration degreasing milk solution as protective agent freeze-drying, thallus is ground
Bacterium powder is ground without influence.Comprehensively consider freezing dry process survival rate, the influence degree and tabletting that protective agent grinds bacterium powder
Situations such as bacterium powder activity change being likely to occur in journey, therefore select 100g/L aqueous trehalose and 150g/L degreasing milk solution
5494 freeze-dried vaccine powder of lactobacillus plantarum CGMCC is prepared for protective agent, further investigates tableting processes to probiotics and microelement
Dietary supplements in microbial activity variation.
Embodiment 4: influence of the tableting pressure to the dietary supplements hardness of probiotics and microelement
The dietary supplements preparation process of probiotics and microelement is shown in attached drawing 1.
It is cold with 100g/L trehalose and 150g/L skimmed milk according to the component design of dietary supplements and the technique of setting
2 kinds of 5494 bacterium powder of freeze-drying lactobacillus plantarum CGMCC of dry protective agent preparation are main component, with antioxidant, micro member
Element, auxiliary material are with the mixing of facing-up method.Tabletting is distinguished with three pressure gear 1.2t, 2.4t, 3.6t pressure of tablet press machine, and weight is
250mg±15mg.The dietary supplements made is detected into hardness (ordinary tablet 3-5kg) with hardness tester, and carries out high-altitude and falls
Experiment (takes 10 dietary supplements to be placed in the vacancy 2M high, makes its free-falling, if dietary supplements does not occur obvious slight crack or disconnected
Fragmentation is split, then illustrates hardness qualification, every 3 repeated experiments), the results are shown in Table 5,
5 tableting pressure of table influences the dietary supplements hardness of probiotics and microelement
Note: different letters have significant difference (P < 0.05) between representing group
By table 5 it is found that after carrying out tabletting with 1.2t pressure, the dietary supplements hardness of two kinds of bacterium powder production is only respectively
2.23kg and 2.46kg, not up to 3-5kg standard.While high-altitude falls experiment and sliver phenomenon occurs, therefore 1.2t pressure is not
Preparation suitable for dietary supplements.And after carrying out tabletting with 3.6t pressure, the dietary supplements hardness of two kinds of bacterium powder production is
More than 6.00kg, and exceed national regulation 3-5kg standard.Therefore 3.6t pressure is unsuitable for the preparation of dietary supplements.And with
After 2.4t pressure carries out tabletting, the dietary supplements hardness of two kinds of bacterium powder production is respectively 4.17kg, 4.22kg, is met the requirements,
High-altitude, which falls to test, simultaneously does not occur sliver phenomenon.Therefore, 2.4t pressure is suitable for the preparation of dietary supplements, selects 2.4t pressure
Prepare dietary supplements.
Embodiment 5: tableting processes are on the active influence of lactobacillus plantarum CGMCC 5494 in dietary supplements
The dietary supplements preparation process of probiotics and microelement is shown in attached drawing 1.
According to the component design (table 1) of dietary supplements and the technique of setting, will with 100g/L trehalose and 150g/L,
200g/L, 250g/L skimmed milk be 4 kinds of 5494 bacterium powder of freeze-drying lactobacillus plantarum CGMCC of cold dry protective agent preparation be mainly at
Point, with antioxidant, microelement, auxiliary material with the mixing of facing-up method, by mixed powder through tablet press machine with 2.4t pressure tabletting, weight
Amount is 250mg ± 15mg.Mixed powder is detected into number of viable with the method for plate culture count, is denoted as W0.With flat after tabletting
Plate colony counting method detects the number of viable in dietary supplements, is denoted as W.Then survival rate are as follows: survival rate=W/W0×
100%.The results are shown in Table 6:
Influence of 6 tableting processes of table to 5494 freeze-dried vaccine powder microbial activity of lactobacillus plantarum CGMCC
Note: different letters have significant difference (P < 0.05) between representing group
Sheeting operation is larger to the activity and survival rate of different 5494 bacterium powder of lactobacillus plantarum CGMCC.Pass through table 6
It is found that 200g/L, 250g/L skimmed milk is that the bacterium powder of protective agent production is probiotics damage after main ingredient carries out tabletting with 150g/L
Hurt larger, 5494 survival rate of lactobacillus plantarum CGMCC is less than 3% in the dietary supplements of probiotics and microelement, survival volume
Less than 2 × 109cfu/g.The bacterium powder made by protective agent of 10% trehalose is probiotics and micro member after main ingredient carries out tabletting
5494 survival rate of lactobacillus plantarum CGMCC can reach 19.61% in the dietary supplements of element, and survival volume can reach 8.3 ×
109cfu/g.Comparison result explanation, relative to skimmed milk, trehalose can provide more effectively for lactobacillus plantarum CGMCC 5494
Protection, damage of the CGMCC 5494 under the severe processing environment of high pressure is reduced, using trehalose as protectant CGMCC
5494 freeze-dried vaccine powder, are more suitable for the production of the dietary supplements of probiotics and microelement.
Embodiment 6: influence of the tableting processes to antioxidant in the dietary supplements of probiotics and microelement is tested
It (is slightly changed) referring to " restoring the measurement of type ascorbic acid in GBT 5009.159-2003 food " the method,
Mixed powder and reduced form ascorbic acid content in dietary supplements after tabletting before measurement tabletting.
0.1g/L ascorbic acid standard uses the preparation of solution: precision weighs 0.2000g ascorbic acid, and 20mL is added
The dissolution of 2mol/L acetic acid solution, complete soln is moved into 100mL brown volumetric flask, and distilled water dilution is settled to scale mixing,
5.0mL is drawn with pipette precision to be placed in 100mL brown volumetric flask, 2mol/L acetic acid solution is added, and distilled water is diluted to quarter
Degree mixes.
Sample treatment: precision weighs before 250mg tabletting dietary supplements after mixed powder or 250mg tabletting, is accurate to
0.001g is placed in 50mL brown volumetric flask.The acetic acid solution that 10mL 2mol/L is added provides acidic environment, and 220g/L is added
Potassium ferrocyanide solution and each 7.5mL of 106g/L acetic acid zinc solution, using generate zinc ferrocyanide reaction protein precipitation, it is double
It steams water and is settled to scale, mix.Complete soln is transferred in 50mL centrifuge tube, 3000r/min is centrifuged 10min, takes supernatant
0.22 μm of film is crossed, it is spare.
The preparation of blank sample: it takes and sample treatment same concentrations, same amount of acetic acid solution, acetic acid zinc solution, ferrous iron
Potassium cyanide solution does reagent blank.Supernatant is prepared by the method for sample treatment.
The drafting of standard curve: precision draws 0.1,0.2,0.4,0.6,0.8,1.0,1.5,2.0mL ascorbic acid standard
Using solution, it is respectively placed in 10mL colorimetric cylinder.Add the disodium ethylene diamine tetra-acetic acid solution of 1.5mL 0.25mol/L,
The acetic acid solution of 1mL0.5mol/L is added the fast blue salts B solution colour developing of 1.25mL 2g/L, adds distilled water to be diluted to scale, mix
It is even.After placing 3min under room temperature (20 DEG C -25 DEG C), move into 1cm cuvette.Absorbance is measured at wavelength 420urn, draws mark
Directrix curve.The standard curve of drafting is shown in attached drawing 2.
The measurement of reduced form ascorbic acid content: taking membrane sample solution and each 5mL of blank solution in 10mL colorimetric cylinder,
Add the disodium ethylene diamine tetra-acetic acid solution of 1.5mL 0.25mol/L, 1.25mL 2g/ is added in the acetic acid solution of 1mL 0.5mol/L
The fast blue salts B solution of L develops the color, and distilled water is added to be diluted to scale, mixes.After placing 3min under room temperature (20 DEG C -25 DEG C), move into
In 1cm cuvette.Absorbance is measured at wavelength 420urn and standard curve compares, and finds reduced form ascorbic acid content
Influence of the dietary supplements tableting processes of 7 probiotics of table and microelement to reduction type ascorbic acid
By table 7 it is found that mixed powder detected level is up to 15.59mg/g before tabletting, dietary supplements detected level after tabletting
Up to 15.45mg/g, recall rate is up to 99.10% before and after tabletting.Illustrate that tableting processes almost do not have reduction type ascorbic acid
Have an impact.
Embodiment 7: storage requirement is on the active influence of lactobacillus plantarum CGMCC 5494 in dietary supplements
By the dietary supplements of the probiotics and microelement that are made in embodiment 5 using 10% trehalose as protective agent with aluminium
Foil bag vacuum heat-seal packaging, be protected from light, keep away it is wet, keep away oxygen, stored under the conditions of being placed in 4 DEG C and 20 DEG C, measurement store 15 days, 30 days, 90
It, 5494 number of viable of lactobacillus plantarum CGMCC after 180 days, investigate storage temperature and period of storage to probiotics and micro
The active influence of the dietary supplements of element.Existed with the dietary supplements that the method for plate culture count detects probiotics and microelement
Viable count when storing initial, is denoted as C0.Storage measures probiotics and microelement after a certain period of time, with the method for plate culture count
Dietary supplements viable count, be denoted as C, then store survival rate are as follows: storage survival rate=C/C0× 100%.As a result such as table 8,
Shown in table 9:
8 dietary supplements of table, 5494 activity change of lactobacillus plantarum CGMCC in storage process under the conditions of 4 DEG C
9 dietary supplements of table, 5494 activity change of lactobacillus plantarum CGMCC in storage process at 20 °C
By table 8, table 9 is stored under the conditions of 4 DEG C it is found that store under the conditions of compared to 20 DEG C, probiotics and microelement
The activity decline of dietary supplements is slower.5494 viable count of lactobacillus plantarum CGMCC declines a number after storage 1 month
Magnitude, viable count is still greater than 10 after storage 6 months6CFU/g.And the meal supplement of probiotics and microelement at 20 °C
Faster, 5494 viable count of lactobacillus plantarum CGMCC has dropped two orders of magnitude, storage 6 after storage 1 month for the activity decline of agent
After a month viable count only residue 103CFU/g illustrates that low-temperature storage is more advantageous to the meal supplement for keeping probiotics and microelement
The activity and extension shelf life of agent.
Embodiment 8: influence of the storage requirement to antioxidant in dietary supplements
By the dietary supplements of the probiotics and microelement that are made in embodiment 5 using 10% trehalose as protective agent with aluminium
Foil bag vacuum heat-seal packaging, be protected from light, keep away it is wet, keep away oxygen, stored under the conditions of being placed in 4 DEG C and 20 DEG C.According to the method in embodiment 5,
It measures in the dietary supplements of the probiotics and microelement after storing 15 days, 30 days, 90 days, 180 days and restores type ascorbic acid
Content.Storage temperature and period of storage are investigated to the shadow for restoring type ascorbic acid in the dietary supplements of probiotics and microelement
It rings.The results are shown in Table 10:
Reduced form ascorbic acid content changes 10 dietary supplements of table in storage process under the conditions of 4 DEG C, 20 DEG C
Note: different letters have significant difference (P < 0.05) between representing group
By table 10 it is found that reduced form is anti-bad in the dietary supplements of probiotics and microelement between each group after storage 15 days
Hematic acid is decreased significantly compared with when storing initial, but as the content of the extension reduction type ascorbic acid of period of storage is not shown
Difference is write, illustrates that restore type ascorbic acid does not degrade during storage 15 to 180 days.Illustrate the packet of aluminium foil bag vacuum heat-seal
Although dress can starvation, a little drop that a little oxygen causes reduction type ascorbic acid may have been remained in packaging process
Solution.But the packaging of aluminium foil bag vacuum heat-seal, is protected from light, keep away it is wet, keep away oxygen preservation condition prevent reduction type ascorbic acid continuation
Degradation is conducive to protect the antioxidant in the dietary supplements of probiotics and microelement, extends shelf life.
Embodiment 9: dietary supplements acts on the intervention reduction of lead content in lead exposure Mice Body
Health male C57BL/6 mouse 40 for taking weight 20-25g, is randomly divided into 4 groups: blank control group, lead exposure mould
Dietary supplements intervention group, the DMSA group of type group, probiotics and microelement, every group of 10 mouse.Experiment 61 days by a definite date, blank
Normal water is given in 56 days sterile PBS solutions of daily stomach-filling 0.3mL before control group, last not stomach-filling in five days.Lead exposure model group
The first 56 days sterile PBS solutions of daily stomach-filling 0.3mL give leaded drinking-water and (lead acetate are dissolved in drinking water, makes its concentration
1g Pb/L is freely drunk for mouse), normal water is given in last not stomach-filling in five days.The diet of probiotics and microelement is mended
Fill 56 days daily stomach-filling 0.3mL probiotics and microelement before agent intervention group meal supplement agent solution (in Example 5 with
It is molten that the dietary supplements of a piece of probiotics and microelement that 10% trehalose is prepared for protective agent is dissolved in the sterile PBS of 0.6mL
In liquid), leaded drinking-water is given, normal water is given in last not stomach-filling in five days.Daily stomach-filling 0.3mL is sterile within DMSA group first 56 days
PBS solution, gives leaded drinking-water, and the DMSA solution of last 5 days daily stomach-filling 0.3mL 5mg/mL gives normal water.Experiment
After put to death mouse, take blood, liver, kidney, brain.Precision weighs blood, liver, kidney, and brain weight is put into after pure nitric acid is added
It is cleared up in Hyperfrequency waves eliminating stove, time 20min.Obtained digestion solution uses atomic absorption spectroscopy determination lead content.Knot
Fruit is as shown in table 11:
11 mice organs of table, blood lead content
Note: the group for indicating different letters indicates significant difference (P < 0.05).
By table 11 it is found that comparison blank control group, lead exposure model group, probiotics and microelement dietary supplements
The liver of intervention group, DMSA (traditional heavy metal detoxification agent) group, kidney, brain, blood lead content is it is found that probiotics and microelement
Dietary supplements, DMSA can significantly reduce mouse liver, kidney, brain, lead content in blood.Illustrate probiotics and micro
The dietary supplements of element has the function of reducing lead content in Mice Body.
Embodiment 10: dietary supplements alleviates lead exposure oxidative damage intervention experiment
Zoopery is carried out by grouping described in embodiment 9, mouse is put to death after experiment, takes blood, liver, kidney,
Brain.Superoxide dismutase (SOD), catalase are measured according to the method that Bioengineering Research Institute's kit is built up in Nanjing
(CAT), reductive glutathione (GSH), glutathione peroxidase (GSH-Px), malonaldehyde (MDA) content or level.
Test result such as table 12, table 13, shown in table 14:
12 dietary supplements of table leads to the intervention relaxation effect of liver oxidative damage to lead exposure poison
Note: different letters have significant difference (P < 0.05) between representing group
13 dietary supplements of table leads to the intervention relaxation effect of kidney oxidative damage to lead exposure poison
Note: different letters have significant difference (P < 0.05) between representing group
14 dietary supplements of table leads to the intervention relaxation effect of brain oxidative damage to lead exposure poison
Note :-indicate to be not detected；Different letters have significant difference (P < 0.05) between representing group
Superoxide dismutase (SOD), catalase (CAT) are antioxidases important in body, and oxidation is answered
Swash one of damage markers.Reduced glutathione (GSH) is the important antioxidant of the intracorporal one kind of biology.Malonaldehyde
It (MDA) is a kind of lipid peroxide, content can reflect the level of lipid peroxidation and cellular damage degree of body.Pass through
Table 12, table 13, table 14 is it is found that compared to blank control group, and the activity of the SOD of lead exposure model group, CAT significantly reduce, and GSH contains
Amount is remarkably decreased, and MDA content significantly rises.And lead exposure model group is compared, the dietary supplements of probiotics and microelement is dry
Pre- organize significantly has restored liver, kidney, and SOD activity caused by lead exposure reduces in brain tissue, has significantly restored liver, kidney group
The activity for knitting middle CAT reduces, and significantly improves liver, kidney, and the GSH content in brain tissue reduces MDA content.Two mercapto fourths
Diacid (DMSA) is a quasi-tradition heavy metal competitiveness antidote, comparison DMSA group discovery, the diet of probiotics and microelement
Replenishers effect ratio DMSA in terms of alleviating oxidativestress damage caused by lead exposure is stronger.In summary as a result, explanation is prebiotic
The dietary supplements of bacterium and microelement effectively alleviates oxidativestress damage caused by lead exposure.
Embodiment 11: dietary supplements, which alleviates aminolevulinate dehydratase activity in lead exposure Mice Body, reduces experiment
Zoopery is carried out by grouping described in embodiment 9, mouse is put to death after experiment, takes blood, after 4 DEG C of standing 2h
3000r/min is centrifuged 10min, takes upper serum.Ammonia is measured according to the method for Shanghai Chuan Xiang Biotechnology Co., Ltd kit
Base levulic acid dehydratase (ALAD) activity, test result are as shown in Table 15:
15 dietary supplements of table is to the alleviation that aminolevulinate dehydratase (ALAD) activity reduces caused by lead exposure poison
Effect
Note: different letters have significant difference (P < 0.05) between representing group
Aminolevulinate dehydratase participates in the biosynthesis of body ferroheme, directly related with body hemopoietic system.It is logical
Table 15 is crossed it is found that the activity of lead exposure model group ALAD significantly reduces compared to blank control group.And probiotics and microelement
Dietary supplements intervention group has significantly restored the activity of ALAD, and effect is suitable with tradition heavy metal detoxification agent DMSA.Illustrate prebiotic
The dietary supplements of bacterium and microelement effectively alleviates the work of aminolevulinate dehydratase caused by lead exposure (ALAD)
Property damage.
Embodiment 12: dietary supplements alleviates zinc protoporphyrin content in lead exposure Mice Body and rises experiment
Zoopery is carried out by grouping described in embodiment 9, mouse is put to death after experiment, takes blood, after 4 DEG C of standing 2h
3000r/min is centrifuged 10min, takes upper serum.Zinc is measured according to the method for Shanghai Chuan Xiang Biotechnology Co., Ltd kit
Protoporphyrin (ZPP) content, test result are as shown in table 16:
Table 16: the relaxation effect that dietary supplements rises amino zinc protoporphyrin content caused by lead exposure poison
Note: different letters have significant difference (P < 0.05) between representing group
The content of zinc protoporphyrin (ZPP) is one of index of lead poisoning in blood.By table 16 it is found that comparing blank control
Group, lead exposure model group ZPP content significantly rise.And the dietary supplements intervention group of probiotics and microelement significantly reduces
ZPP content, effect are suitable with tradition heavy metal detoxification agent DMSA.Illustrate that the dietary supplements of probiotics and microelement is effective
The content for alleviating zinc protoporphyrin caused by lead exposure (ZPP) rises, and alleviates lead poisoning.
Embodiment 13: dietary supplements alleviates lead exposure intelligent of ageing mice injury experiment
Step-through test is utilized mouse and becomes dark habit, keeps away concealed installation and sets and is divided into light room and darkroom.Light room illumination is 25W, darkroom
It is not provided with illumination, there is the connection of the channel 3cm between light room darkroom, it can random open and close.Light room, darkroom bottom are paved with copper grid, secretly
Room base copper grid can be powered, voltage 24V.Mouse enters darkroom and is shocked by electricity.Experiment is divided into training, formally tests two parts.
Training makes mouse possess electric shock memory, and end is formally tested afterwards for 24 hours, formal experimental observation mouse incubation period (mouse first
The secondary time completely into darkroom), into the errors number in darkroom, investigate the ability of learning and memory of mouse.The specific method is as follows:
Zoopery is carried out by grouping described in embodiment 9, mouse step-through test is carried out after stomach-filling.Step-through test first is in the sky
Noon 10:00 starts to train.Training time is 3min, and all mouse are individually placed among bright room, body back to channel after starting
Not face hidden door, channel is closed between light room darkroom.Channel between light room darkroom is opened after adapting to 1min, starts simultaneously at timing.
Mouse enters behind darkroom shocked by electricity after take out immediately.The next morning 10:00 starts formally to test, and formal experimental period is
Electric current is not added in 5min, darkroom.All mouse are individually placed among bright room, face is not dark for body back to channel after experiment starts
, channel is closed between light room darkroom.Channel between light room darkroom is opened after adapting to 1min, starts simultaneously at timing.Record 5min
Interior mouse incubation period and the errors number for entering darkroom.Experimental result is as shown in table 17:
Relaxation effect of 17 dietary supplements of table to intellectual damage caused by lead exposure poison
Note: different letters have significant difference (P < 0.05) between representing group
By table 17 it is found that comparing blank control group, lead exposure model group incubation period is obviously shortened, on errors number is obvious
It rises.The ability of learning and memory of mouse receives damage.And the dietary supplements intervention group of probiotics and microelement is significantly restored
Shorter latencies, the intellectual damage that errors number rises, effect are than traditional heavy metal detoxification agent DMSA more stronger.Illustrate probiotics
The ability of learning and memory of mouse is effectively restored with the dietary supplements of microelement.
Embodiment 14 prepares according to the following ratio while the dietary supplements containing profitable probliotics and microelement
(1) the lactobacillus plantarum CGMCC after spreading cultivation and washing is resuspended using 10% trehalose as freeze drying protectant
5494, make viable bacteria concentration up to 1011CFU/mL or more is freeze-dried after preculture 60min at 37 DEG C of temperature and is lyophilized
Bacterium powder.
(2) according to parts by weight, by 35.9% microcrystalline cellulose, 4% low-substituted hydroxypropyl cellulose, 0.1%
Superfine silica gel powder, 2% vitamin C, 7.5% calcium carbonate, 0.5% zinc acetate, 50% bacterium powder, mixing are mixed with facing-up method
It is even, obtain fine powder product.
(3) directly use tablet press machine with 2.4t pressure tabletting powder obtained in the previous step, obtained tablet weight is 250mg
±15mg。
The dietary supplements active component content obtained by this method is high, shelf life is long；Prebiotic bacterial content can reach 9.91
×109Cfu/g, viable count is still greater than 10 after storing 6 months at 4 DEG C6CFU/g；Ascorbic acid content is up to 19.25mg/g, 20
DEG C storage 6 months after still have 90% or more retention rate；The lead that can reduce the internal organs such as liver in Mice Body, kidney, brain simultaneously contains
It is living to alleviate oxidativestress damage caused by the lead poisoning of Mice Body internal cause, aminolevulinate dehydratase for the lead content of amount and blood
Property reduce, zinc protoporphyrin content rises, alleviate because intelligence caused by lead poisoning reduces.
Embodiment 15 prepares according to the following ratio while the dietary supplements containing profitable probliotics and microelement
(1) the lactobacillus plantarum CGMCC after spreading cultivation and washing is resuspended using 10% trehalose as freeze drying protectant
5494, make viable bacteria concentration up to 1011CFU/mL or more is freeze-dried after preculture 60min at 37 DEG C of temperature and is lyophilized
Bacterium powder.
(2) according to parts by weight, by 56.7% microcrystalline cellulose, 6% low-substituted hydroxypropyl cellulose, 0.3%
Superfine silica gel powder, 1% vitamin C, 5.7% calcium carbonate, 0.3% zinc acetate, 30% bacterium powder, mixing are mixed with facing-up method
It is even, obtain fine powder product.
(3) directly use tablet press machine with 2.4t pressure tabletting powder obtained in the previous step, obtained tablet weight is 250mg
±15mg。
The dietary supplements active component content obtained by this method is high, shelf life is long；Prebiotic bacterial content can reach 6.82
×109Cfu/g, viable count is still greater than 10 after storing 6 months at 4 DEG C6CFU/g；Ascorbic acid content is up to 9.77mg/g, 20
DEG C storage 6 months after still have 90% or more retention rate；The lead that can reduce the internal organs such as liver in Mice Body, kidney, brain simultaneously contains
It is living to alleviate oxidativestress damage caused by the lead poisoning of Mice Body internal cause, aminolevulinate dehydratase for the lead content of amount and blood
Property reduce, zinc protoporphyrin content rises, alleviate because intelligence caused by lead poisoning reduces.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (4)
1. a kind of dietary supplements containing profitable probliotics and microelement simultaneously, which is characterized in that the dietary supplements be with
The probiotics bacterial powder that freeze-drying obtains is main ingredient, is aided with antioxidant and microelement, is added after proper auxiliary materials with powder
It is prepared through pressed disc method；The probiotics is the lactobacillus plantarum (Lactobacillus that deposit number is CGMCC 5494
Plantarum), it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on November 29th, 2011
The heart；
The antioxidant is vitamin C；Microelement is calcium and zinc；Auxiliary material is one or more usually used in tablets
Filler, adhesive, disintegrating agent and lubricant；
The dietary supplements, according to parts by weight containing the microcrystalline cellulose of 35.9-56.7%, the low-substituted hydroxypropyl of 4-6%
Base cellulose, the superfine silica gel powder of 0.1-0.3%, the vitamin C of 1-2%, the calcium carbonate of 5.7-7.5%, the acetic acid of 0.3-0.5%
Zinc, the bacterium powder of 30-50%；
The viable bacteria content of lactobacillus plantarum CGMCC 5494 reaches 10 in the dietary supplements9CFU/g or more；
The freeze-drying is using the degreasing milk solution of the trehalose of 100-150g/L or 100-250g/L as frozen-dried protective
Agent；
It is through tablet press machine with 2.4t pressure tabletting that the pressed disc method, which carries out tabletting, and obtained tablet weight is 250mg ± 15mg.
2. dietary supplements according to claim 1, which is characterized in that the freeze-drying is the trehalose with 100g/L
As freeze drying protectant.
3. dietary supplements according to claim 1, the probiotics bacterial powder the preparation method is as follows: by lactobacillus plantarum
CGMCC 5494 expands culture, centrifugation, washing after activating, freeze drying protectant is then added, thallus is resuspended, reach cell concentration
1011CFU/mL or more, pre-freeze after mixing, is then freeze-dried.
4. dietary supplements according to claim 1, the dietary supplements, according to parts by weight containing 46.5% it is micro-
Crystalline cellulose, 5% low-substituted hydroxypropyl cellulose, 0.1% superfine silica gel powder, 1.6% vitamin C, 6.4% carbonic acid
Calcium, 0.4% zinc acetate, 40% bacterium powder.
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