CN108531417A - A kind of preparation method and product of the intestinal beneficial bacterium decomposing dietary fiber - Google Patents
A kind of preparation method and product of the intestinal beneficial bacterium decomposing dietary fiber Download PDFInfo
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- CN108531417A CN108531417A CN201810179129.1A CN201810179129A CN108531417A CN 108531417 A CN108531417 A CN 108531417A CN 201810179129 A CN201810179129 A CN 201810179129A CN 108531417 A CN108531417 A CN 108531417A
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Abstract
The present invention discloses a kind of preparation method and product of the intestinal beneficial bacterium decomposing dietary fiber, belongs to probio preparing technical field.The preparation method of the intestinal beneficial bacterium provided by the invention for decomposing dietary fiber includes the following steps：(1) caprophyl is once filtered, excrement slag is removed；(2) secondary filter recycles caprophyl；(3) caprophyl of recycling is transferred in culture solution；(4) shaking table shakes, and is incubated overnight；(5) it centrifuges, abandons supernatant, recycle beneficial bacterium.Product prepared by the preparation method of the intestinal beneficial bacterium provided by the invention for decomposing dietary fiber is the beneficial bacterium product mainly for dietary fiber decomposition or the weaker part population of absorbability, and associated beneficial bacterial strain, the enteron aisle stable state for adjusting and maintaining special population are screened from the caprophyl of healthy population.
Description
Technical field
The present invention relates to probio preparing technical field, the preparation of specifically a kind of intestinal beneficial bacterium for decomposing dietary fiber
Method.
Background technology
Hundreds of microorganism is inhabited in human intestine, exercises the important physiological functions such as digestion, nutrition, immune, symbiosis,
Numerous studies show that enteric microorganism has great influence to function of intestinal canal and physiological health.Fruit rich in dietary fiber and vegetable
Dish can be short chain fatty acids such as acetate, propionic acid by the bacterial decomposition in enteron aisle although cannot directly be digested by human body
Salt or butyrate etc., and then the utilization that is absorbed by the body adjust health and physiological metabolism.And to upset,gastro-intestinal, dietary fiber
The weaker special population of capacity of decomposition then needs corresponding specific beneficial microbial inoculum to adjust, digestion work(of the enhancing enteron aisle to dietary fiber
Energy.And existing caprophyl transplanting instantly, majority is for the disorder of the intestinal microecologies such as inflammatory bowel disease or intestinal irritable syndrome
Crowd plays more notable curative effect, has more researching value and meaning to the caprophyl of visible healthy population.For meals
Fiber hydrolization or the weaker part population of absorbability are eaten, then lacks corresponding beneficial bacterium product in the market, more lacks from health
Associated beneficial bacterial strain, the investigative technique of the enteron aisle stable state for adjusting and maintaining special population are screened in the caprophyl of crowd.
Invention content
Therefore, the technical problem to be solved in the present invention be to overcome it is in the prior art lack decomposed for dietary fiber or
The beneficial bacterium product of the weaker part population of absorbability, and associated beneficial bacterial strain is screened from the caprophyl of healthy population, it uses
In the defect of the investigative technique for the enteron aisle stable state for adjusting and maintaining special population, to provide a kind of enteron aisle decomposing dietary fiber
The preparation method and product of beneficial bacterium.
For this reason, the present invention provides the following technical solutions：
A kind of preparation method for the intestinal beneficial bacterium decomposing dietary fiber, includes the following steps：
(1) caprophyl is once filtered, excrement slag is removed；
(2) secondary filter recycles caprophyl；
(3) caprophyl of recycling is transferred in culture solution；
(4) shaking table shakes, and is incubated overnight；
(5) it centrifuges, abandons supernatant, recycle beneficial bacterium.
It is as follows：
(1) healthy excrement is collected, sterile water is added and shakes up, filters out excrement slag；
(2) secondary filter is carried out by the filter device equipped with 0.2 μm of filter membrane again, recycled on caprophyl to filter membrane；
(3) filter membrane with caprophyl in step (2) is added in inulin culture solution, seals culture solution, is then transferred to
In 37 ° of constant incubators, shaking table oscillation；
After (4) 24 hours, step (3) processed culture solution is transferred in 50mL centrifuge tubes, 8000rpm high speed centrifugations,
Supernatant is abandoned, beneficial bacterium is recycled；
(5) LB culture solutions are added, 80% glycerine shakes up beneficial bacterium mixed liquor, is preserved in -80 ° of refrigerators.
The inulin culture solution ingredient is as follows：Inulin, peptone, NaCl.
The proportioning of each ingredient of inulin culture solution is as follows：The inulin culture solution of 1 volume unit, including 20 parts by weight are oligomeric
Fructose, 10g peptones (provide nitrogen source), and 10 parts by weight NaCl, NaOH are adjusted to 7.0, unit of weight g, volume unit L.
The inulin culture liquor need to be at 120 °, 20min high pressure sterilizations.
The weight percent concentration of the inulin culture solution is：2-10%.
The raw material proportioning of the LB culture solutions is as follows：
0.5 parts by weight of peptone, 0.25 parts by weight of yeast extract, 0.5 parts by weight of sodium chloride add 50 parts by volume of water, weight
Amount unit is g, volume unit L.
In step (5), the volume ratio of the LB culture solutions and 80% glycerine is 1:1.
The obtained intestinal beneficial bacterium for decomposing dietary fiber is directly prepared by the preparation method.
Inulin and banaina are all rich in oligofructose.Oligofructose is a kind of water-soluble dietary fiber, and long-term use can be with
Serum cholesterol is reduced, lipid-metabolism is improved, is confirmed through animal and human experimentation.Oligofructose can be by beneficial bacteriums such as Bifidobacteriums
It is utilized, fermentation generates lactic acid, can promote absorption of the human body to minerals with minerals such as dissolving calcium, magnesium, iron.Human body is taken in
After oligofructose, internal profitable strain bifidobacteria can inhibit external source pathogenic bacteria and the intrinsic putrefactivebacteria of enteral such as Salmonella
The growth and breedings such as bacterium reduce the growth and accumulation of enteral corrupt substance, promote intestines peristalsis, prevent constipation and diarrhea.Oligomeric fruit
Sugar is a kind of excellent water-soluble dietary fiber, can effectively reduce the quantity of serum cholesterol, triglyceride, free fatty,
It has a better role for a series of angiocardiopathies such as hypertension, artery sclerosis because of caused by blood fat height.
The same banaina for being rich in dietary fiber, can also be decomposed by some intestinal beneficial bacteriums, do control group herein, with than
Compared with the yield for preparing beneficial bacterium with inulin culture solution.
Technical solution of the present invention has the following advantages that：
1. a kind of preparation method of intestinal beneficial bacterium decomposing dietary fiber provided by the invention is by optimizing dietary fiber
Culture solution, directed screening and acquisition intestinal beneficial bacterium.
2. a kind of preparation method of intestinal beneficial bacterium decomposing dietary fiber provided by the invention, which can reach, quickly prepares spy
Determine the intestinal beneficial microbial inoculum of crowd, and can further use same culture conditions on the basis of the beneficial bacterium being originally taken,
Expand beneficial bacterium production.
3. product is main prepared by a kind of preparation method of intestinal beneficial bacterium decomposing dietary fiber provided by the invention
For dietary fiber decomposition or the beneficial bacterium product of the weaker part population of absorbability, and sieved from the caprophyl of healthy population
Phase selection is closed beneficial to bacterial strain, the enteron aisle stable state for adjusting and maintaining special population.
Specific implementation mode
It is to preferably further understand the present invention to provide following embodiments, it is not limited to the best embodiment party
Formula is not construed as limiting present disclosure and protection domain, anyone under the inspiration of the present invention or by the present invention and its
The feature of his prior art be combined and obtain it is any with the present invention it is same or similar as product, all fall within the present invention
Within protection domain.
Specific experiment step or condition person are not specified in embodiment, according to routine experiment described in document in the art
The operation of step or condition can carry out.Reagents or instruments used without specified manufacturer, being can be by acquisition purchased in market
Conventional reagent product.
Inulin (Inulin) enzyme-linked immunologic detecting kit：It is available commercially from the Shanghai bio tech ltd Run Yu, specification
48T/96T。
The specific ingredient of inulin culture solution：Inulin, peptone, NaCl
One of formula：1L inulin culture solutions, including 20g oligofructose (providing carbon source, the energy for microorganism), 10g albumen
Peptone (provides nitrogen source), and 10g NaCl (provide microorganism growing environment, maintain osmotic balance), and it is (suitable that NaOH is adjusted to 7.0
Acid or alkali environment).Then, solution is at 120 °, 20min high pressure sterilizations.
Banaina culture solution ingredient：Banaina, peptone, NaCl
Formula：1L banana culture solutions, including 20g banainas (providing carbon source, the energy for microorganism), 10g peptones (provide
Nitrogen source), 10g NaCl (provide microorganism growing environment, maintain osmotic balance), and 7.0 (suitable soda acids are adjusted to NaOH
Environment).Then, solution is at 120 °, 20min high pressure sterilizations.
Embodiment 1
A kind of preparation method for the intestinal beneficial bacterium decomposing dietary fiber, includes the following steps：
(1) a small amount of excrement of healthy volunteer is collected in beaker, and sterile water is added and shakes up, excrement is filtered out by filter paper
Slag；
(2) secondary filter is carried out by the filter device equipped with 0.2 μm of filter membrane again, recycled on caprophyl to filter membrane；
(3) from the enteric microorganism being enriched in caprophyl on filter membrane, half is cut with the scissors of sterilizing, is put into 50mL, weight hundred
It is 2% inulin culture solution to divide specific concentration, and the filter membrane in (2) is added in culture solution, seals culture solution, is then transferred to 37 °
In constant incubator, shaking table oscillation.After 24 hours, culture solution is transferred in 50mL centrifuge tubes, 8000rpm high speed centrifugations are abandoned
Supernatant recycles beneficial bacterium；
(4) 2mL LB culture solutions are added, 80% glycerine of 2mL shakes up beneficial bacterium mixed liquor, is preserved in -80 ° of refrigerators.
Embodiment 2
A kind of preparation method for the intestinal beneficial bacterium decomposing dietary fiber, includes the following steps：
(1) a small amount of excrement of healthy volunteer is collected in beaker, and sterile water is added and shakes up, excrement is filtered out by filter paper
Slag；
(2) secondary filter is carried out by the filter device equipped with 0.2 μm of filter membrane again, recycled on caprophyl to filter membrane；
(3) from the enteric microorganism being enriched in caprophyl on filter membrane, half is cut with the scissors of sterilizing, is put into 50mL weight percents
Filter membrane in (2) is added in culture solution the inulin culture solution that specific concentration is 5%, seals culture solution, is then transferred to 37 ° of perseverances
In warm incubator, shaking table oscillation.After 24 hours, culture solution is transferred in 50mL centrifuge tubes, 8000rpm high speed centrifugations are abandoned
Clearly, beneficial bacterium is recycled；
(4) 2mL LB culture solutions are added, 80% glycerine of 2mL shakes up beneficial bacterium mixed liquor, is preserved in -80 ° of refrigerators.
Embodiment 3
A kind of preparation method for the intestinal beneficial bacterium decomposing dietary fiber, includes the following steps：
(1) a small amount of excrement of healthy volunteer is collected in beaker, and sterile water is added and shakes up, excrement is filtered out by filter paper
Slag；
(2) secondary filter is carried out by the filter device equipped with 0.2 μm of filter membrane again, recycled on caprophyl to filter membrane；
(3) from the enteric microorganism being enriched in caprophyl on filter membrane, half is cut with the scissors of sterilizing, is put into 50mL weight percents
Filter membrane in (2) is added in culture solution the inulin culture solution that specific concentration is 10%, seals culture solution, is then transferred to 37 ° of perseverances
In warm incubator, shaking table oscillation.After 24 hours, culture solution is transferred in 50mL centrifuge tubes, 8000rpm high speed centrifugations are abandoned
Clearly, beneficial bacterium is recycled；
(4) 2mL LB culture solutions are added, 80% glycerine of 2mL shakes up beneficial bacterium mixed liquor, is preserved in -80 ° of refrigerators.
Comparative example 1
(1) it is 10% inulin culture solution to use 50mL weight percent concentrations, adds 0.2 μm of sterile filter membrane, sealing training
Nutrient solution is then transferred in 37 ° of constant incubators, shaking table oscillation.After 24 hours, culture solution is transferred in 50mL centrifuge tubes,
8000rpm high speed centrifugations abandon supernatant, recycle beneficial bacterium；
After (2) 24 hours, the culture solution of experimental group is transferred in 50mL centrifuge tubes, supernatant is abandoned in 8000rpm high centrifugations,
Recycle beneficial bacterium.
(3) 2mL LB culture solutions are added, 80% glycerine of 2mL shakes up beneficial bacterium mixed liquor, is preserved in -80 ° of casees.
Comparative example 2
A kind of preparation method for the intestinal beneficial bacterium decomposing dietary fiber, includes the following steps：
(1) a small amount of excrement of healthy volunteer is collected in beaker, and sterile water is added and shakes up, excrement is filtered out by filter paper
Slag；
(2) secondary filter is carried out by the filter device equipped with 0.2 μm of filter membrane again, recycled on caprophyl to filter membrane；
(3) from the enteric microorganism being enriched in caprophyl on filter membrane, half is cut with the scissors of sterilizing, is put into the banaina of 50mL
Culture solution seals culture solution with masking foil, is then transferred in 37 ° of constant incubators, shaking table shaken cultivation；
After (4) 24 hours, the culture solution of experimental group is transferred in 50mL centrifuge tubes, 8000rpm high speed centrifugations are abandoned
Clearly, beneficial bacterium is recycled；
(5) 2mL LB culture solutions are added, 80% glycerine of 2mL shakes up beneficial bacterium mixed liquor, is preserved in -80 ° of refrigerators.
Experimental example 1：
The content of the experimental group and control group inulin before and after microorganism is added is detected and compared by inulin detection kit
Variation.
Experimental example 2
HPLC detects the production concentration that beneficial bacterium decomposes
Assay is carried out to the lactic acid of generation by high performance liquid chromatography (HPLC), to assess profitable strain to diet
The resolution ratio of fiber.Meanwhile sterile inulin culture solution is set, it is cultivated under similarity condition with experimental group, as blank control.
The lactic acid concn content of the inulin group (embodiment 2), banana group (comparative example 2) and the control group (comparative example 1) that measure is respectively
10.2mg/mL, 5.7mg/mL and 0mg/mL.Illustrate beneficial bacterium inulin group than banana group more can fast decoupled dietary fiber, into
Row self-reproduction, the beneficial bacterium yield higher accordingly obtained are more suitable for the extension culture and screening of beneficial bacterium.
Experimental example 3
UV spectrophotometer measuring is beneficial to bacteria concentration
It is sucked out in 25mL to centrifuge tube, is centrifuged, after abandoning supernatant, that recycles has from the inulin culture solution of 50mL
Beneficial bacteria class, addition 25mL sterile waters are dissolved and are diluted, then the bacterium solution of 1mL is therefrom sucked out with liquid-transfering gun, add 1mL sterile waters
It carries out secondary dilution and bacteria suspension is made, ultraviolet absorption value of this bacteria suspension at OD600nm is detected by spectrophotometer, is denoted as
OD1=0.851.It to the inulin culture solution of remaining 25mL, is centrifuged, abandons supernatant, obtained sediment beneficial to mushroom, be added to
Identical inulin culture solution carries out secondary be incubated overnight (condition of culture is with for the first time) to beneficial bacterium.It uses and experimental group (embodiment again
2) after same method dilution bacterium solution, the ultraviolet absorption value of the beneficial bacterium solution after culture is surveyed, OD2=1.483 is denoted as.By comparing
The difference of OD1 and OD2, OD2 are significantly greater than OD1, show to increase after culture beneficial to bacteria concentration, to demonstrate having of filtering out
Bacteria group can rely on inulin culture solution growth and breeding.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (9)
1. a kind of preparation method for the intestinal beneficial bacterium decomposing dietary fiber, which is characterized in that include the following steps：
(1) caprophyl is once filtered, excrement slag is removed；
(2) secondary filter recycles caprophyl；
(3) caprophyl of recycling is transferred in culture solution；
(4) shaking table shakes, and is incubated overnight；
(5) it centrifuges, abandons supernatant, recycle beneficial bacterium.
2. preparation method according to claim 1, which is characterized in that be as follows：
(1) healthy excrement is collected, sterile water is added and shakes up, filters out excrement slag；
(2) secondary filter is carried out by the filter device equipped with 0.2 μm of filter membrane again, recycled on caprophyl to filter membrane；
(3) filter membrane with caprophyl in step (2) is added in inulin culture solution, seals culture solution, is then transferred to 37 ° of perseverances
In warm incubator, shaking table oscillation；
After (4) 24 hours, step (3) processed culture solution is transferred in 50mL centrifuge tubes, and 8000rpm high speed centrifugations are abandoned
Clearly, beneficial bacterium is recycled；
(5) LB culture solutions are added, 80% glycerine shakes up beneficial bacterium mixed liquor, is preserved in -80 ° of refrigerators.
3. preparation method according to claim 2, which is characterized in that the inulin culture solution ingredient is as follows：Inulin, albumen
Peptone, NaCl.
4. preparation method according to claim 3, which is characterized in that the proportioning of each ingredient of inulin culture solution is as follows：1
The inulin culture solution of volume unit, including 20 parts by weight oligofructose, 10g peptones, 10 parts by weight NaCl are adjusted to NaOH
7.0, unit of weight g, volume unit L.
5. preparation method according to claim 4, which is characterized in that the inulin culture liquor need to be at 120 °, 20min
High pressure sterilization.
6. preparation method according to claim 5, which is characterized in that the weight percent concentration of the inulin culture solution
For：2-10%.
7. preparation method according to claim 6, which is characterized in that the raw material proportioning of the LB culture solutions is as follows：
0.5 parts by weight of peptone, 0.25 parts by weight of yeast extract, 0.5 parts by weight of sodium chloride add 50 parts by volume of water, weight list
Position is g, volume unit L.
8. preparation method according to claim 7, which is characterized in that in step (5), the LB culture solutions and described 80%
The volume ratio of glycerine is 1:1.
9. directly preparing the obtained intestinal beneficial for decomposing dietary fiber by any preparation methods of claim 1-8
Bacterium.
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Cited By (2)
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| CN109504727A (en) * | 2018-12-04 | 2019-03-22 | 山东中医药大学 | The method and detection method of reduced form glucorphanin are prepared using intestinal bacteria metabolism |
| CN112553118A (en) * | 2020-12-24 | 2021-03-26 | 上海理工大学 | Method for enriching beneficial bacteria by using prebiotics |
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