CN108853475A - Antibacterial suppository of a kind of acidification enteron aisle and preparation method thereof - Google Patents
Antibacterial suppository of a kind of acidification enteron aisle and preparation method thereof Download PDFInfo
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- CN108853475A CN108853475A CN201811144926.2A CN201811144926A CN108853475A CN 108853475 A CN108853475 A CN 108853475A CN 201811144926 A CN201811144926 A CN 201811144926A CN 108853475 A CN108853475 A CN 108853475A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
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- A—HUMAN NECESSITIES
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/02—Suppositories; Bougies; Bases therefor; Ovules
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Abstract
The invention belongs to functional suppository preparation technical fields, more particularly to antibacterial suppository of a kind of acidification enteron aisle and preparation method thereof, mixed fatty glycerides are dissolved by heating in water-bath, fermentation liquid and monostearate are sequentially added, is stirred in addition, gelatin is added after mixing, continue stirring to emulsus, it pours into the bolt film for coating glycerol, is cooled and shaped after temperature reduction, demoulding obtains finished product；Contain nisin in the fermentation liquid.The present invention generates the organic matters such as nisin and lactic acid using streptococcus lactis fermented whey powder, and acidification enteron aisle effectively inhibits spoilage organisms in enteron aisle to breed, and neutralizing ammonium ion in enteron aisle reduces its absorption strength, improves intestinal microenvironment.
Description
Technical field
The invention belongs to functional suppository preparation technical fields, and in particular to a kind of antibacterial suppository of acidification enteron aisle and its preparation
Method.
Background technique
Nisin (Nisin) is by Lactococcus lactis subsp. lactis (Loctococcus Lctis
Subsp.Lactis the polypeptide with antibacterial action that specific bacterial strain generates in).Nisin can inhibit most of leather
Growth of Lan Shi positive bacteria, including meat poisoning bacillus, heat-resisting spoilage organisms, bacillus stearothermophilus, clostridium sporogenes etc., and it is right
Saccharomycete and mould are invalid.But under certain condition, such as：Freezing, heating reduce pH value and through EDTA and other surfactants
Processing, also has lethal effect to part Gram-negative bacteria.The application of nisin has nothing compared with other preservatives
The characteristics of poison, dosage be few, easy to use, favorable anti-corrosion effect, is more and more valued by people.
The production of streptococcus lactis peptide is mainly with streptococcus lactis (Streptococcus lactic) or Lactococcus lactis
(Laclococcusla ctis) is obtained as production bacterium by the method for microbial fermentation.According to the requirement of its nutrient with
Proportion relation completes the approach and control methods of biosynthesis, and then expands nisin application range, therefore culture medium
Formulating it is especially significant.
Whey is the byproduct for producing cheese, casein etc., is cream yellow-green liquid remaining after enzyme or skyr.It is logical
Often production 1kg cheese can obtain 9kg whey.Backward in technique due to China's fermentation industry unreasonable structure, a large amount of wheys cannot close
Reason is utilized and is discarded, so that the development and utilization day of whey is aobvious urgent.Contain in cream 55% nutritional ingredient in whey, wherein albumen
About 1%, fat 0.4%, total reducing sugar 3%~5% (lactose accounts for 99.8%), in addition there are multivitamin and minerals.Lactic acid chain
The effect of the fermentation of coccus peptide is mainly influenced by carbon source and nitrogen source, inorganic salts, cation, surfactant and inhibitor
Type and content, which produce it, also to have a significant impact.
Summary of the invention
In order to solve the above-mentioned technical problems, the present invention provides generate nisin using streptococcus lactis fermented whey powder
The organic matters such as bacterium peptide and lactic acid, acidification enteron aisle effectively inhibit spoilage organisms in enteron aisle to breed, and neutralizing ammonium ion in enteron aisle reduces its suction
Concentration is received, intestinal microenvironment is improved.
The invention is realized in this way providing a kind of preparation method for being acidified the antibacterial suppository of enteron aisle, include the following steps：It will
Mixed fatty glycerides dissolve by heating in water-bath, sequentially add fermentation liquid and monostearate, stir in addition,
Gelatin is added after mixing, after continuing stirring to emulsus, pours into the bolt film for coating glycerol, is cooled to after temperature reduction
Type, demoulding obtain finished product；Contain nisin in the fermentation liquid.
Further, the preparation method of the fermentation liquid includes the following steps：
1) prepared by whey medium：Whey powder is hydrolyzed into obtain whey solution with 6% (W/V), adjusting pH is 6.8, takes whey molten
Liquid 1000ml adds soy oligosaccharides fructose 30g, yeast extract solution 2.5g, soybean protein powder soln 10g, Tween80 solution respectively
0.2g, tricalcium phosphate solution 0.8g, wherein soy oligosaccharides fructose mass concentration is 30%, and yeast extract concentration of polymer solution is
0.1%, soyabean protein powder mass concentration is that 2%, Tween80 mass concentration is 0.01%, and the mass concentration of tricalcium phosphate is
1%, whey medium is made after mixing；
2) prepared by fermentation liquid：Whey medium made from step 1) is sterilized 30min at 63 DEG C, is distributed into 250mL's
It is 100mL per bottled liquid measure in triangular flask, the streptococcus lactis for accessing 5g ferments, and 32 DEG C of fermentation temperature, time 15-
20h surveyed the pH and residual sugar of one time fermentation liquid, until pH value is between 3-4 every 2 hours during the fermentation.
Further, the mixed fatty glycerides are high hydroxyl value fatty glyceride and low hydroxyl value fatty glyceride
Mass ratio is 3:2 mixed fatty glycerides.
Further, the mass ratio of the mixed fatty glycerides and the fermentation liquid is 3:1, the fatty acid mixed
Glyceride, gelatin, monostearate mass ratio be 16：1.5：1.
Further, the temperature that the mixed fatty glycerides dissolve by heating in water-bath is 60 DEG C, and temperature reduces
It is poured into the bolt film for coating glycerol after to 50 DEG C.
Further, the best prescription composition of raw materials of the acidification antibacterial suppository of enteron aisle is：Fermentation liquid, fatty acid mixed are sweet
Grease, monostearate, gelatin quality are 1200g altogether, and suppository 400 processed altogether, every 3g, every contain nisin 2.5
×105RU/kg。
The present invention also provides the antibacterial suppositorys of acidification enteron aisle of above-mentioned method preparation.
Compared with the prior art, the advantages of the present invention are as follows：By generating the organic matters such as nisin and lactic acid, acid
Changing enteron aisle effectively inhibits spoilage organisms in enteron aisle to breed, and neutralizing ammonium ion in enteron aisle reduces its absorption strength, improves intestinal microenvironment.
The growth of caecum stool pathogenic bacteria can be effectively inhibited using intestinal canal administration method, and there is the function of sterilization and bacterium processed；Cream
Acid can change intestinal microenvironment, and reductions that liquid pH value occurs enhances the acidification of enteron aisle, coerce the high density increment of ammonia with
Endotoxic generation；There is certain auxiliary therapeutic action to anti-pre- and treatment hepatic encephalopathy.
Detailed description of the invention
Fig. 1 is influence result figure of the different lactose concns to M2 strain fermentation in whey solution；
Fig. 2 is influence result figure of the different soyabean protein powder concentration to M2 strain fermentation；
Fig. 3 is influence result figure of the different yeast extract concentration to M2 strain fermentation；
Fig. 4 is influence result figure of the different Tween80 concentration to M2 strain fermentation；
Fig. 5 is influence result figure of the different tricalcium phosphate concentration to M2 strain fermentation.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, below with reference to embodiment, to the present invention
It is further elaborated.It should be appreciated that specific embodiment described herein is used only for explaining the present invention, it is not used to
Limit the present invention.
Technical solution of the present invention in order to obtain, applicant spy is to each ingredient in whey medium to streptococcus lactis
Following research has been done in the influence of fermentation character, has selected optimum fermentation condition, and streptococcus lactis selects M2 bacterial strain：
1, the research of whey fermentation characteristic
Residual sugar and pH value change in the pure whey fermentation of table 1
It can be seen that M2 bacterial strain has certain Fermented in pure whey medium by data in table 1, with prolonging for time
Long, residual sugar and pH are gradually decreased, and after fermentation 16 hours, residual sugar and pH are no longer reduced, it is contemplated that in fermentation 16 hours or so
Measure the antibacterial circle diameter of fermentation liquid.
2, influence of the various concentration lactose to M2 strain fermentation
Whey powder is made into the whey solution of 1%, 3%, 6%, 9% concentration respectively, the obtained cream in whey medium
Sugared content is respectively 7.19%, 21.56%, 43.14%, 64.71%, measures antibacterial circle diameter, the result is shown in Figure 1 after the 16h that ferments.
As seen from Figure 1, M2 bacterial strain can in the whey of different lactose concns fermenting and producing streptococcus lactis peptide, inhibition zone
Diameter is smaller than the diameter under 7.19% and 21.56% concentration under 43.14% and 64.71% concentration, shows that high lactose is dense
Degree has inhibiting effect to the fermenting and producing of thalli growth and streptococcus lactis peptide.So optimal lactose concn is 21.56% or so.
But since the nutritional ingredient in whey is inadequate, the requirement of microorganism growth cannot be fully met, antibacterial circle diameter is low, so
Optimize whey medium in following tests, it is made to be more suitable for the fermenting and producing of streptococcus lactis peptide.
3, influence of the various concentration soyabean protein powder to M2 strain fermentation
Based on being 21.56% whey by lactose concn, the soybean protein powder soln of various concentration is added, fermentation 16 is small
Shi Hou measures antibacterial circle diameter with supernatant, as a result sees Fig. 2.
As shown in Figure 2, when soybean protein powder soln concentration is 1% and 2%, antibacterial circle diameter is bigger, continues growing big
Legumin powder concentration, antibacterial circle diameter decline, illustrating that concentration is excessive has inhibiting effect to streptococcus lactis peptide.So choosing big
The concentration of legumin powder solution is 2% or so.
4, influence of the different yeast extract solution concentrations to M2 strain fermentation
It can promote the synthesis of bacteriocin containing some amino acid in yeast extract, the whey for being 21.56% with lactose concn,
Based on adding 2% soybean protein powder soln, then the yeast extract solution of various concentration is added, is measured with fermented supernatant fluid antibacterial
As a result loop diameter is shown in Fig. 3.
By Fig. 3 data it is found that antibacterial circle diameter obviously increases after addition yeast extract solution, illustrate soyabean protein powder and yeast
The compound nitrogen source of cream composition is more advantageous to the generation of streptococcus lactis peptide.But as yeast extract solution concentration improves, antibacterial circle diameter becomes
Change unobvious, this may be because immobilized cell gel beads abundant proliferation before fermentation, immobilized cell fermenting
Only a small amount of in journey slowly to increase, nitrogen source is mainly used for the physiological activity of cell itself, comprehensively considers fermentation costs and selects the concentration to be
0.1% yeast extract solution additive amount.
5, influence of the difference Tween80 concentration to M2 strain fermentation
The whey for being 21.56% with lactose concn, adds 2% soybean protein powder soln and 0.1% yeast extract solution
Based on, then the Tween80 solution of various concentration is added, antibacterial circle diameter is measured with fermented supernatant fluid, as a result sees Fig. 4.
By Fig. 4 data it is found that antibacterial circle diameter obviously increases after addition Tween80, between 0~0.1, with
The increase antibacterial circle diameter of Tween80 concentration increases.It is because Tween80 is as a kind of surfactant, is heat-resisting water-soluble
The Ester of property long chain fatty acids, can reduce the surface tension between bacterial body and culture medium contact surface, can stimulate a variety of born of the same parents
The generation of exoenzyme can influence the membrane permeability of certain micro-organisms cell, and nutriment is promoted to enter cell and streptococcus lactis peptide exclusion body
Outside.Furthermore Tween80 can promote the dissolution for being adsorbed on the bacteriocin of cell surface, but the excessive concentration of Tween80, inhibition zone
Diameter decline, it is 0.01% that comprehensive fermentation costs consideration, which selects the concentration of Tween80,.
6, influence of the different tricalcium phosphate solution concentrations to M2 strain fermentation
The most thorny issue is calcium alginate gel and PO during Immobilization in Sodium Alginate4 -3Tricalcium phosphate is formed, is made
Calcium alginate gel structure is destroyed, and PO4 -3It is that cell is indispensable.The general PO for using low concentration at present4 -3Or increase chlorine
The amount for changing calcium, makes calcium alginate gel be unlikely to rupture.Scherer etc. is used and a certain amount of tricresyl phosphate is directly added in matrix
Calcium allows microbial cell to draw PO from tricalcium phosphate4 -3, can prevent the rupture of gel particle from dissolving.According to Wang etc., card
It draws progress embedded immobilization cell after glue addition tricalcium phosphate to have many advantages, if pH is adjusted, enhances cell viability,
Cell loading amount is improved, yield etc. is increased.Therefore, in the calcium chloride and 0.01% of the fermentation medium addition 1% optimized above
Potassium dihydrogen phosphate based on, investigate addition tricalcium phosphate to complex carrier embedded immobilization M2 bacterial strain production streptococcus lactis peptide
It influences.As a result see Fig. 5.
By Fig. 5 data it is found that antibacterial circle diameter increases after addition tricalcium phosphate, in 1g/L, antibacterial circle diameter is maximum, after
The continuous additive amount antibacterial circle diameter for increasing tricalcium phosphate becomes smaller, so taking the additive amount of tricalcium phosphate solution is 1g/L.
7, the determination of whey sterilization method
The comparison of 2 two kinds of sterilization methods of table
Lactalbumin is denaturalized at high temperature, and whey medium becomes buff, and has lumpy precipitate, fermenting property drop
It is low.So 63 DEG C are chosen in test, 30min sterilizing.
Embodiment,
The present embodiment provides a kind of preparation methods for being acidified the antibacterial suppository of enteron aisle, include the following steps：
1) mixed fatty glycerides for taking high hydroxyl value fatty glyceride and low hydroxyl value fatty glyceride to form, will
Mixed fatty glycerides 60 DEG C of heating for dissolving in water-bath；
2) fermentation liquid and monostearate are sequentially added, is stirred in addition, the preparation method of fermentation liquid includes following step
Suddenly：
A, prepared by whey medium：Whey powder is hydrolyzed into obtain whey solution with 6% (W/V), lactose concn is 22% at this time,
Adjusting pH is 6.8, takes whey solution 1000ml, and addition yeast extract solution 2.5g, soybean protein powder soln 10g, Tween80 are molten respectively
Liquid 0.2g, tricalcium phosphate solution 0.8g, wherein yeast extract concentration of polymer solution is 0.1%, and soyabean protein powder mass concentration is
2%, Tween80 mass concentration are 0.01%, and the mass concentration of tricalcium phosphate is 1%, and whey medium is made after mixing；
B, prepared by fermentation liquid：Whey medium made from step 1) is sterilized 30min at 63 DEG C, is distributed into 250mL's
It is 100mL per bottled liquid measure in triangular flask, the streptococcus lactis for accessing 5g ferments, and 32 DEG C of fermentation temperature, time 15-
20h surveyed the pH and residual sugar of one time fermentation liquid, until pH value is between 3-4 every 2 hours during the fermentation.
3) gelatin is added after mixing, after continuing stirring to emulsus,
4) it pours into the bolt film for coating glycerol, is cooled and shaped after temperature is reduced to 50 DEG C, demoulding obtains finished product；Wherein,
Fermentation liquid, mixed fatty glycerides, monostearate, gelatin quality are 1200g altogether, suppository 400 processed altogether, every 3g, often
Grain contains nisin 2.5 × 105RU/kg。
Nisin activity measurement：
Indicate the preparation of bacteria suspension：One, staphylococcus aureus inclined-plane is taken, 10mL sterile water is added, lawn is all scraped
Under, it pours into the triangular flask of the 90mL sterile water equipped with bead, shakes and bacteria suspension is made as stoste, stoste is diluted to
10-1Dilution (10-1The bacteria suspension concentration of dilution is about 109Cfu/mL or OD600Value is near 0.35).
Fermentation liquor pretreatment：It is 2.5 that fermentation liquid is adjusted pH value with the HCl of 10mol/L under stiring, is then added at 90 DEG C
Hot 30min, 5000r/min are centrifuged 12min, remove microorganism collection supernatant.
Diffusion method is quantified using Oxford cup：In the nutrient agar melted on superclean bench with sterilizing Big caliber straw absorption
20mL injects internal diameter for 90mm, and in the plate of high 16-17mm, 10mL instruction bacterium solution is poured into after to be solidified (with 45~50 DEG C of battalion
Feeding agar is made into, bacteria concentration 109A/mL), and make its coating uniformly, it is to be solidified after place Oxford cup (internal diameter 6.0 ±
0.1mm, high 8.0 ± 0.1mm), the distance between Oxford cup is sufficiently large, in order to avoid inhibition zone is overlapped, is started after standing 5~10min
Fermentation liquid is added dropwise, is carefully placed into incubator, after 30 DEG C of cultures for 24 hours, surveying antibacterial circle diameter size with vernier caliper is 18.25mm.
Claims (7)
1. a kind of preparation method for being acidified the antibacterial suppository of enteron aisle, which is characterized in that include the following steps：By mixing-in fat acid glycerol
Ester dissolves by heating in water-bath, sequentially adds fermentation liquid and monostearate, stirs in addition, is added after mixing
Gelatin after continuing stirring to emulsus, is poured into the bolt film for coating glycerol after temperature reduction, is cooled and shaped, demoulding obtains finished product；
Contain nisin in the fermentation liquid.
2. the preparation method of the acidification antibacterial suppository of enteron aisle described in accordance with the claim 1, which is characterized in that the system of the fermentation liquid
Preparation Method includes the following steps：
1) prepared by whey medium：Whey powder is hydrolyzed into obtain whey solution with 6% (W/V), adjusting pH is 6.8, takes whey solution
1000ml adds soy oligosaccharides fructose 30g, yeast extract solution 2.5g, soybean protein powder soln 10g, Tween80 solution respectively
0.2g, tricalcium phosphate solution 0.8g, wherein soy oligosaccharides fructose mass concentration is 30%, and yeast extract concentration of polymer solution is
0.1%, soyabean protein powder mass concentration is that 2%, Tween80 mass concentration is 0.01%, and the mass concentration of tricalcium phosphate is
1%, whey medium is made after mixing；
2) prepared by fermentation liquid：Whey medium made from step 1) is sterilized 30min at 63 DEG C, is distributed into the triangle of 250mL
It is 100mL per bottled liquid measure in bottle, the streptococcus lactis for accessing 5g ferments, and 32 DEG C of fermentation temperature, time 15-20h,
The pH and residual sugar for surveying one time fermentation liquid in fermentation process every 2 hours, until pH value is between 3-4.
3. the preparation method of the acidification antibacterial suppository of enteron aisle described in accordance with the claim 1, which is characterized in that the fatty acid mixed
Glyceride is high hydroxyl value fatty glyceride and low hydroxyl value fatty glyceride mass ratio is 3:2 mixed fatty glycerides.
4. the preparation method of the acidification antibacterial suppository of enteron aisle described in accordance with the claim 1, which is characterized in that the fatty acid mixed
The mass ratio of glyceride and the fermentation liquid is 3:1, the mixed fatty glycerides, gelatin, monostearate mass ratio
It is 16：1.5：1.
5. the preparation method of the acidification antibacterial suppository of enteron aisle described in accordance with the claim 1, which is characterized in that the fatty acid mixed
The temperature that glyceride dissolves by heating in water-bath is 60 DEG C, and temperature is poured into after being reduced to 50 DEG C in the bolt film for coating glycerol.
6. the preparation method of the acidification antibacterial suppository of enteron aisle described in accordance with the claim 1, which is characterized in that the acidification enteron aisle suppression
The best prescription composition of raw materials of bacterium suppository is：Fermentation liquid, mixed fatty glycerides, monostearate, gelatin quality are altogether
1200g, suppository 400 processed altogether, every 3g, every contain nisin 2.5 × 105RU/kg。
7. according to the antibacterial suppository of acidification enteron aisle of any method preparation of claim 1-6.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1663573A (en) * | 2004-03-04 | 2005-09-07 | 青岛东海药业有限公司 | A stable and safe microecological formulation, its preparation and usage |
| CN1899604A (en) * | 2005-07-19 | 2007-01-24 | 长春金赛药业有限责任公司 | Rectal suppository of protein medicines and its preparing method and use in systemic disease therapy |
| WO2009135945A1 (en) * | 2008-05-09 | 2009-11-12 | University College Cork-National University Of Ireland, Cork | Nisin derivatives and the use thereof |
| CN102018707A (en) * | 2010-11-29 | 2011-04-20 | 昆明邦宇制药有限公司 | Medicinal composition for treating diarrhea and preparation thereof |
| CN102753142A (en) * | 2009-12-02 | 2012-10-24 | 贝蒂纳·海尔 | Suppository for rectal, vaginal or urethral administration containing a probiotic, an antibiotic and an unsaturated non- esterified fatty acid |
-
2018
- 2018-09-29 CN CN201811144926.2A patent/CN108853475B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1663573A (en) * | 2004-03-04 | 2005-09-07 | 青岛东海药业有限公司 | A stable and safe microecological formulation, its preparation and usage |
| CN1899604A (en) * | 2005-07-19 | 2007-01-24 | 长春金赛药业有限责任公司 | Rectal suppository of protein medicines and its preparing method and use in systemic disease therapy |
| WO2009135945A1 (en) * | 2008-05-09 | 2009-11-12 | University College Cork-National University Of Ireland, Cork | Nisin derivatives and the use thereof |
| CN102753142A (en) * | 2009-12-02 | 2012-10-24 | 贝蒂纳·海尔 | Suppository for rectal, vaginal or urethral administration containing a probiotic, an antibiotic and an unsaturated non- esterified fatty acid |
| CN102018707A (en) * | 2010-11-29 | 2011-04-20 | 昆明邦宇制药有限公司 | Medicinal composition for treating diarrhea and preparation thereof |
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