CN110201099A - The method of Pediococcus pentosaceus pure-blood ferment Medicated Leaven - Google Patents
The method of Pediococcus pentosaceus pure-blood ferment Medicated Leaven Download PDFInfo
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Abstract
The present invention provides a kind of Medicated Leaven pure-blood ferment methods, it is fermented in raw material needed for inoculation Pediococcus pentosaceus is fermented to traditional Medicated Leaven；Raw material needed for the tradition Medicated Leaven ferments need to be through 100 DEG C or more high-temperature sterilizations.The present invention also provides the obtained Medicated Leaven products of this method.Medicated Leaven of the invention has the ability of outstanding promotion gastric emptying.Its effect is higher than well the Medicated Leaven that spontaneous fermentation obtains, also above bacillus subtilis, absidia corymbifera, aspergillus flavus and aspergillus niger pure breed fermentation gained Medicated Leaven.
Description
Technical field
The present invention relates to Chinese medicine Medicated Leavens to process field.
Background technique
Medicated Leaven is a kind of traditional Chinese medicine, by polygonum flaccidum, sweet wormwood, Siberian cocklebur grass, rde bean, semen armeniacae amarae, wheat bran, flour fermentation system
It is standby to form.Medicated Leaven has strengthening the spleen and stomach, the effect to help digestion in adjusting；For weakness of the spleen and the stomach, retention of food and drink, feeling of stuffiness in chest abdominal distension, children
Dyspepsia.
Currently, Medicated Leaven fermentation is that traditional natural ferments, miscellaneous bacteria is more in fermented sample, and different environment also can be to six minds
Qu Zhiliang is affected greatly.
Summary of the invention
The object of the present invention is to provide a kind of pure-blood ferment methods of Medicated Leaven.
Technical solution of the present invention includes:
A kind of Medicated Leaven pure-blood ferment method, it is carried out in raw material needed for inoculation Pediococcus pentosaceus is fermented to Medicated Leaven
Fermentation；Raw material needed for the tradition Medicated Leaven ferments need to be through 100 DEG C or more high-temperature sterilizations；
Raw material needed for the Medicated Leaven ferments includes: Siberian cocklebur grass, sweet wormwood, polygonum flaccidum, rde bean, semen armeniacae amarae, wheat bran and flour.
Medicated Leaven pure-blood ferment method as the aforementioned, the high-temperature sterilization are 121 DEG C of sterilizing 20min.
Medicated Leaven pure-blood ferment method as the aforementioned, raw material needed for the tradition Medicated Leaven ferments are as follows: 4~6 weight of Siberian cocklebur grass
Amount part, 4~6 parts by weight of sweet wormwood, 4~6 parts by weight of polygonum flaccidum, 0.5~1.5 parts by weight of rde bean, 0.5~1.5 parts by weight of semen armeniacae amarae,
45~55 parts by weight of wheat bran, 20~30 parts by weight of flour；
The semen armeniacae amarae, rde bean are the coarse powder that can cross No. 2 sieves；
The Siberian cocklebur grass, sweet wormwood, polygonum flaccidum are to be mixed in a manner of water extract with rde bean, semen armeniacae amarae, wheat bran, flour；
Medicated Leaven pure-blood ferment method as the aforementioned, the weight of the water extract are 6.5 times of primary dose.
Medicated Leaven pure-blood ferment method as the aforementioned, the inoculum concentration of the corresponding Pediococcus pentosaceus of every g raw material be (1~3) ×
105A thallus；
Preferably, the inoculum concentration of the corresponding Pediococcus pentosaceus of every g raw material is 2 × 105A thallus.
Medicated Leaven pure-blood ferment method as the aforementioned, it is plus cotton plug ferments in gnotobasis；
Its fermentation condition is:
35~38 DEG C of temperature；
And/or humidity 70~80%；
And/or 4~7 days.
Medicated Leaven pure-blood ferment method as the aforementioned, fermentation condition is:
37 DEG C of temperature；
And/or humidity 75%；
And/or 4 days.
The aforementioned resulting Medicated Leaven of Medicated Leaven pure-blood ferment method.
There are aroma in Medicated Leaven as the aforementioned, partial region there are flocculence white colony.
The invention has the following beneficial effects:
1) products obtained therefrom smell of the present invention is preferable.Medicated Leaven fragrance component is mainly ethyl linoleate, ethyl palmitate, and
In the content of this 2 kinds of ingredients, the present invention is higher than the Medicated Leaven content of spontaneous fermentation.
2) products obtained therefrom of the present invention has the ability of outstanding promotion gastric emptying.Its effect is higher than well what spontaneous fermentation obtained
Medicated Leaven, also above bacillus subtilis, absidia corymbifera, aspergillus flavus and aspergillus niger pure breed fermentation gained Medicated Leaven.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
Above content of the invention is described in further detail again below by way of specific embodiment.But it should not be by this
The range for being interpreted as the above-mentioned theme of the present invention is only limitted to example below.All technologies realized based on above content of the present invention are equal
Belong to the scope of the present invention.
Detailed description of the invention
Fig. 1: the outside drawing of Medicated Leaven obtained by spontaneous fermentation.
Fig. 2: the outside drawing of Medicated Leaven obtained by Pediococcus pentosaceus pure-blood ferment.
Fig. 3: the outside drawing of Medicated Leaven obtained by bacillus subtilis pure-blood ferment.
Fig. 4: the outside drawing of Medicated Leaven obtained by absidia corymbifera pure-blood ferment.
Fig. 5: the outside drawing of Medicated Leaven obtained by aspergillus flavus pure-blood ferment.
Fig. 6: the outside drawing of aspergillus niger pure breed fermentation gained Medicated Leaven.
Fig. 7: the Wei Entu of ingredient in Medicated Leaven obtained by different fermentations mode.
Specific embodiment
Some materials source involved by this part is as follows:
1 instrument
2 reagents
The Medicated Leaven pure-blood ferment method of the invention of embodiment 1
1. prepared by softwood (material)
1.5g semen armeniacae amarae, the mixing of 1.5g rde bean, cross No. 2 sieves and are ground into coarse powder, mix with 30g flour, 55g wheat bran, then
It is sub-packed in 250mL wide-mouth bottle, jumps a queue, sealed with brown paper, 121 DEG C of sterilizing 20min.
Siberian cocklebur grass 4g, sweet wormwood 4g, polygonum flaccidum 4g are taken, 12 times of amount water are added, is impregnated 30 minutes, is decocted 1 hour, with 200 mesh filter clothes
Sheet frame filtration, filtrate are condensed into clear cream (6.5 times of amount water of crude drug in whole), 121 DEG C of sterilizing 20min.
Raw material after above-mentioned sterilizing is sufficiently mixed after letting cool under ultraviolet lamp, obtains softwood.
2. fermentation
Aseptically, taking 10ml concentration is 3 × 106Thallus/mL Pediococcus pentosaceus suspension, it is sterilized with oneself
Softwood uniformly mixes, and clogs wide-mouth bottle bottleneck with cotton, sets 35 DEG C, fermented and cultured 6d in the climatic chamber of humidity 70%.Hair
After ferment, material is taken out and is lyophilized in freeze drier spare.
The Medicated Leaven pure-blood ferment method of the invention of embodiment 2
1. prepared by softwood (material)
1g semen armeniacae amarae, the mixing of 1g rde bean, cross No. 2 sieves and are ground into coarse powder, mix with 25g flour, 50g wheat bran, then dispense
It in 250mL wide-mouth bottle, jumps a queue, is sealed with brown paper, 121 DEG C of sterilizing 20min.
Siberian cocklebur grass 5g, sweet wormwood 5g, polygonum flaccidum 5g are taken, 12 times of amount water are added, is impregnated 30 minutes, is decocted 1 hour, with 200 mesh filter clothes
Sheet frame filtration, filtrate are condensed into clear cream (6.5 times of amount water of crude drug in whole), 121 DEG C of sterilizing 20min.
Raw material after above-mentioned sterilizing is sufficiently mixed after letting cool under ultraviolet lamp, obtains softwood.
2. fermentation
Aseptically, taking 10ml concentration is 2 × 106Thallus/mL Pediococcus pentosaceus suspension, it is sterilized with oneself
Softwood uniformly mixes, and clogs wide-mouth bottle bottleneck with cotton, sets 37 DEG C, fermented and cultured 4d in the climatic chamber of humidity 75%.Hair
After ferment, material is taken out and is lyophilized in freeze drier spare.
The Medicated Leaven pure-blood ferment method of the invention of embodiment 3
1. prepared by softwood (material)
0.5g semen armeniacae amarae, the mixing of 0.5g rde bean, cross No. 2 sieves and are ground into coarse powder, mix with 20g flour, 45g wheat bran, then
It is sub-packed in 250mL wide-mouth bottle, jumps a queue, sealed with brown paper, 121 DEG C of sterilizing 20min.
Siberian cocklebur grass 6g, sweet wormwood 4g, polygonum flaccidum 5g are taken, 12 times of amount water are added, is impregnated 30 minutes, is decocted 1 hour, with 200 mesh filter clothes
Sheet frame filtration, filtrate are condensed into clear cream (6.5 times of amount water of crude drug in whole), 121 DEG C of sterilizing 20min.
Raw material after above-mentioned sterilizing is sufficiently mixed after letting cool under ultraviolet lamp, obtains softwood.
2. fermentation
Aseptically, taking 10ml concentration is 0.82 × 106Thallus/mL Pediococcus pentosaceus suspension, with oneself through going out
The softwood of bacterium uniformly mixes, and clogs wide-mouth bottle bottleneck with cotton, sets 38 DEG C, fermented and cultured in the climatic chamber of humidity 80%
7d.After fermentation, material is taken out and is lyophilized in freeze drier spare.
Beneficial effects of the present invention will be further illustrated in the form of experimental example below.
The comparison of 1 different microorganisms pure-blood ferment Medicated Leaven of experimental example and traditional zymotic Medicated Leaven
1. fermentation process
1.1 traditional zymotic
Semen armeniacae amarae, rde bean mixing, cross No. 2 sieves and are ground into coarse powder, mix with flour, wheat bran；Separately take polygonum flaccidum, sweet wormwood, grey
Auricled Hedyotis Herb adds 12 times of amount water, impregnates 30 minutes, decocts 1 hour, is filtered with 200 mesh filter cloth sheet frames, and filtrate is condensed into clear cream (crude drug in whole
6.5 times of amount water), it is mixed thoroughly while hot with above-mentioned medicinal powder, the i.e. scattered softwood for the agglomerating touching held is made, in 37 DEG C, the perseverance of 75% humidity
Ferment four days in constant temperature and humidity case, it is dry to get.Prescription: Siberian cocklebur grass 5g, sweet wormwood 5g, polygonum flaccidum 5g, rde bean 1g, semen armeniacae amarae 1g, wheat
Bran 50g, flour 25g.
The preparation of 1.2 pure-blood ferment softwoods (material)
Semen armeniacae amarae, rde bean mixing, cross No. 2 sieves and are ground into coarse powder, mix with flour, wheat bran, then are sub-packed in 250mL wide-mouth
In bottle, jumps a queue, sealed with brown paper；Sweet wormwood, peppery Liao, Siberian cocklebur grass are same as above after decocting and concentrating loaded on conical flask, are jumped a queue, the above raw material
It is set after being let cool under 121 DEG C of sterilizing 20min, ultraviolet lamp in high-pressure steam sterilizing pan together, it is spare that softwood is made.Raw material prescription with
Traditional zymotic is identical.
1.3 potato culture PDA
It weighs 4.3g potato dextrose agar to be dissolved in 100mL pure water, boil to dissolution.Through 121 DEG C of 20min after packing
High pressure sterilization is spare.The culture medium is for cultivating absidia corymbifera.
1.4 beef-protein medium
Beef extract 0.3g is weighed, peptone 1g, NaC 1.5g, agar 1.5g, water 100mL boil hydrotropy solution.It is passed through after packing
121 DEG C of 20min high pressure sterilizations are spare.The culture medium is for cultivating bacillus subtilis.
1.5 wort agar medium
Wort agar powder 4g is weighed, with water 100mL, boils hydrotropy solution.It is standby through 121 DEG C of 20min high pressure sterilizations after packing
With.The culture medium is for cultivating aspergillus flavus, aspergillus niger.
1.6 MRS broth bouillons
MRS culture medium 5g is taken, adds pure water 100mL, stirring is to being completely dissolved.Through 121 DEG C of 20min high pressure sterilizations after packing
It is spare.The culture medium is for cultivating Pediococcus pentosaceus.
1.7 pure-blood ferment
1.7.1 the preparation of spore suspension
Pediococcus pentosaceus, bacillus subtilis, aspergillus niger, aspergillus flavus and absidia corymbifera are brought back to life respectively, are inoculated into respectively
It on ready sterilising medium plate, sets in incubator and cultivates, wherein Pediococcus pentosaceus (30 DEG C), bacillus subtilis (28
DEG C), aspergillus niger, aspergillus flavus and absidia corymbifera (37 DEG C) are rinsed spore/thallus with aqua sterilisa when flora growth is luxuriant
Get off, count (bacterium is counted with thallus, and fungi is with spore count) using microscopic dyeing method, and it is 2 × 10 that concentration, which is made,6Spore
(or thallus) mL-1Spore/thallus suspension liquid it is spare.
1.7.2 pure-blood ferment
Aseptically, the above-mentioned spore/thallus suspension liquid of 10ml is taken respectively, is uniformly mixed with oneself sterilized softwood,
Wide-mouth bottle bottleneck is clogged with cotton, sets 37 DEG C, fermented and cultured 4d in the climatic chamber of humidity 75%.After fermentation, material is taken
It is lyophilized out and in freeze drier spare.
The comparison of 2 fermented samples
2.1 nature and flavor compare
Medicated Leaven character description is blocky or coarse granule shape, surface yellow, there is the mould clothing of canescence, slightly wine aroma or aging
Gas, mildly bitter flavor.Normal fermentation is mostly fermentation emphasis with " surface is all over raw yellow-white or the mould clothing of canescence ", and fermented sample has more
Distinctive smell after fermentation, so the superiority and inferiority of fermented sample can be compared according to the surface texture of sample after fermentation and smell.
2.2 effects that help digestion compare
2.2.1 the preparation of medical fluid
Spontaneous fermentation (ZR), Pediococcus pentosaceus fermentation (WTPQ), fermentation of bacillus subtilis (KCYB), umbrella branch are ploughed respectively
The sample of mould fermentation (SZLT), aspergillus flavus fermentation (HQM) and fermentation of Aspergillus niger (HEI), takes Medicated Leaven sample appropriate respectively, with
10 times of amount water mix, and impregnate 30min, twice, each 1h, 200 mesh filtered through gauze filtrate will merge water-bath refluxing extraction twice
Afterwards, concentrated by rotary evaporation is to required concentration.Digestion-promoting stomachic is ground into a powder plus required concentration is made in water.
2.2.2 influence of the different samples to normal mouse functions of intestines and stomach
Mouse is pressed into weight, gender is randomly divided into 8 groups, every group 10.Respectively blank group, ZR group, WTPQ group, KCYB
Group, SZLT group, HQW group, HEI group, digestion-promoting stomachic group.Healthy SPF grades of KM mouse 60 are taken, weighing is random by weight, gender
It is divided into 6 groups, every group 10.After animal conventinal breeding 3 days, wherein model control group gives physiological saline, remaining several groups are given phase
The medical fluid answered, continuous gavage 7 days.
Be deprived of food but not water 10 hours before experiment, after the last administration after 0.5 hour each group give 0.05% carbon powder 0.3ml/
Only, cervical dislocation is distinguished after 20min to put to death, take cardia to rectum end stomach and intestine immediately, stomach is taken from pyloric sphincter, by it
It is put into chopping in 8ml normal saline solution to stir evenly, stands 1h, 3000rmin at room temperature-1It is centrifuged 15min, supernatant is in 546nm
Place surveys absorbance (A value) and is the residual quantity of carbon powder in stomach, and puts to death immediately after in addition taking 4 mouse to give carbon powder suspension, takes
Stomach surveys the benchmark absorbance that its absorbance is carbon powder.Gastric emptying rate (Gastric emptying) %=(carbon powder/A in 1-A stomach
Benchmark carbon powder) × 100%.It takes out small intestine and separates mesenterium, clip pylorus to the intestinal tube in ileocecum portion is placed on pallet, gently
Small intestine is pulled into straight line, the distance and small intestine overall length that measurement carbon powder advances.Intestinal propulsive rate (Intestinal
Transit) distance/small intestine overall length × 100% that %=carbon powder advances
2.2.3 statistical procedures
Experimental data unified expression are as follows: use mean+SD (x ± s), Data Analysis Software SPSS
21.0 softwares.As a result analysis is compared with t inspection.Analyzing result is that difference has statistical significance with P < 0.05.
2.3 GC-MS analysis
2.3.1 chromatographic condition
DB-5MS quartz capillary column [30m × 0.25mm × 0.25 μm], injector temperature: 280 DEG C.The method of sample introduction
Method: temperature program: 60 DEG C of the column temperature most started, with 20 DEG C of min-1200 DEG C are warming up to, then with 10 DEG C of min-1It is warming up to 280
DEG C keep 20min；Carrier gas: high-pure helium, flow velocity 1mLmin-1；Input mode: split sampling；Split ratio: 50: 1；Sample volume: 1 μ
L。
2.3.2 Mass Spectrometry Conditions
Chromatography and mass spectrometer interface temperature: 280 DEG C；Ionization mode: electron bombardment ionization source (EI)；Monitoring mode: full scan；Scanning
Range: 35~550aum；Ionizing energy: 70eV；Solvent delay: 2min；Ion source temperature: 280 DEG C；Compose library: INST2005.
2.3.1 sample preparation
Medicated Leaven each sample powder 20.0g (80 mesh) is weighed respectively, and precision weighs, with petroleum ether (60~90 DEG C) 200mL
Reflux 3h obtains petroleum ether extract, and recycling design is redissolved to doing with chloroform, and being made into concentration is 5mgmL-1Solution adds a small amount of
Anhydrous sodium sulfate dehydration and drying, sealing are used as test solution.
3 experimental results
3.1 appearance character results
With the appearance character and Odor Evaluations of Medicated Leaven after fermentation, after different strains fermentation and the fermentation of traditional zymotic technique
Sample be compared, different fermentations sample can grow white colony or white mould, and only bacterial fermentation and spontaneous fermentation just has
Wine aroma, and mold fermentation sample is old mould smell, see Table 1 for details.
1 different fermentations sample property result of table
3.2 different samples influence result to normal mouse functions of intestines and stomach
As shown in Table 2, traditional zymotic and the Medicated Leaven of different strain fermentation can promote normal mouse compared with blank group
Alvine pushing rate and gastric emptying rate, SZLT and HEI group can significantly increase normal gastric emptying rate (P < 0.05), but to small intestine
It promotes without obvious effect, but with trend is increased, other groups play the role of significantly increasing gastric emptying, alvine pushing rate (P <
0.01)。
2 alvine pushing rate gastric emptying rate result of table
Remarks: compared with model group, * indicates that P < 0.05, * * indicate P < 0.01
3.3 GC-MS analyze result
Testing result is shown in Table 3, detected 76 kinds of compounds in six samples altogether, wherein ZR, WTPQ, KCYB, SZLT,
HQM, HEI respectively detect 31,30,31,34,30,36 kind, and it is myristic acid, palmitinic acid first respectively that their shared ingredients, which have 14 kinds,
Ester, palmitoleic acid, palmitinic acid, ethyl palmitate, Heptadecanoic acide, deoxidation qinghaosu, ethyl linoleate, ethyl stearte,
Paullinic acid, the own ester of palmitinic acid, BETA- rosasterol oxide, squalene, clionasterol.And following six kinds of ingredients have
Exist in four to five kinds of fermentation methods: pentadecanoic acid, elaidic acid, methyl linoleate, linoleic acid, stearic acid, campesterol.It is trans-
9- octadecenoic acid methyl ester, C18: 2,20 acid of linolenic acid, 9,17 2 olefine aldehydr of (9Z)-ten eight carbon, n-heptadecane, cetyl ten
Six seven kinds of alkanoic acid ester ingredients occur at least three kinds of fermentation methods, caused by the difference or different fermentations mode between them
It influences.Each component is overlapped situation and sees Fig. 7.
3 GC-MS testing result of table
To sum up, the method for Pediococcus pentosaceus pure-blood ferment Medicated Leaven of the invention, can obtain purebred with other microorganisms
The tunning combination for fermenting different.Fermented product of the invention is fragrant in heavy wine, and without obvious old musty, smell is easy to be connect by people
By.Fermented product of the invention gastric emptying also with higher.The product that method and this method of the invention obtains has
Good application prospect.
Claims (10)
1. a kind of Medicated Leaven pure-blood ferment method, which is characterized in that it is former needed for inoculation Pediococcus pentosaceus is fermented to Medicated Leaven
In material, ferment；Raw material needed for the tradition Medicated Leaven ferments need to be through 100 DEG C or more high-temperature sterilizations；
Raw material needed for the Medicated Leaven ferments includes: Siberian cocklebur grass, sweet wormwood, polygonum flaccidum, rde bean, semen armeniacae amarae, wheat bran and flour.
2. Medicated Leaven pure-blood ferment method as described in claim 1, it is characterised in that: the high-temperature sterilization is 121 DEG C of sterilizings
20min。
3. Medicated Leaven pure-blood ferment method as described in claim 1, it is characterised in that:
Raw material needed for the tradition Medicated Leaven ferments are as follows: 4~6 parts by weight of Siberian cocklebur grass, 4~6 parts by weight of sweet wormwood, 4~6 weight of polygonum flaccidum
Part, 0.5~1.5 parts by weight of rde bean, 0.5~1.5 parts by weight of semen armeniacae amarae, 45~55 parts by weight of wheat bran, 20~30 weight of flour
Part；
The semen armeniacae amarae, rde bean are the coarse powder that can cross No. 2 sieves；
The Siberian cocklebur grass, sweet wormwood, polygonum flaccidum are to be mixed in a manner of water extract with rde bean, semen armeniacae amarae, wheat bran, flour.
4. Medicated Leaven pure-blood ferment method as claimed in claim 3, it is characterised in that:
The weight of the water extract is 6.5 times of primary dose.
5. Medicated Leaven pure-blood ferment method as claimed in claim 3, it is characterised in that:
The inoculum concentration of the corresponding Pediococcus pentosaceus of every g raw material is (1~3) × 105A thallus.
6. Medicated Leaven pure-blood ferment method as claimed in claim 5, it is characterised in that:
The inoculum concentration of the corresponding Pediococcus pentosaceus of every g raw material is 2 × 105A thallus.
7. Medicated Leaven pure-blood ferment method as described in claim 1, it is characterised in that: it is plus cotton plug is sent out in gnotobasis
Ferment；
Its fermentation condition is:
35~38 DEG C of temperature；
And/or humidity 70~80%；
And/or 4~7 days.
8. Medicated Leaven pure-blood ferment method as claimed in claim 7, which is characterized in that its fermentation condition is:
37 DEG C of temperature；
And/or humidity 75%；
And/or 4 days.
9. by any resulting Medicated Leaven of Medicated Leaven pure-blood ferment method of claim 1~8.
10. Medicated Leaven as claimed in claim 9, which is characterized in that there are flocculence white bacterium for the Medicated Leaven partial region
It falls, there is aroma.
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