CN107595907A - Kefir grain fermented ginseng liquid - Google Patents
Kefir grain fermented ginseng liquid Download PDFInfo
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Abstract
The invention discloses a kind of Kefir grains fermented ginseng liquid and kluyveromyces marxianus fermented ginseng liquid, Kefir grain fermented ginseng liquid, it is kefir grains to be inoculated into ginseng pulp to ferment by weight percentage, rare saponin(e Rg3 improves 134.7 % after fermentation, ginsenoside Rd improves 104.5%, while adds ginseng fruit flavor and barbecue fragrance；Kluyveromyces marxianus fermented ginseng liquid, it is kluyveromyces marxianus to be inoculated into ginseng pulp to ferment by weight percentage, ginseng sapoglycoside Rg 3 content is 3.31mg/g after fermentation, while produces special fragrance；The present invention utilizes Kefir grain fermented ginsengs, edible safety, while improves ginseng mouthfeel, improves the content of rare saponin(e, makes that ginseng medical value is higher, intestinal absorption ability is strong.
Description
Technical field
The invention belongs to fermentation engineering field, and in particular to Kefir grain fermented ginsengs liquid, kluyveromyces marxianus
Fermented ginseng liquid.
Background technology
Ginsenoside is ginseng（Panax ginsengC. A. Mey. entum）One of middle primary bioactive components,
Belong to tetracyclic triterpene saponin(e, and saponin(e chemical constitution is by aglycon skeleton and glycosyl, the one kind for being connected to be formed by Glc- is sugared
Glycosides compound, because different aglycon skeletons forms different ginsenosides from different sugar side chains, ginsenoside has
The multiple efficacies such as anti-aging, anti-oxidant, antitumor and anti-inflammatory.How much ginsenoside content in ginseng is grown naturally can be divided into point
For main ginsenoside and rare ginsenoside, rare ginsenoside（Such as ginseng sapoglycoside Rg 3, ginseng saponin Rh 2, Ginsenoside compound K
Deng）There is stronger pharmacological activity antitumor etc., by the ginsenoside of rich content in ginseng（Rb1、Rb2、Rc、Rd、
Re, Rg1, Rf etc.）It is converted into the rare ginsenoside that medical value is higher, intestinal absorption ability is stronger（Rh1、Rh2、F1、F2、
Rg3, Rb3, Rh2, compound K etc.）One of emphasis as modern study.Rare ginsenoside Rg3 has suppression tumour thin
Born of the same parents' propagation, infiltration, transferance, suppress vascular endothelial cell proliferation effect of stomach cancer induction etc. and receive much concern.Ginseng sapoglycoside Rg 3
The suppression of tumour cell, generated by suppressing tumor neovasculature first.It can be produced during low dose application, it is swollen to be best suitable for prevention
Knurl occurs and the Preventive of tumour, and without the common toxic side effect of other cancer therapy drugs, completely can be long-term use of.Rg3
Propagation, the infiltration of tumour oncocyte, inducing apoptosis of tumour cell can also be suppressed in larger metering.But the ginseng grown naturally
Middle ginseng sapoglycoside Rg 3 content is few, and the purpose of conversion can be reached by chemical method, enzyme process and microbial method.Microbial method turns
Change principle and essentially consist in Institute of Micro-biology's secretionβEnzyme system based on-glucuroide converts to ginsenoside.Shao Wei etc. exists
《GS0202 bacterium production ginsenoside-βThe reaction condition of-glucuroide condition and its enzyme》Middle report, utilizeβ- glucuroide
By-GLc keys the hydrolysis of C-21 positions two of ginsenoside Rb1, and then it is converted into rare ginsenoside Rg3；Yan Ping Xiong etc. exists《It is black
The saponin(e Changeement of aspergillus solid fermentation pseudo-ginseng》In, find after fermentation of Aspergillus niger ginseng, can also generate ginsenoside
Rg3, and obtain preferable changing effect.
Kefir granule (Kefir Grain) is the strain of Kefir grains, and Kefir grains are that a kind of edible of multi-cultur es symbiosis is micro-
Living resources, it is commonly used to fermentation cow's milk, sheep breast etc. and yoghurt product is made, compared with common yoghurt product, Kefir grains breast maximum
Feature is that carbon dioxide can be produced in fermentation process so that the product has the unique oral sensations of soda, so the product and quilt
Referred to as posset or kefyr.There are some researches show contain in Kefir grain zymotic fluidsβAbundant enzyme system including-glucuroide, its
Inβ- glucuroide is one of important enzyme for converting ginsenoside, but is rarely had currently with the research of Kefir grain fermented ginsengs
Report.
The content of the invention
The invention aims to solve the problems, such as that ginseng taste is bad, and provide a kind of features good taste, ginseng sapoglycoside Rg 3 content
High, the Kefir grain fermented ginseng liquid and kluyveromyces marxianus fermented ginseng liquid of edible safety.
Kefir grain fermented ginseng liquid, it is prepared by following methods：
By weight percentage, 3-6%kefir grains are inoculated into ginseng pulp, ferment 60-84h under the conditions of 26-30 DEG C；
Described fermentation time 72h；
Described ginseng pulp is by the min of ginseng steaming 15-25, adds water to be beaten, high-temperature sterilization.
Kluyveromyces marxianus fermented ginseng liquid, it is prepared by following methods：
By weight percentage, 2-6% kluyveromyces marxianus are inoculated into ginseng pulp, ferment 36- under the conditions of 26-30 DEG C
72h；
Described percentage by weight is 3%；
Described fermentation time is 48h；
Described ginseng pulp is by the min of ginseng steaming 15-25, adds water to be beaten, high-temperature sterilization；
Its deposit number of described kluyveromyces marxianus is CGMCC 13907.
The invention provides a kind of Kefir grains fermented ginseng liquid and kluyveromyces marxianus fermented ginseng liquid.Kefir
Grain fermented ginseng liquid, is kefir grains to be inoculated into ginseng pulp to ferment by weight percentage, and rare saponin(e Rg3 is improved after fermentation
134.7 %, ginsenoside Rd improve 104.5%；Kluyveromyces marxianus fermented ginseng liquid, by weight percentage will be
Kluyveromyces marxianus, which are inoculated into ginseng pulp, to ferment, and ginseng sapoglycoside Rg 3 content is 3.31mg/g after fermentation；The present invention utilizes
Kefir grain fermented ginsengs, edible safety, while ginseng mouthfeel is improved, the content of rare saponin(e is improved, makes the medicinal valency of ginseng
Value is higher, intestinal absorption ability is strong.Special fragrance is produced after kluyveromyces marxianus fermented ginseng, it is non-to people's one kind
Chang Yuyue impression；Fragrance component after kefir grain fermented ginsengs is analyzed, it is found that the ginseng after fermentation adds fruit flavor
With barbecue fragrance, while its bad flavor is reduced.
Brief description of the drawings
Fig. 1 is producedβThe culture shape and morphological feature of-glucuroide bacteria strain；A. c21 bacterial strains are in improvement aesculin training
Support the form in base；B. form of the c21 bacterial strains in seed culture medium；C. c21 bacterial strains are in microscope（×100）Under shape
State；
Fig. 2 strains to be tested（C21）The PCR in 26S rDNA D1/D2 regions；
Phylogenetic trees of the Fig. 3 according to 26SrDNA sequences；
Fig. 4 ginseng water ratio, inoculum concentration, fermentation time influence on ginseng sapoglycoside Rg 3.
Embodiment
The Kefir grains fermented ginseng of embodiment 1 is starched
The complete fresh ginseng of no disease and pests harm is selected, is cleaned up, steams 20 min, according to ginseng water quality ratio（g/g）1:2、5000
More crawls of rpm/min amount to 3 min mashing, 110 DEG C of 20 min of sterilizing.By kefir grains 5%（w/w）Inoculum concentration be inoculated into people
In ginseng slurry, 3d is cultivated under the conditions of 28 DEG C；The sterilized water of equal volume is added under same culture conditions, as non-fermented ginseng blank
Control group.Freeze after fermentation ends, be then dried using vacuum refrigeration immediately, sample is vacuum-packed after drying, at -4 DEG C
Under store for future use.HPLC methods are combined the conversion situation of ginseng sapoglycoside Rg 3 in analysis ginseng pulp with TLC methods.
Described HPLC method analysis conditions：C18 chromatographic columns (4.6 mm × 250 mm, 5 μm)；35 DEG C of column temperature；Sample size 10
μL；The mL min of flow velocity 1-1；Mobile phase A is the aqueous solution containing 5 % phosphoric acid, B is acetonitrile, and elution requirement is 0~45 min, 33 %
B, 45~80 min, 49 %B；Detection wavelength is 203 nm.
Described TLC methods, it is that solvent is chloroform-methanol-water on tlc silica gel plate by ginsenoside point sample
(10:5:1 V/V/V), spray 20 % sulfuric acid-ethanol solution is after 110 DEG C of 5 min of colour developing.Its gray scale is scanned with thin-layer chromatogram scanner,
Calculate saponin(e conversion ratio.
Ginseng sapoglycoside Rg 3 has a variety of path for transformation, wherein a path for transformation is ginsenoside Rb1 → Rd → Rg3.This reality
The change of ginsenoside Rb1, Rd and Rg3 in fermented ginseng slurry are determined in test, as a result as shown in table 1.It will be apparent that kefir grains ferment
Rg3 content is considerably increased afterwards, and rare saponin(e Rg3 improves 134.7 %；Meanwhile ginsenoside Rb1's ratio is not in fermented ginseng
Fermentation group reduces 41.3 %, and ginsenoside Rd improves 104.5%.Therefore it may be speculated that kefir grains fermented ginseng converts people
It is Rb1 → Rd → Rg3 to join the possible path for transformation of saponin(e Rg3, is shownβ- glucuroide plays important work in the path for transformation
With.
Note：All data are average value ± standard deviation (n=3) in table, and it is to account for ginseng to do to be related to content of ginsenoside
Weight.
Embodiment 2 is producedβThe screening of the bacterial strain of-glucuroide
Kefir grains are fermented at 28 DEG C after ginseng pulp 48h, and it is pressed into 10-3、10-4、10-5、10-6Four gradient dilutions, respectively take 50
μ L bacterium solutions are spread evenly across improvement esculin medium, 25 DEG C of 48 h of culture, and the hydrolysis spot that black occurs in periphery of bacterial colonies is then productionβ- glucuroide bacterial strain.Kefir grain fermented ginseng bacterium solutions be coated on improvement esculin medium on cultivate 36 h after, filter out
Produce the bacterial strain C21 of black hydrolysis spot（Figure 1A）, judge it for productionβ- glucuroide bacterium strain.The bacterial strain is accessed into solid seed
Observation finds that bacterium colony is creamy white, is smooth, be opaque after culture medium, and surface is smooth, and bacterium colony is easily provoked（Figure 1B）.Microscope（×
100）As shown in Figure 1 C, Preliminary Identification result is one plant of production saccharomycete to lower observation form result.
Described improvement aesculin agarose media, it is 1 g aesculins, 0.5 g ironic citrates, 2 g yeast extracts, 0.5g
Peptone, 20 g agar, through 121 DEG C of min of high pressure steam sterilization 20；Seed culture based formulas is 10 g dusty yeasts, 20 g albumen
Peptone, 20 g glucose, 20 g agar, 1000 mL distilled water, it is adjusted to pH 6.0, through 121 DEG C of min of high pressure steam sterilization 20.
Embodiment 3 is producedβThe Molecular Identification of the bacterial strain of-glucuroide
Method for identifying molecules：The sequence analysis in 26S rDNA D1/D2 areas.Strain to be tested DNA extractions use SK8257 kits
Operation；The use of primer is NL1（5'-GCATATCAATAAGCGGAGGAAAAG-3'）And NL4（5'-GGTCCGTGTTT
CAAGACGG-3'）, by Shanghai Sheng Gong limited companies synthetic primer and sequencing, obtained strains DNA sequence dna information exists
The upper Blast of NCBI compare analysis.PCR reaction systems（25 μL）, including genomic DNA（20-50 ng/μL）0.5 μ L, 10 ×
Buffer（Contain Mg2+）2.5 μ L, dNTP（Each 2.5 mM）1 μ L, μ L of enzyme 0.2, F（10 μM）0.5 μ L, R（10 μM）0.5
μ L, add distilled water to 25 μ L；PCR response procedures are 94 DEG C of min of pre-degeneration 4,94 DEG C of 45 s of extension, 30 circulations；55 ℃
Extend 45 s, 30 circulations；72 DEG C of 1 min of extension, 30 circulations；72 DEG C are repaired 1 min of extension；4 DEG C of terminating reactions.PCR
Amplified production is detected with 1 % agarose gel electrophoresis, and deposition condition is 150 V, 100 mA, 20 min, uses gel imager
Observe result.
Using NL1 and NL4 pair of primers amplification strain to be tested 26S rDNA nearly 5 ' ends D1/D2 regions, amplification is shown in figure
2.As shown in Figure 2, amplified production through electrophoresis detection occur 578 bp fluorescent bands, it is consistent with expected results, clip size with
The fragment of report（500~600 bp）It is consistent.The D1/D2 regions of strain to be tested are sequenced, homology sequence is carried out in GenBank
Row search（BLAST Search）, strain to be tested is as a result shown compared with kluyveromyces marxianus sequence, and homology is up to 99%
（Fig. 3）, therefore further checking strain to be tested is kluyveromyces marxianus（Kluyveromyces marxianus）.The bacterium
Kind it is a very important strain in Kluyveromyces, because of its high temperature resistant, growth rate is fast and that security is high etc. is many excellent
Gesture and be applied to such as enzyme, ethanol industrial biotechnology field.Microorganism conversion ginsenoside reaction mechanism is commonly considered as
Glycosyl in enzyme hydrolysis ginsenoside caused by microorganism, rare saponin(e is obtained so as to cause the change of structure, such as ginseng soap
Glycosides Rg3.Result of study shows at present, and microorganism conversion Rg3 enzyme is mainlyβ- glucuroide, and utilize microorganism to produceβ- Portugal
The research that polyglycoside enzyme is applied to ginsenoside conversion is most for fungies such as aspergillus nigers.Because the inulinase-producing activity of Aspergillus niger strain can be with
The growth of holding time and reduce, be unfavorable for storing, the application in food processing is restricted.Containing more in kefir grains
The kind microorganism such as lactic acid bacteria and saccharomycete, but during kefir grains fermentation full constituent ginseng, find productionβMicro- life of-glucuroide
Thing is kluyveromyces marxianus.Kluyveromyces marxianus strong adaptability, Marx in kefir grain fermented ginsengs can be speculated
Kluyveromyces become dominant bacteria, it is suppressed that the growth of lactic acid bacteria.By the bacterial strain（Specific name：Kluyveromyces
marxianus）It is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center（CGMCC）, deposit number is
CGMCC 13907, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, it is preserved on March 21st, 2017.
The extraction of total ginsenoside in the full constituent fermented ginseng of embodiment 4 slurry
By isolated kluyveromyces marxianus bacterial strain（C21）With 5%（W/W）Addition is inoculated into full ginseng culture medium,
The h of static gas wave refrigerator 48 under the conditions of 28 DEG C.The sterilized water that equal volume is added under same culture conditions is used as non-fermented ginseng blank pair
According to group.Freeze after fermentation ends, be then dried using vacuum refrigeration immediately, sample is vacuum-packed after drying, at -4 DEG C
Store for future use.
Take fermented ginseng to starch the g of freeze-dried powder 1, using soxhlet extraction, after chloroform is heated to reflux 3 h, discard trichlorine
Methane, solvent is volatilized, moved into 100 mL conical flasks, add 50 mL water-saturated n-butanols, after standing overnight, be ultrasonically treated（Work(
The W of rate 250, the Hz of frequency 50）30 min, the mL of filtrate 25 is taken after filtering, is placed in evaporating dish and is evaporated, added in dried object a small amount of
Methanol solution is used to detect after being transferred to 5 mL volumetric flask constant volumes after being completely dissolved.Each ginsenoside mark product are dissolved in 100% methanol
0.2 mg/mL solution is made to analyze for saponin(e.
The kluyveromyces marxianus bacterium fermented ginseng of embodiment 5 improves ginseng sapoglycoside Rg 3 process optimization
Ginseng water ratio, inoculum concentration, fermentation time are investigated to converting ginseng sapoglycoside Rg 3 content（mg/g）Influence.Ginseng sapoglycoside Rg 3 contains
Amount（mg/g）Represent that rare ginsenoside Rg3 contents account for ginseng dry weight ratio.To convert ginseng sapoglycoside Rg 3 content（mg/g）For
Performance assessment criteria, the suitable fermentation condition of each single factor test is determined, term of reference is provided to convert the condition optimizing of ginseng sapoglycoside Rg 3,
Each factor setting 3 of experimental design is parallel.On the basis of screening obtained bacterial strain conversion ginseng Rg3 single factor experiments, adopt
With center combination experiment Box-Behnken design experiments factor and level（It is shown in Table 2）, result of the test Design Expert
8.1 analyses, determine influence of the reciprocation between each factor and factor to ginseng sapoglycoside Rg 3 content, optimize its process conditions.
From the most important several factors of ferment effect are influenceed, ginseng water ratio, fermentation time and inoculum concentration are set out, and have studied conversion ginseng
Saponin(e Rg3 effect（See Fig. 4）.As a result show, ginseng water ratio is 1:2-1:In the range of 2.6, with the increase of ratio, ginsenoside
Ascendant trend is presented in Rg3 contents, when ginseng water ratio is 1:It is 3.22 ± 0.18 mg/g that content of ginsenoside, which reaches maximum, when 2.6,
Subsequent ginseng sapoglycoside Rg 3 content has declined, but remains above original state, may be in the state of the fixation of ginseng pulp amount, ginseng
Water is more relative than the content of higher ginseng to be reduced, and microbial fermentation substrate is reduced, and then causes content of ginsenoside to decline.Investigate horse
Found in gram influence of this kluyveromyces inoculum concentration to rare saponin(e conversion ratio, with the increase of inoculum concentration, ginseng sapoglycoside Rg 3
Content present first increase after reduction trend, when inoculum concentration be 3% when content of ginsenoside reach peak 3.02 ± 0.25
Mg/g, the content of subsequent ginseng sapoglycoside Rg 3 have declined, and this is probably the accumulation due to saccharomycete, caused cometabolism
Ginseng sapoglycoside Rg 3 is changed into other saponin(es by product.In fermentation time 1-9 days, as fermentation time increases, ginsenoside
Rg3 contents are presented first rise after downward trend, and stepped decline, when fermenting between when being 3 days, ginseng sapoglycoside Rg 3
Content reaches the mg/g of maximum 3.13 ± 0.11, why this variation tendency can be presented, it may be possible to due to fermentation time mistake
Long, moisture gradually decreases, and causes viable count to reduce, and because nutriment is reduced, secondary metabolite accumulation, influences ginseng
Saponin(e Rg3 accumulation.
According to Box-Behnken center combination experimental design principles, comprehensive single factor experiment result, the water of 3 factor 3 is carried out
Flat response interview is tested, as a result as shown in table 3.Variance analysis is carried out to data, the results showed that experiment uses quadratic regression equation
Model highly significant, it is statistically meaningful.The factor that contrast influences ginseng sapoglycoside Rg 3 content is ginseng water ratio（A）＞
Yeast-inoculated amount（B）＞ fermentation times（C）.By the Treatment Analysis of data, the predicted maximum for trying to achieve response surface R is 3.3298
Mg/g, ginseng water ratio now is 1：2.65th, fermentation time 3d, the % of kluyveromyces marxianus inoculum concentration 2.94.For testing model
Accuracy, using fermentation test is carried out under optimal conditions of fermentation after optimization, experiment three times obtains ginseng sapoglycoside Rg 3 content and is averaged
It is worth for 3.31 ± 0.04 mg/g.Predicted value approaches with actual value, and the zymotechnique for illustrating the optimization is effective.
Note：Ginseng sapoglycoside Rg 3 content（mg/g）Represent, account for ginseng dry weight ratio.
The present invention is with kefir grains and from the isolated Kluyveromyces marxianus bacterial strain of kefir grains（C21）Fermentation is complete
Component ginseng, discovery can significantly improve ginseng sapoglycoside Rg 3 content, after optimized zymotechnique ginseng sapoglycoside Rg 3 content with not
Ginseng sapoglycoside Rg 3 is compared and improves 3.48 times in fermented ginseng.Special perfume (or spice) is produced after kluyveromyces marxianus fermented ginseng
Gas, give people a kind of very pleasant impression；Fragrance component after kefir grain fermented ginsengs is analyzed, after finding fermentation
Ginseng adds fruit flavor and barbecue fragrance, while reduces its bad flavor.
Claims (8)
1. Kefir grain fermented ginseng liquid, it is prepared by following methods：
By weight percentage, 3-6%kefir grains are inoculated into ginseng pulp, ferment 60-84h under the conditions of 26-30 DEG C.
2. Kefir grains fermented ginseng liquid according to claim 1, it is characterised in that：Described fermentation time 72h.
3. Kefir grains fermented ginseng liquid according to claim 1 or 2, it is characterised in that：Described ginseng pulp is by people
Ginseng steams 15-25 min, adds water to be beaten, high-temperature sterilization.
4. kluyveromyces marxianus fermented ginseng liquid, it is prepared by following methods：
By weight percentage, 2-6% kluyveromyces marxianus are inoculated into ginseng pulp, ferment 36- under the conditions of 26-30 DEG C
72h。
5. kluyveromyces marxianus fermented ginseng liquid according to claim 4, it is characterised in that：Described weight percent
Than for 3%.
6. kluyveromyces marxianus fermented ginseng liquid according to claim 5, it is characterised in that：Described fermentation time
For 48h.
7. kluyveromyces marxianus fermented ginseng liquid according to claim 6, it is characterised in that：Described ginseng pulp
By the min of ginseng steaming 15-25, to add water to be beaten, high-temperature sterilization.
8. kluyveromyces marxianus fermented ginseng liquid according to claim 7, it is characterised in that：Described Marx gram
Yeast is tieed up in Shandong, and its deposit number is CGMCC 13907.
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| CN112980913A (en) * | 2021-04-14 | 2021-06-18 | 北京欣康研医药科技有限公司 | A method for preparing baicalein from baicalin by fermentation |
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Application publication date: 20180119 |