CN107502631A - A kind of production method of candida utili β D glucans - Google Patents
A kind of production method of candida utili β D glucans Download PDFInfo
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- C12P19/00—Preparation of compounds containing saccharide radicals
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Abstract
The present invention discloses a kind of production method of candida utili β D glucans.This method is removed the preparation of the murphy juice of the deproteinized after the murphy juice of potato starch, the murphy juice aqueous solution of deproteinized, sterilizing first, is then inoculated with candida utili bacterium（Candida utilis）CICC 1769 is fermented, the intracellular that the filtering fermentation liquor of gained obtains contains the candida utili bacterium thalline of candida utili β D glucans, then extracted using high-temp extracting and ultrasonication synergy broken wall, then through centrifuging, washing, being dried in vacuo, finally give purity and reach as high as 97%, yield reaches as high as 19%, and yield reaches as high as 87% candida utili β D glucans.Workload greatly reduces in the production method, while reduces production cost, turns waste into wealth simultaneously, meets green production demand.
Description
Technical field
The invention belongs to field of biology, is related to a kind of production method of candida utili callose.
Background technology
Candida utili is that one kind can be used as food additive by many national authentications together with brewer's yeast, kluyveromyces
Add the microorganism of agent, and the microorganism available for health food that China Health department allows, using industrial wastes, training
Any growth factor need not be added in base by, which supporting, to grow.
Yeast beta-dextran is the structural polysaccharide of yeast cell wall.They have different functional characteristics, such as medical treatment, system
The industries such as medicine, food, cosmetics.They can also be as the bio-modification agent of immune response.In addition, they are also beneficial to
Antimicrobial antiphlogistic reacts；Promote the absorption of mould toxin；Reduce the effect such as cholesterol particles in low-density lipoprotein；Also have anti-
Cancer, anti-mutagenesis, characteristic that is anti-oxidant and promoting wound healing.
The cultivation method for saccharomycete of domestic production yeast callose is mainly using preparation carbon nitrogen source or yeast leaching at present
Go out powder peptone dextrose culture-medium, the shortcomings of culture medium cost is high so be present, and if preparing carbon-nitrogen ratio in production process, that
Because the nutritional conditions such as carbon-nitrogen ratio needed for barms are relatively complicated, therefore the problems such as research work amount is larger be present, and
Yield, yield and the purity of yeast callose is all than relatively low in existing most of document, respectively only up to 80%, 17% and
86%。
The content of the invention
It is high the invention aims to solve production cost present in above-mentioned yeast callose production method,
The technical problems such as research work amount is big and a kind of production method of candida utili callose is provided, the production method can
Using by the use of discarded murphy juice after deproteinized as culture medium, so as to solve culture yeasts bacterium in the prior art to utilize it is a large amount of
Carbon source and nitrogen source and heuristic process is relatively complicated, so as to which workload greatly reduce, while reduce production cost, same to time-varying
Waste be changed into values, meets green production demand, and finally gives the candida utili of high yield, and candida utili β-D- Portugals gather
The yield of sugar reaches as high as 87%, and yield reaches as high as 19%, and purity reaches as high as 97%.
Technical scheme
A kind of production method of candida utili callose, specifically includes following steps：
（1）, remove potato starch murphy juice preparation
Potato is smashed with pulverizer, obtains mashed potatoes, the distilled water of 10 times of potato quality is then added in mashed potatoes, fully
Stir and evenly mix, 20min is centrifuged under the conditions of 10000r/min and removes precipitation, the precipitation is potato starch, and obtained filtrate places 5
It produces the murphy juice for removing potato starch；
（2）, deproteinized the murphy juice aqueous solution preparation
By step（1）The murphy juice of the removal potato starch of gained is filtered by 8 layers of gauze, is added and is steamed in the filtrate of gained
The addition of distilled water, wherein distilled water is 5 times of gained filtrate volume, and it is to be stripped 1h at 121 DEG C then to control temperature, is obtained
To extract；
The murphy juice of above-mentioned removal potato starch can use the discarded murphy juice of farina factory to substitute；
The extract of above-mentioned gained is filtered with model SHZ- Ш types vacuum pump using circulatory waters under -0.1MPa vacuum,
To remove protein, the distilled water of 10 times of the filtrate volume of gained is incorporated as after suction filtration in the filtrate of gained, after stirring and evenly mixing
To the murphy juice aqueous solution of deproteinized；
（3）, deproteinized after sterilizing murphy juice preparation
Regulating step（2）The pH of the murphy juice aqueous solution of the deproteinized of gained is 4-8, then adds glycerine, and temperature is controlled after mixing
Spend and carry out sterilizing 15min for 121 DEG C, the murphy juice of the deproteinized after being sterilized；
It is the NaOH aqueous solution that mass percent concentration is 1% to adjust the alkali used in the pH of the murphy juice aqueous solution of deproteinized, and acid is
Mass percent concentration is 0.1% HCl/water solution；
The addition of above-mentioned glycerine is the 5-25% of the murphy juice aqueous solution volume for the deproteinized that pH is 4-8；
（4）, inoculation fermentation
To step（3）The candida utili bacterium of glycerol tube preservation is inoculated with the murphy juice of deproteinized after the sterilizing of gained
（Candida utilis）CICC 1769, it is 24-32 DEG C then to control temperature, and rotating speed is that 100-200r/min carries out fermentation training
24h is supported, obtains zymotic fluid；
Inoculum concentration presses the candida utili bacterium that 1-8mL glycerol tube preservations are accessed in the deproteinized murphy juice per 100mL after sterilizing
（Candida utilis）CICC 1769；
Per the candida utili bacterium of 1mL glycerol tube preservations（Candida utilis）In CICC 1769, the Candida utilis that contains
Saccharomycete（Candida utilis）The bacterium numbers of CICC 1769 are 1.6 × 109CFU；
（5）, by step（4）The filtering fermentation liquor of gained, filtrate is removed, obtained filter cake is that intracellular contains candida utili
The candida utili bacterium thalline of callose；
（6）, under the conditions of 121 DEG C, using distilled water by step（5）The intracellular of gained contains candida utili callose
Candida utili bacterium thalline carry out extraction 30min so that candida utili somatic cells carry out breaking-wall cell, then will
The leaching liquor of gained is 20KHz in supersonic frequency by ultrasonic cell disruption instrument, carries out supersound process 60min under 800W, so
15min is centrifuged under 10000r/min afterwards, the precipitation of gained is washed with distilled water, until efflux is clarified, terminates washing,
Then it is -20 DEG C to control temperature again, and vacuum is that 10Pa carries out vacuum freeze drying, produces candida utili β-D- Portugals and gathers
Sugar.
Candida utili callose obtained by above-mentioned production method, because there is general yeast β-D- Portugals to gather for it
The characteristic of sugar, therefore the quality of food can be improved, the source of dietary fiber in food is can serve as, as a kind of very excellent
Food additives have very big development prospect.Further, the candida utili callose of gained can significantly increase water
The non-specific immune systems of animal are produced, the resistance that aquatic livestock can be greatly enhanced in aquaculture is applied, is a kind of nothing
The application approach of public hazards, natural green.Further, the candida utili callose of gained can utilize its immunological regulation
Activity promotes the regeneration of skin, adjusts the growth of cell, then reaches the action effect of anti-aging, i.e., the production protein of above-mentioned gained
Candida callose can be used in food, feed, cosmetics and aquaculture.
Further, the candida utili callose obtained by aforementioned production method, due to because not dropped in human body
The enzyme of beta glucan is solved, so they play the role of natural trophic fiber.Research shows that the diet of high microsteping can reduce
Cholesterol, and reduce chronic disease and the cardiopathic incidences such as arthritis.When beta glucan enters oral cavity, will start to inhale
Water expands.The dietary fiber of beta glucan can increase the haulage time of enteron aisle, play the role of to slow down intestinal absorption, therefore can show
Ground reducing blood lipid, hypoglycemic and raising immunocompetence are write, to there is the trouble of the diseases such as diabetes, obesity, constipation and high cholesterol
Person plays the role of good auxiliary treatment.
Further, the candida utili callose obtained by aforementioned production method, due to can be with stimulating innate immunity
System, greatly improved so as to reach the ability of resistance bacterium, fungi and virus etc..Further, candida utili β-D-
Glucan by activating T cell, macrophage and NK, be able to can also stimulate T lymphocytes differentiation and
Activation, the change of simultaneously activated pathway complement is influenceed, be finally reached the effect of enhancing body cell and humoral immunity, therefore the production protein
Candida callose has liver protecting, protection stomach lining, improves the healthcare functions such as microcirculation, strengthen immunity.
Beneficial effects of the present invention
The production method of a kind of candida utili callose of the present invention, due to the desizing that is utilized in production process
After testing, pH value 4.82, COD value is 29.8g O to murphy juice2/ L and BOD5Value be 15.6g O2/ L, formed sediment close to potato
PH, COD, BOD of the discarded murphy juice of powder factory5Index, therefore the production of the candida utili callose of the present invention
Method can be produced with the raw material that the discarded murphy juice of farina factory is culture medium, so as to alleviate discarded potato
Problem of environmental pollution caused by juice, realization are turned waste into wealth, and so as to reduce the production cost of yeast callose, and are produced
Process operation is simple, is easy to large-scale production.
Further, the production method of a kind of candida utili callose of the invention, by institute in production process
The intracellular obtained contains the candida utili bacterium thalline of candida utili callose by using high-temp extracting and ultrasound
The extracting method of broken wall synergy, finally gives the candida utili yeast callose of high yield, high-purity, its is pure
Degree reaches as high as 97%, and yield reaches as high as 19%, and yield reaches as high as 87%.
Brief description of the drawings
The infrared spectrogram of Fig. 1 candida utili calloses；
The ultraviolet full scan collection of illustrative plates of Fig. 2 candida utili calloses.
Embodiment
The present invention is expanded on further below by specific embodiment and with reference to accompanying drawing, but is not intended to limit the present invention.
Strain used is candida utili in various embodiments of the present invention（Candida utilis）, bacterium numbering CICC
1769, buy in Chinese industrial Microbiological Culture Collection administrative center, it is in Chinese industrial Microbiological Culture Collection administrative center
Preservation, deposit number：ATCC 9950；Preservation organization address：The building of Jiuxianqiao, Chaoyang District, Beijing City Road No. 24 the 6th, postcode：
100015, date of supply：On July 13rd, 2015.
INSTRUMENT MODEL and manufacturer's information used are as follows in various embodiments of the present invention：
Ultrasonic cell disruption instrument, model JY99-IIDN, NingBo XinZhi Biology Science Co., Ltd's production；
Vacuum pump using circulatory water, model SHZ- Ш types, the sub- flourish biochemical instrument production in Shanghai.
Fourier infrared spectrograph, TENSOR series, German Bruker companies；
Ultraviolet specrophotometer, UV8000 types, Shanghai Yuan Xi Instrument Ltd..
The measure of candida utili callose content, with glucose as a standard, is adopted in various embodiments of the present invention
The candida utili callose content of gained in each embodiment is determined with phend-sulphuric acid（Bibliography：Song Bo, Zhao
Gorge, the extracting and developing and activity rating [J] Chinese Sea medicines of a kind of Soft Corals polysaccharide of Li Guoqiang, 2016,35
(5):1-6.DOI:10.13400/j.cnki.cjmd.2016.05.001.）；
The purity of candida utili callose in various embodiments of the present invention（%）The quality ÷ productions of=yeast callose
Quality × 100% of gained Thick many candies in protein candida cell wall；
The yield of candida utili callose（%）=candida utili callose quality ÷ the productions actually obtained
Quality × 100% of protein candidiasis thalline；
The yield of candida utili callose（%）=candida utili callose quality ÷ the productions actually obtained
Quality × 100% for the callose that protein candidiasis contain in theory into the cell.
Embodiment 1
A kind of production method of candida utili callose, specifically includes following steps：
（1）, remove potato starch murphy juice preparation
Take 40.0g potato pulverizers to smash, obtain mashed potatoes, 400mL distilled water is then added in mashed potatoes, is sufficiently stirred
Mix, it is to remove potato starch that 20min is centrifuged under the conditions of 10000r/min and removes precipitation, the filtrate of the removal potato starch of gained
Place the murphy juice for producing for 5 days and removing potato starch；
After testing the pH value of the murphy juice of above-mentioned gained be 4.82, COD value be 29.8g O2/ L and BOD5Value be 15.6g O2/
L；
（2）, deproteinized the murphy juice aqueous solution preparation
By 280mL steps（1）The murphy juice of the removal potato starch of gained is filtered by 8 layers of gauze, is taken obtained by 100mL
Filtrate, distilled water 500mL is added, be stripped 1h at being 121 DEG C in temperature, obtain extract；
The extract of above-mentioned gained is filtered with vacuum pump using circulatory water under -0.1MPa vacuum, to remove protein, is taken
The filtrate of gained, adds 2000mL distilled water, is mixed, the murphy juice for obtaining deproteinized is water-soluble thereto after 200mL is filtered
Liquid；
（3）, deproteinized after sterilizing murphy juice preparation
The HCl/water solution regulating step that the NaOH aqueous solution and mass percent concentration that are 1% with mass percent concentration are 0.1%
（2）The pH of the murphy juice aqueous solution of the deproteinized of gained is 4, then adds glycerine, controls temperature to be gone out for 121 DEG C after mixing
Bacterium 15min, the murphy juice of the deproteinized after being sterilized；
The addition of above-mentioned glycerine is the 25% of the murphy juice aqueous solution volume for the deproteinized that pH is 4；
（4）, inoculation fermentation
To step（3）The candida utili bacterium of glycerol tube preservation is inoculated with the murphy juice of deproteinized after the sterilizing of gained
（Candida utilis）CICC 1769, it is 24 DEG C then to control temperature, and rotating speed is that 100r/min carries out fermented and cultured 24h, is obtained
To zymotic fluid；
Inoculum concentration presses the candida utili bacterium for accessing 1mL glycerol tube preservations in the murphy juice of the deproteinized after sterilizing per 100mL
（Candida utilis）CICC 1769；
Per the candida utili bacterium of 1mL glycerol tube preservations（Candida utilis）In CICC 1769, the Candida utilis that contains
Saccharomycete（Candida utilis）The bacterium numbers of CICC 1769 are 1.6 × 109CFU（CFU represents the viable bacteria in unit volume
Number）；
（5）, by step（4）The zymotic fluid of gained is filtered with Buchner funnel, removes filtrate, obtained filter cake is that intracellular contains
There is the candida utili bacterium thalline of candida utili callose；
After measured, calculated by every lmL zymotic fluids, it is false to obtain the production protein that 8.20mg intracellulars contain candida utili callose
Silk saccharomycete；
Contain the candida utili bacterium 0.0986mg containing callose of candida utili callose per mg intracellulars；
Contain candida utili callose 0.5394mg in per mL zymotic fluids；
（6）, under the conditions of 121 DEG C, calculate in mass ratio, intracellular contains the Candida utilis ferment of candida utili callose
Female bacterium thalline：Distilled water is 15：100 ratio, with distilled water by step（5）The intracellular of gained contains candida utili β-D-
The candida utili bacterium thalline of glucan carries out extraction 30min, so that candida utili thalline carries out breaking-wall cell, then
In frequency it is 20KHz by ultrasonic cell disruption instrument, power is to carry out supersound process 60min under 800W, then in 10000r/
15min is centrifuged under min, the precipitation of gained is washed with distilled water, until efflux is clarified, is terminated washing, is then controlled again
Temperature is -20 DEG C, and vacuum is that 10Pa carries out vacuum freeze drying, produces candida utili callose.
After measured, the yield of the candida utili callose of above-mentioned gained be 82%, yield 13%, purity is
96%。
The candida utili callose of above-mentioned gained is measured using Fourier infrared spectrograph, gained
The infrared spectrogram of candida utili callose is as shown in figure 1, the glucan as can be seen from Figure 1 extracted exists
890cm-1Neighbouring peak is β-type, in 3200cm-1Neighbouring peak is the specific peak absorbance peak of glycosidic bond, has been indicated above passing through
It is the yeast dextran that glucosides key type is beta configuration that this method, which obtains sample,；
The candida utili callose of above-mentioned gained is measured using ultraviolet specrophotometer, the production protein of gained is false
The ultraviolet spectrogram of silk yeast callose as shown in Fig. 2 from figure 2 it can be seen that in the range of 190 ~ 400nm of wavelength,
The impurity such as protein and nucleic acid are not contained in candida utili callose, it is higher to be indicated above gained sample purity.
Embodiment 2
A kind of production method of candida utili callose, specifically includes following steps：
（1）, remove potato starch murphy juice preparation
The step of with embodiment 1（1）;
（2）, deproteinized the murphy juice aqueous solution preparation
The step of with embodiment 1（2）；
（3）, deproteinized after sterilizing murphy juice preparation
The HCl/water solution regulating step that the NaOH aqueous solution and mass percent concentration that are 1% with mass percent concentration are 0.1%
（2）The pH of the murphy juice aqueous solution of the deproteinized of gained is 8, then adds glycerine, controls temperature to be gone out for 121 DEG C after mixing
Bacterium 15min, the murphy juice of the deproteinized after being sterilized；
The addition of above-mentioned glycerine is the 15% of the murphy juice aqueous solution volume for the deproteinized that pH is 8；
（4）, inoculation fermentation
To step（3）The candida utili bacterium of glycerol tube preservation is inoculated with the murphy juice of deproteinized after the sterilizing of gained
（Candida utilis）CICC 1769, it is 28 DEG C then to control temperature, and rotating speed is that 150r/min carries out fermented and cultured 24h, is obtained
To zymotic fluid；
Inoculum concentration presses the candida utili bacterium that 4mL glycerol tube preservations are accessed in the deproteinized murphy juice per 100mL after sterilizing
（Candida utilis）CICC 1769；
Per the candida utili bacterium of 1mL glycerol tube preservations（Candida utilis）In CICC 1769, the Candida utilis that contains
Saccharomycete（Candida utilis）The bacterium numbers of CICC 1769 are 1.6 × 109CFU；
（5）, by step（4）The zymotic fluid of gained is filtered with Buchner funnel, removes filtrate, obtained filter cake is that intracellular contains
There is the candida utili bacterium thalline of candida utili callose；
After measured, calculated by every lmL zymotic fluids, receive to obtain 10.16mg（Dry weight）Intracellular contains candida utili callose
Candida utili bacterium；
Contain the candida utili bacterium 0.1039mg containing callose of candida utili callose per mg intracellulars；
Contain candida utili callose 0.7988mg in per mL zymotic fluids；
（6）, under the conditions of 121 DEG C, calculate in mass ratio, intracellular contains the Candida utilis ferment of candida utili callose
Female bacterium thalline：Distilled water is 15：100 ratio, with distilled water by step（5）The intracellular of gained contains candida utili β-D-
The candida utili bacterium thalline of glucan carries out extraction 30min, so that candida utili thalline carries out breaking-wall cell, then
In frequency it is 20KHz by ultrasonic cell disruption instrument, power is to carry out supersound process 60min under 800W, then in 10000r/
15min is centrifuged under min, the precipitation of gained is washed with distilled water, until efflux is clarified, is terminated washing, is then controlled again
Temperature is -20 DEG C, and vacuum is that 10Pa carries out vacuum freeze drying, produces candida utili callose.
After measured, the yield of the candida utili callose of above-mentioned gained be 87%, yield 19%, purity is
97%。
Embodiment 3
A kind of production method of candida utili callose, specifically includes following steps：
（1）, remove potato starch murphy juice preparation
The step of with embodiment 1（1）;
（2）, deproteinized the murphy juice aqueous solution preparation
The step of with embodiment 1（2）；
（3）, deproteinized after sterilizing murphy juice preparation
The HCl/water solution regulating step that the NaOH aqueous solution and mass percent concentration that are 1% with mass percent concentration are 0.1%
（2）The pH of the murphy juice aqueous solution of the deproteinized of gained is 7, then adds glycerine, controls temperature to be gone out for 121 DEG C after mixing
Bacterium 15min, the murphy juice of the deproteinized after being sterilized；
The addition of above-mentioned glycerine is the 10% of the murphy juice aqueous solution volume for the deproteinized that pH is 7；
（4）, inoculation fermentation
To step（3）The candida utili bacterium of glycerol tube preservation is inoculated with the murphy juice of deproteinized after the sterilizing of gained
（Candida utilis）CICC 1769, it is 30 DEG C then to control temperature, and rotating speed is that 200r/min carries out fermented and cultured 24h, is obtained
To zymotic fluid；
Inoculum concentration presses the candida utili bacterium that 2mL glycerol tube preservations are accessed in the deproteinized murphy juice per 100mL after sterilizing
（Candida utilis）CICC 1769；
Per the candida utili bacterium of 1mL glycerol tube preservations（Candida utilis）In CICC 1769, the Candida utilis that contains
Saccharomycete（Candida utilis）The bacterium numbers of CICC 1769 are 1.6 × 109CFU；
（5）, by step（4）The zymotic fluid of gained is filtered with Buchner funnel, removes filtrate, obtained filter cake is that intracellular contains
There is the candida utili bacterium thalline of candida utili callose；
After measured, calculated by every lmL zymotic fluids, it is false to obtain the production protein that 9.23mg intracellulars contain candida utili callose
Silk saccharomycete；
Contain 0.0998mg containing callose in the candida utili bacterium of candida utili callose per mg intracellulars；
Contain candida utili callose 0.6729mg in every mL zymotic fluids i.e. obtained by production method of the invention；
（6）, under the conditions of 121 DEG C, calculate in mass ratio, intracellular contains the Candida utilis ferment of candida utili callose
Female bacterium thalline：Distilled water is 15：100 ratio, with distilled water by step（5）The intracellular of gained contains candida utili β-D-
The candida utili bacterium thalline of glucan carries out extraction 30min, so that candida utili thalline carries out breaking-wall cell, then
In frequency it is 20KHz by ultrasonic cell disruption instrument, power is to carry out supersound process 60min under 800W, then in 10000r/
15min is centrifuged under min, the precipitation of gained is washed with distilled water, until efflux is clarified, is terminated washing, is then controlled again
Temperature is -20 DEG C, and vacuum is that 10Pa carries out vacuum freeze drying, produces candida utili callose.
After measured, the yield of the candida utili callose of above-mentioned gained be 83%, yield 17%, purity is
97%。
Embodiment 4
A kind of production method of candida utili callose, specifically includes following steps：
（1）, remove potato starch murphy juice preparation
The step of with embodiment 1（1）;
（2）, deproteinized the murphy juice aqueous solution preparation
The step of with embodiment 1（2）；
（3）, deproteinized after sterilizing murphy juice preparation
The HCl/water solution regulating step that the NaOH aqueous solution and mass percent concentration that are 1% with mass percent concentration are 0.1%
（2）The pH of the murphy juice aqueous solution of the deproteinized of gained is 6, then adds glycerine, controls temperature to be gone out for 121 DEG C after mixing
Bacterium 15min, the murphy juice of the deproteinized after being sterilized；
The addition of above-mentioned glycerine is the 20% of the murphy juice aqueous solution volume for the deproteinized that pH is 7；
（4）, inoculation fermentation
To step（3）The candida utili bacterium of glycerol tube preservation is inoculated with the murphy juice of deproteinized after the sterilizing of gained
（Candida utilis）CICC 1769, it is 32 DEG C then to control temperature, and rotating speed is that 200r/min carries out fermented and cultured 24h, is obtained
To zymotic fluid；
Inoculum concentration presses the candida utili bacterium that 8mL glycerol tube preservations are accessed in the deproteinized murphy juice per 100mL after sterilizing
（Candida utilis）CICC 1769；
Per the candida utili bacterium of 1mL glycerol tube preservations（Candida utilis）In CICC 1769, the Candida utilis that contains
Saccharomycete（Candida utilis）The bacterium numbers of CICC 1769 are 1.6 × 109CFU；
（5）, by step（4）The zymotic fluid of gained is filtered with Buchner funnel, removes filtrate, obtained filter cake is that intracellular contains
There is the candida utili bacterium thalline of candida utili callose；
After measured, calculated by every lmL zymotic fluids, it is false to obtain the production protein that 7.98mg intracellulars contain candida utili callose
Silk saccharomycete；
Contain the candida utili bacterium of candida utili callose, 0.0993mg containing callose per mg intracellulars；
Contain candida utili callose 0.6236mg in every mL zymotic fluids i.e. obtained by production method of the invention；
（6）, under the conditions of 121 DEG C, calculate in mass ratio, intracellular contains the Candida utilis ferment of candida utili callose
Female bacterium thalline：Distilled water is 15：100 ratio, with distilled water by step（5）The intracellular of gained contains candida utili β-D-
The candida utili bacterium thalline of glucan carries out extraction 30min, so that candida utili thalline carries out breaking-wall cell, then
In frequency it is 20KHz by ultrasonic cell disruption instrument, power is to carry out supersound process 60min under 800W, then in 10000r/
15min is centrifuged under min, the precipitation of gained is washed with distilled water, until efflux is clarified, is terminated washing, is then controlled again
Temperature is -20 DEG C, and vacuum is that 10Pa carries out vacuum freeze drying, produces candida utili callose.
After measured, the yield of the candida utili callose of above-mentioned gained be 83%, yield 15%, purity is
96%。
Embodiment 5
A kind of production method of candida utili callose, specifically includes following steps：
（1）, remove potato starch murphy juice preparation
The step of with embodiment 1（1）;
（2）, deproteinized the murphy juice aqueous solution preparation
The step of with embodiment 1（2）；
（3）, deproteinized after sterilizing murphy juice preparation
The HCl/water solution regulating step that the NaOH aqueous solution and mass percent concentration that are 1% with mass percent concentration are 0.1%
（2）The pH of the murphy juice aqueous solution of the deproteinized of gained is 5, then adds glycerine, controls temperature to be gone out for 121 DEG C after mixing
Bacterium 15min, the murphy juice of the deproteinized after being sterilized；
The addition of above-mentioned glycerine is the 5% of the murphy juice aqueous solution volume for the deproteinized that pH is 7；
（4）, inoculation fermentation
To step（3）The candida utili bacterium of glycerol tube preservation is inoculated with the murphy juice of deproteinized after the sterilizing of gained
（Candida utilis）CICC 1769, it is 26 DEG C then to control temperature, and rotating speed is that 100r/min carries out fermented and cultured 24h, is obtained
To zymotic fluid；
Inoculum concentration presses the candida utili bacterium that 6mL glycerol tube preservations are accessed in the deproteinized murphy juice per 100mL after sterilizing
（Candida utilis）CICC 1769；
Per the candida utili bacterium of 1mL glycerol tube preservations（Candida utilis）In CICC 1769, the Candida utilis that contains
Saccharomycete（Candida utilis）The bacterium numbers of CICC 1769 are 1.6 × 109CFU；
（5）, by step（4）The zymotic fluid of gained is filtered with Buchner funnel, removes filtrate, obtained filter cake is that intracellular contains
There is the candida utili bacterium thalline of candida utili callose；
After measured, calculated by every lmL zymotic fluids, it is false to obtain the production protein that 8.91mg intracellulars contain candida utili callose
Silk saccharomycete；
Contain the candida utili bacterium of candida utili callose, 0.0991mg containing callose per mg intracellulars；
Contain candida utili callose 0.6021mg in every mL zymotic fluids i.e. obtained by production method of the invention；
（6）, under the conditions of 121 DEG C, calculate in mass ratio, intracellular contains the Candida utilis ferment of candida utili callose
Female bacterium thalline：Distilled water is 15：100 ratio, with distilled water by step（5）The intracellular of gained contains candida utili β-D-
The candida utili bacterium thalline of glucan carries out extraction 30min, so that candida utili thalline carries out breaking-wall cell, then
In frequency it is 20KHz by ultrasonic cell disruption instrument, power is to carry out supersound process 60min under 800W, then in 10000r/
15min is centrifuged under min, the precipitation of gained is washed with distilled water, until efflux is clarified, is terminated washing, is then controlled again
Temperature is -20 DEG C, and vacuum is that 10Pa carries out vacuum freeze drying, produces candida utili callose.
After measured, the yield of the candida utili callose of above-mentioned gained be 83%, yield 14%, purity is
96%。
In summary, the production method of a kind of candida utili callose of the invention, due in production process
The potato juice raw material of the desizing utilized pH value 4.82, is 29.8g O after testing2/ L and BOD5Value be 15.6g O2/ L,
PH, COD, BOD of its murphy juice discarded close to farina factory5Index, therefore the candida utili of the present invention
The production method of callose can be given birth to by the use of the discarded murphy juice of farina factory as the raw material of culture medium
Production, simultaneously because the intracellular of gained contains the candida utili bacterium thalline of candida utili callose in production process
The extracting method that is acted synergistically by using high-temp extracting and ultrasonication, the high yield finally given, high-purity, high yield pulp1
Candida utili yeast callose, its yield reach as high as 19%, and its purity reaches as high as 97%, and yield reaches as high as
87%。
Above example is merely to illustrate present disclosure, and in addition, the present invention also has other embodiment, in every case
Those skilled in the art are because of technical inspiration involved in the present invention, and the technology for using equivalent substitution or equivalent deformation mode to be formed
Scheme is all fallen within protection scope of the present invention.
Claims (8)
1. a kind of production method of candida utili callose, it is characterised in that specifically include following steps：
（1）, remove starch murphy juice preparation
The beans pulverizer that fetches earth is smashed, and obtains mashed potatoes, and the distilled water of 10 times of mass ratioes is then added in mashed potatoes, is sufficiently stirred
Mix, 20min is centrifuged under the conditions of 10000r/min and removes potato starch, obtained filtrate is placed 5 days, obtains removing potato starch
Murphy juice；
（2）, deproteinized the murphy juice aqueous solution preparation
By step（1）The murphy juice of the removal potato starch of gained is filtered by 8 layers of gauze, is added and is steamed in the filtrate of gained
The addition of distilled water, wherein distilled water is 5 times of gained filtrate volume, and it is to be stripped 1h at 121 DEG C then to control temperature, is obtained
To extract；
The extract of above-mentioned gained is filtered with vacuum pump using circulatory water under -0.1MPa vacuum, the filter of gained after suction filtration
The distilled water of 10 times of the filtrate volume of gained is incorporated as in liquid, the murphy juice aqueous solution of deproteinized is obtained after stirring and evenly mixing；
（3）, deproteinized after sterilizing murphy juice preparation
Regulating step（2）The pH of the murphy juice aqueous solution of the deproteinized of gained is 4-8, then adds glycerine, and temperature is controlled after mixing
Spend and carry out sterilizing 15min for 121 DEG C, the murphy juice of the deproteinized after being sterilized；
It is the NaOH aqueous solution that mass percent concentration is 1% to adjust the alkali used in the pH of the murphy juice aqueous solution of deproteinized, and acid is
Mass percent concentration is 0.1% HCl/water solution；
The addition of above-mentioned glycerine is the 5-25% of the murphy juice aqueous solution volume for the deproteinized that pH is 4-8；
（4）, inoculation fermentation
To step（3）The candida utili bacterium of glycerol tube preservation is inoculated with the murphy juice of deproteinized after the sterilizing of gained
（Candida utilis）CICC 1769, it is 24-32 DEG C then to control temperature, and rotating speed is that 100-200r/min carries out fermentation training
24h is supported, obtains zymotic fluid；
Inoculum concentration presses the candida utili bacterium that 1-8mL glycerol tube preservations are accessed in the deproteinized murphy juice per 100mL after sterilizing
（Candida utilis）CICC 1769；
Per the candida utili bacterium of 1mL glycerol tube preservations（Candida utilis）In CICC 1769, the Candida utilis that contains
Saccharomycete（Candida utilis）The bacterium numbers of CICC 1769 are 1.6 × 109CFU；
（5）, by step（4）The filtering fermentation liquor of gained, filtrate is removed, obtained filter cake is that intracellular contains candida utili
The candida utili bacterium thalline of callose；
（6）, under the conditions of 121 DEG C, using distilled water by step（5）The intracellular of gained contains candida utili callose
Candida utili bacterium thalline carry out extraction 30min so that candida utili somatic cells carry out breaking-wall cell, then will
The leaching liquor of gained is 20KHz in supersonic frequency by ultrasonic cell disruption instrument, carries out supersound process 60min under 800W, so
15min is centrifuged under 10000r/min afterwards, the precipitation of gained is washed with distilled water, until efflux is clarified, terminates washing,
Then it is -20 DEG C to control temperature again, and vacuum is that 10Pa carries out vacuum freeze drying, produces candida utili β-D- Portugals and gathers
Sugar.
A kind of 2. cultural method of high yield candida utili callose as claimed in claim 1, it is characterised in that
Step（1）The middle murphy juice for removing starch is substituted with the discarded murphy juice of farina factory.
A kind of 3. cultural method of high yield candida utili callose as claimed in claim 1, it is characterised in that：
Step（3）In：Regulating step（2）The pH of the murphy juice aqueous solution of the deproteinized of gained is 7-8, and the addition of glycerine is pH
For the 10-15% of the murphy juice aqueous solution volume of 7-8 deproteinized；
Step（4）In：It is 28-30 DEG C to control temperature, and rotating speed is that 150-200r/min is fermented；Inoculum concentration is gone out by every 100mL
The candida utili bacterium of 2-4mL glycerol tube preservations is accessed in deproteinized murphy juice after bacterium（Candida utilis）CICC
1769。
A kind of 4. cultural method of high yield candida utili callose as claimed in claim 3, it is characterised in that：
Step（3）In：Regulating step（2）The pH of the murphy juice aqueous solution of the deproteinized of gained is 7, and the addition of glycerine is that pH is
The 10% of the murphy juice aqueous solution volume of 7 deproteinized；
Step（4）In：It is 30 DEG C to control temperature, and rotating speed is that 200r/min is fermented；Inoculum concentration is gone as after every 100mL sterilizings
The candida utili bacterium of 2mL glycerol tube preservations is accessed in albumen murphy juice（Candida utilis）CICC 1769.
A kind of 5. cultural method of high yield candida utili callose as claimed in claim 3, it is characterised in that：
Step（3）In：Regulating step（2）The pH of the murphy juice aqueous solution of the deproteinized of gained is 8, and the addition of glycerine is that pH is
The 15% of the murphy juice aqueous solution volume of 8 deproteinized；
Step（4）In：It is 28 DEG C to control temperature, and rotating speed is that 150r/min is fermented；Inoculum concentration is gone as after every 100mL sterilizings
The candida utili bacterium of 4mL glycerol tube preservations is accessed in albumen murphy juice（Candida utilis）CICC 1769.
A kind of 6. cultural method of high yield candida utili callose as claimed in claim 1, it is characterised in that：
Step（3）In：Regulating step（2）The pH of the murphy juice aqueous solution of the deproteinized of gained is 7, and the addition of glycerine is that pH is
The 20% of the murphy juice aqueous solution volume of 7 deproteinized；
Step（4）In：It is 32 DEG C to control temperature, and rotating speed is that 200r/min is fermented；Inoculum concentration is gone as after every 100mL sterilizings
The candida utili bacterium of 8mL glycerol tube preservations is accessed in albumen murphy juice（Candida utilis）CICC 1769.
A kind of 7. cultural method of high yield candida utili callose as claimed in claim 1, it is characterised in that：
Step（3）In：Regulating step（2）The pH of the murphy juice aqueous solution of the deproteinized of gained is 7, and the addition of glycerine is that pH is
The 5% of the murphy juice aqueous solution volume of 7 deproteinized；
Step（4）In：It is 26 DEG C to control temperature, and rotating speed is that 100r/min is fermented；Inoculum concentration is gone as after every 100mL sterilizings
The candida utili bacterium of 6mL glycerol tube preservations is accessed in albumen murphy juice（Candida utilis）CICC 1769.
A kind of 8. cultural method of high yield candida utili callose as claimed in claim 1, it is characterised in that：
Step（3）In：Regulating step（2）The pH of the murphy juice aqueous solution of the deproteinized of gained is 4, and the addition of glycerine is that pH is
The 25% of the murphy juice aqueous solution volume of 4 deproteinized；
Step（4）In：It is 24 DEG C to control temperature, and rotating speed is that 100r/min is fermented；Inoculum concentration is gone as after every 100mL sterilizings
The candida utili bacterium of 1mL glycerol tube preservations is accessed in albumen murphy juice（Candida utilis）CICC 1769.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110656080A (en) * | 2019-11-22 | 2020-01-07 | 顾霆 | Directional culture method of yeast cells |
| CN111635916A (en) * | 2020-06-28 | 2020-09-08 | 上海应用技术大学 | Pretreatment method for yeast beta-glucan extraction |
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| CN102337309A (en) * | 2011-09-28 | 2012-02-01 | 吴力克 | Potato residue culture medium |
| CN106397628A (en) * | 2016-10-11 | 2017-02-15 | 上海应用技术大学 | Method for extracting beta-D-glucan from cell walls of candida utilis |
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| CN102337309A (en) * | 2011-09-28 | 2012-02-01 | 吴力克 | Potato residue culture medium |
| CN106397628A (en) * | 2016-10-11 | 2017-02-15 | 上海应用技术大学 | Method for extracting beta-D-glucan from cell walls of candida utilis |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110656080A (en) * | 2019-11-22 | 2020-01-07 | 顾霆 | Directional culture method of yeast cells |
| CN111635916A (en) * | 2020-06-28 | 2020-09-08 | 上海应用技术大学 | Pretreatment method for yeast beta-glucan extraction |
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