CN107523595A - A kind of environment-friendly preparation method thereof of high-purity oligomate - Google Patents
A kind of environment-friendly preparation method thereof of high-purity oligomate Download PDFInfo
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Abstract
The invention discloses a kind of environment-friendly preparation method thereof of high-purity oligomate, it is prepared by the fermentation of the whole yeast cells including the endocellular enzyme containing beta galactosidase, also using ethanol solution permeabilized treatment whole yeast cells, and galactooligosaccharide (yeast purposes one) is prepared using permeability yeast cells catalysing lactose, also obtain high-purity oligomate (yeast purposes two) using whole yeast cells reaction purification galactooligosaccharide crude product.Permeability yeast cells and whole yeast cells, which all repeat, in this method recycles, prepared by the high-purity oligomate green circulatory for realizing " primary yeast, two kinds of purposes ".The galactooligosaccharide product purity that the present invention is obtained reaches as high as 99%, and technique is simple, does not add harmful substance, and required equipment cost is low, is easy to large-scale industry popularization.
Description
Technical field
The invention belongs to functional oligose preparing technical field, is related to a kind of green preparation of high-purity oligomate
Prepared by method, including the fermentation of the whole yeast cells of the endocellular enzyme containing beta galactosidase, also using ethanol solution permeability
Whole yeast cells are handled, and galactooligosaccharide is prepared using permeability yeast cells catalysing lactose, also using yeast
Full cell effect purifying galactooligosaccharide crude product obtains high-purity oligomate, and in this method permeability yeast cells and
Whole yeast cells are all repeatable to be recycled, and the high-purity oligomate green for realizing " primary yeast, two kinds of purposes " is followed
It is prepared by ring.
Background technology
Galactooligosaccharide (galacto-oligosaccharides, GOS) is a kind of important prebiotic factor, can effectively be promoted
The Bifidobacterium propagation entered in enteron aisle, have prevent constipation, improve mineral absorption and lipid-metabolism, promote vitamin generation,
Immunity, anti-aging and the function such as antitumor are improved, is a kind of important functional food additives.GOS is generally passed through by lactose
The transglucosidation catalysis system of beta galactosidase (β-D-galactoside galactohydrolases, EC 3.2.1.23)
.But because beta galactosidase is also a kind of glycoside hydrolase in itself, in GOS synthetic reaction, not only there is breast
Sugar catalyzes and synthesizes target product GOS reaction by transglucosidation, and also there is lactose to be hydrolyzed generation glucose and gala
The side reaction of sugar, this not only reduces target product GOS yield, and makes in GOS crude products simultaneously that there is glucose, galactolipin
With the product such as unreacted lactose, prepare for the purifying of follow-up high-purity GOS products and bring difficulty.
Mainly there are membrane separation process, chromatography, ethanol precipitation and supercritical fluid for GOS purification process at present
Extraction etc..These methods are high to equipment requirement, and complex operation and cost height, usual products obtained therefrom purity is no more than 65%, no
Beneficial to large-scale production high-purity GOS.
The content of the invention
To solve above-mentioned purifying problem, the present invention develops a kind of yeast fermentation method reaction purification GOS new technology.Yeast
Flourish can be carried out using glucose, galactolipin and lactose as carbon source, be it is a kind of can apply in field of food to people
The microorganism of class safety.And yeast, under suitable condition of culture, growth metabolism speed is fast, and the cycle is short, it is easy to industrial big rule
Mould produces.Whole yeast cells are added in GOS crude products, its metabolizable consumption of glucose, galactolipin and lactose can be utilized, but
The characteristics of non-degradable GOS, the miscellaneous sugar component in GOS crude products is removed, improve GOS purity.The present invention is real using a primary yeast
Existing GOS catalysis prepares two kinds of purposes with reaction purification, simple to operate, cost is low, reusable edible, gained GOS product purities
Height, be advantageous to commercial Application and popularization.
Technical scheme is as follows:
A kind of environment-friendly preparation method thereof of high-purity oligomate, comprises the following steps：
1) prepared by the fermentation of the whole yeast cells of the endocellular enzyme containing beta galactosidase；
2) ethanol solution permeabilized treatment whole yeast cells are utilized；
3) galactooligosaccharide is prepared using permeability yeast cells catalysing lactose；
4) high-purity oligomate is obtained using whole yeast cells reaction purification galactooligosaccharide crude product.
The method of the step 1) is：Yeast monoclonal is inoculated into seed liquid culture medium and prepares yeast starter liquid, so
Backward rich lactinated biomass culture medium adds 0.1~10% (v/v) yeast starter liquid, is 16~37 DEG C in temperature, stirring
Rotating speed is 50~500rpm, and pH value is fermented under conditions of being 4~10 to be prepared, and fermentation time be 8~48h, zymotic fluid for concentration 1~
The zymotic fluid of 20g/L whole yeast cells.
Described yeast is selected from Pichia pastoris, saccharomyces cerevisiae, Kluyveromyces lactis or Kluyveromyces fragilis.
Described seed liquid culture medium is to contain 1~30g yeast extracts, 1~30g peptones and 1~10g in every liter of culture medium
Sodium chloride；Described rich lactinated biomass is selected from lactose, whey or whey powder；Described culture medium is in every liter of culture medium
Containing the rich lactinated biomass of 1~100g, 1~30g yeast extracts and 1~30g peptones；Described whole yeast cells contain β-
Galactosidase endocellular enzyme；Also containing the ethanol that concentration is 5~40g/L in described zymotic fluid.
The method of the step 2) is：Zymotic fluid described above is centrifuged, obtains whole yeast cells precipitation
And fermented supernatant fluid, whole yeast cells precipitate the reaction purification for permeabilized treatment and galactooligosaccharide crude product；Using concentration
The whole yeast cells that ethanol solution permeabilized treatment concentration for 20%~50% (v/v) is 1~100g/L, permeabilized treatment temperature
To spend for 0~37 DEG C, the permeabilized treatment time is 5s~5min, centrifuges and obtains Ethanol Treatment liquid and permeability yeast cells,
The 50mM sodium phosphate buffers that 1%~10% (w/v) pH value is 4~10 are added to permeability yeast cells to wash, then are centrifuged and removed
Supernatant is removed, it is every gram of stem cell 1 × 10 to obtain beta galactosidase endocellular enzyme enzyme activity3~4 × 104U permeability yeast is thin
Born of the same parents.
The method of the step 3) is：It is 5~40g/L in permeability barm cell concentration, temperature is 20~50 DEG C, pH value
For 4~10 and under conditions of speed of agitator is 50~500rpm, use permeability yeast cells enzyme law catalysis concentration for 100~
500g/L lactose prepares galactooligosaccharide, after 0.25~18h of catalytic reaction, obtains the oligomeric gala that concentration is 1~150g/L
Sugared crude product, while the glucose that concentration is 50~150g/L and the galactolipin accessory substance that concentration is 20~100g/L are produced, it is catalyzed
Reaction also remains after terminating containing the lactose that concentration is 10~200g/L；Galactooligosaccharide crude product is centrifuged, obtained
Galactooligosaccharide supernatant and permeability yeast cell pellets, galactooligosaccharide supernatant are used for the anti-of high-purity oligomate
It should purify.
The method of the step 4) is：Galactooligosaccharide supernatant described above is carried out to 1 to 10 times of dilution, added
Concentration is 1~100g/L whole yeast cells, is 15~40 DEG C in temperature, pH is 4~10 and speed of agitator is 10~500rpm
Under conditions of carry out fermented and cultured, to purify galactooligosaccharide supernatant, purification time is for consumption lactose, glucose and galactolipin
1~30h, galactooligosaccharide solution after purification is obtained, galactooligosaccharide supernatant and yeast after purification are obtained by centrifuging
Full cell precipitation, galactooligosaccharide supernatant obtain the galactooligosaccharide product that purity is 65%~99% by distillation and concentration.
The described centrifugal condition of the present invention is 1000~15000rpm of centrifugal rotational speed, 1~20min of centrifugation time.
Described fermented supernatant fluid obtains the ethanol solution that concentration is 20~50% (v/v) by distilling, complete for yeast
Cell Permeabilization processing；Described vapo(u)rizing temperature is 60~100 DEG C；Described Ethanol Treatment liquid can in step 2) yeast it is entirely thin
Born of the same parents precipitate permeabilized treatment repeated recycling utilize.
Described permeability yeast cell pellets can prepare galactooligosaccharide repetitive cycling profit by catalysing lactose in step 3)
With.
The described solution of galactooligosaccharide after purification contains ethanol；The described supernatant of galactooligosaccharide after purification is by steaming
20%~50% (v/v) ethanol solution can be obtained by evaporating concentration, available for whole yeast cells permeabilized treatment；Described yeast is entirely thin
Born of the same parents precipitation can in step 4) reaction purification galactooligosaccharide crude product repeated recycling utilize.
The present invention provides a kind of environment-friendly preparation method thereof of high-purity oligomate, compared with prior art with following excellent
Point：
(1) according to the environment-friendly preparation method thereof of high-purity oligomate of the present invention, by " primary yeast, two kinds
Purposes " realizes catalysis preparation and the reaction purification of galactooligosaccharide, and galactooligosaccharide end-product purity can reach 99%, solution
Determine the problem of galactooligosaccharide production cost in the prior art is high, purity is low.
(2) present invention prepares galactooligosaccharide using permeability yeast cells, avoids carrying for beta galactosidase endocellular enzyme
Take and purification step, reduction enzyme activity loss, while permeability yeast cells can be with repeated recycling utilizes, improve enzyme utilizes effect
Rate, reduce the cost of enzyme preparation.
(3) present invention may be used in the fermentation preparatory phase of whole yeast cells and the reaction purification stage of galactooligosaccharide crude product
Ethanol is produced, high-purity oligomate and ethanol synchronous production is realized, reduces production cost.
(4) ethanol that the present invention is obtained can obtain high concentration ethanol product by distillation and concentration, for whole yeast cells
Permeabilized treatment, not only reduce the cost of permeabilized treatment, and avoid the use of other source ethanol, so as to ensure permeability
Change the foodsafety of yeast cells.
(5) whole yeast cells of the present invention, permeability yeast cells and Ethanol Treatment liquid can repetitive cycling profits
With the preparation cost of galactooligosaccharide can be greatly lowered, the pollution to environment can be also avoided in preparation process, is realized
It is prepared by green circulatory.
(6) the environment-friendly preparation method thereof technique of high-purity oligomate of the present invention is simple, does not add harmful substance,
Required equipment cost is low, and product purity is high, is easy to large-scale industry popularization.
Brief description of the drawings
Operating process prepared by the green of Fig. 1 high-purity oligomates (GOS).
Fig. 2 permeability Pichia pastoris catalysis 100g/L lactose prepares GOS timeamplitude map (▼：Lactose；●：It is low
Poly- galactolipin；▲：Glucose；■：Galactolipin).
Timeamplitude map (the ▼ of the full cell effect purifying GOS crude products of Fig. 3 Pichia pastoris：Lactose；●：Galactooligosaccharide；
▲：Glucose；■：Galactolipin).
Fig. 4 permeability brewing yeast cell catalysis 500g/L lactose prepares GOS timeamplitude map (▼：Lactose；●：It is low
Poly- galactolipin；▲：Glucose；■：Galactolipin).
Timeamplitude map (the ▼ of the full cell effect purifying GOS crude products of Fig. 5 saccharomyces cerevisiaes：Lactose；●：Galactooligosaccharide；
▲：Glucose；■：Galactolipin).
Fig. 6 permeable lactic acid saccharomyces Kluyveri cells catalysis 400g/L lactose prepares GOS timeamplitude map (▼：Lactose；
●：Galactooligosaccharide；▲：Glucose；■：Galactolipin).
Fig. 7 permeable lactic acid saccharomyces Kluyveri cells are catalyzed the repeated recycling utilize research (Y for preparing GOSGOS：GOS is total
Percentage composition in sugar).
Timeamplitude map (the ▼ of the full cell effect purifying GOS crude products of Fig. 8 Kluyveromyces lactis：Lactose；●：Oligomeric half
Lactose；▲：Glucose；■：Galactolipin).
Repeated recycling utilize research (the Y of the full cell effect purifying GOS crude products of Fig. 9 Kluyveromyces lactisGOS：GOS is total
Percentage composition in sugar).
Embodiment
Technical scheme is further elaborated with reference to the accompanying drawings and examples, but institute's protection domain of the present invention
Not limited to this.The equivalent substitution done to present invention, or be correspondingly improved, still fall within protection scope of the present invention.
A kind of environment-friendly preparation method thereof (Fig. 1) of high-purity oligomate of the present invention, comprises the steps：
1) prepared by the fermentation of the whole yeast cells of the endocellular enzyme containing beta galactosidase
Yeast monoclonal is inoculated into seed liquid culture medium and prepares yeast starter liquid, then to rich lactinated biomass
Culture medium adds 0.1~10% (v/v) yeast starter liquid, is 16~37 DEG C in temperature, speed of agitator is 50~500rpm, pH value
Prepared to ferment under conditions of 4~10, fermentation time be 8~48h, can must contain concentration for 1~20g/L whole yeast cells
Zymotic fluid.
2) ethanol solution permeabilized treatment whole yeast cells are utilized
Zymotic fluid described above is centrifuged, whole yeast cells precipitation is obtained and fermented supernatant fluid, yeast is complete
Cell precipitation is used for the reaction purification of permeabilized treatment and galactooligosaccharide crude product.Concentration is used as 20%~50% (v/v's)
Ethanol solution permeabilized treatment concentration is 1~100g/L whole yeast cells, and permeabilized treatment temperature is 0~37 DEG C, permeability
Processing time is 5s~5min, centrifuges and obtains Ethanol Treatment liquid and permeability yeast cells, and latter adds 1%~10%
(w/v) the 50mM sodium phosphate buffers that pH value is 4~10 wash, then are centrifuged off supernatant, obtain beta galactosidase intracellular
Enzyme enzyme activity is every gram of stem cell 1 × 103~4 × 104U permeability yeast cells.
3) galactooligosaccharide is prepared using permeability yeast cells catalysing lactose
It is 5~40g/L in permeability barm cell concentration, temperature is 20~50 DEG C, and pH value is 4~10 and speed of agitator is
Under conditions of 50~500rpm, permeability yeast cells enzyme law catalysis concentration is used to be prepared for 100~500g/L lactose oligomeric
Galactolipin, after 0.25~18h of catalytic reaction, concentration can be obtained and be 1~150g/L galactooligosaccharide crude product, while also produced dense
The galactolipin accessory substance for being 20~100g/L for 50~150g/L glucose and concentration is spent, catalytic reaction also contains dense after terminating
Spend the lactose residual for 10~200g/L；Galactooligosaccharide crude product is centrifuged, obtain galactooligosaccharide supernatant and
Permeability yeast cell pellets, the former is used for the reaction purification of high-purity oligomate.
4) high-purity oligomate is obtained using whole yeast cells reaction purification galactooligosaccharide crude product
Galactooligosaccharide supernatant described above is carried out to 1 to 10 times of dilution, adds the ferment that concentration is 1~100g/L
Female full cell, be 15~40 DEG C in temperature, pH be 4~10 and speed of agitator be 10~500rpm under conditions of carry out fermentation training
Support, to purify galactooligosaccharide supernatant, purification time is 1~30h, can be purified for consumption lactose, glucose and galactolipin
Galactooligosaccharide solution afterwards, galactooligosaccharide supernatant after purification and whole yeast cells precipitation are obtained by centrifuging, the former
The galactooligosaccharide product that purity is 65%~99% can be obtained by distillation and concentration.
Embodiment one
It is prepared by the fermentation of the full cell of Pichia pastoris of the endocellular enzyme containing beta galactosidase
The Pichia pastoris strain of preservation is seeded on flat-plate solid LB culture mediums, 30 DEG C of culture 72h, activation single bacterium is made
Fall.Take the single bacterium colony of activation to be seeded in LB liquid medium, 30 DEG C, 160rpm concussion expansion culture 20h, obtain seed culture
Liquid.In 500mL conical flasks, 0.1mL seed culture fluids are accessed in 100mL fermentation mediums, in initial pH value 4.0, rotating speed
50rpm, 48h is cultivated under the conditions of 16 DEG C of temperature, obtain zymotic fluid, be centrifugally separating to obtain in the full cell precipitation of Pichia pastoris and fermentation
Clear liquid, the full cell precipitation of Pichia pastoris are used for the reaction purification of permeabilized treatment and galactooligosaccharide (GOS) crude product, fermentation supernatant
Liquid removes.Fermentative medium formula is as follows：Whey powder 100g/L, peptone 1g/L, yeast extract 1g/L, 121 DEG C of sterilizing 20min.
Utilize the full cell of ethanol solution permeabilized treatment Pichia pastoris
The full cell of Pichia pastoris that concentration is 1g/L is carried out thoroughly under the conditions of 0 DEG C using 20% (v/v) ethanol solution
Propertyization handles 5s, centrifuges and obtains Ethanol Treatment liquid and permeability Pichia pastoris precipitation, Ethanol Treatment liquid removes, to saturating
Property Pichia pastoris precipitation add 1% (w/v) pH value be 4 concentration be 50mmol/L sodium phosphate buffers washing, then from
The heart removes supernatant, obtains permeability Pichia pastoris.
GOS is prepared using permeability Pichia pastoris catalysing lactose
It is 100g/L to prepare lactose solution to mass concentration, adds 5g/L permeability Pichia pastoris, and reaction condition is
20 DEG C, pH 4.0, speed of agitator 50rpm of temperature, take a sample per half an hour, by HPLC detect GOS, lactose, glucose and
Galactose concentration.Experimental result obtains GOS crude products as shown in Fig. 2 after reaction 2.5h, and it contains 2.6% GOS, 19.2%
Remain lactose, 39.9% accessory substance glucose and 38.9% accessory substance galactolipin.20min is centrifuged by 1000rpm, obtains GOS
Supernatant and permeability Pichia pastoris precipitation, GOS supernatants are used for reaction purification, and permeability Pichia pastoris precipitation is removed
Go.
High-purity oligomate is obtained using the full cell effect purifying GOS crude products of Pichia pastoris
It is 100g/L that GOS supernatants are diluted into total carbohydrates concentration, adds the full cell of 1g/L Pichia pastoris, reaction
Condition is 15 DEG C, pH 4, speed of agitator 10rpm, takes a sample within every 3 hours, and GOS, lactose, glucose and half are detected by HPLC
Lactose concn.As shown in figure 3, with the extension of purification time, lactose, glucose and galactolipin are constantly consumed experimental result,
And GOS is not degraded consumption, after purifying 18h, lactose, glucose and galactolipin are almost totally consumed, and realize the pure of GOS
Change.20min is centrifuged by 1000rpm, obtains GOS supernatants after purification and the full cell precipitation of Pichia pastoris, after purification GOS supernatants
Liquid obtains the GOS products that purity is 65% by distillation and concentration, and the full cell precipitation of Pichia pastoris removes.
Embodiment two
It is prepared by the fermentation of the full cell of saccharomyces cerevisiae of the endocellular enzyme containing beta galactosidase
The saccharomyces cerevisiae strain of preservation is seeded on flat-plate solid LB culture mediums, 30 DEG C of culture 72h, activation single bacterium is made
Fall.Take the single bacterium colony of activation to be seeded in LB liquid medium, 30 DEG C, 160rpm concussion expansion culture 20h, obtain seed culture
Liquid.In 500mL conical flasks, 10mL seed culture fluids are accessed in 100mL fermentation mediums, are 10 in initial pH value, rotating speed
For 500rpm, temperature cultivates 8h under the conditions of being 37 DEG C, obtains zymotic fluid, be centrifugally separating to obtain the full cell precipitation of saccharomyces cerevisiae and hair
Ferment supernatant, the full cell precipitation of saccharomyces cerevisiae are used for the reaction purification of permeabilized treatment and GOS crude products, and fermented supernatant fluid removes.
Fermentative medium formula is as follows：Whey 100mL/L, peptone 30g/L, yeast extract 30g/L, 121 DEG C of sterilizing 20min.
Utilize the full cell of ethanol solution permeabilized treatment Pichia pastoris
It is that the full cell of 100g/L saccharomyces cerevisiaes is carried out thoroughly under the conditions of 37 DEG C to concentration using 50% (v/v) ethanol solution
Propertyization handles 5min, centrifuges and obtains Ethanol Treatment liquid and permeability brewing yeast cell precipitation, and Ethanol Treatment liquid removes, to
It is the washing of 50mmol/L sodium phosphate buffers that permeability brewing yeast cell precipitation, which adds the concentration that 10% (w/v) pH value is 10,
Supernatant is centrifuged off again, obtains permeability brewing yeast cell.
GOS is prepared using permeability brewing yeast cell catalysing lactose
It is 500g/L to prepare lactose solution to mass concentration, adds 40g/L permeability brewing yeast cells, and reaction condition is
Temperature 50 C, pH 10.0, speed of agitator 500rpm, take a sample within every 20 minutes, GOS, lactose, glucose are detected by HPLC
And galactose concentration.Experimental result is as shown in figure 4, after reaction 60min, and GOS concentration reaches peak, and GOS crude products contain
20.0% GOS, 37.6% residual lactose, 26.6% accessory substance glucose and 15.8% accessory substance galactolipin.By
15000rpm centrifuges 1min, obtains GOS supernatants and permeability brewing yeast cell precipitation, and GOS supernatants are used for reaction purification,
Permeability brewing yeast cell precipitation removes.
Utilize the full cell effect purifying GOS crude products of saccharomyces cerevisiae
It is 50g/L that GOS supernatants are diluted into total carbohydrates concentration, adds the full cell of 100g/L saccharomyces cerevisiaes, instead
It is 40 DEG C, pH 10.0 to answer condition, speed of agitator 500rpm, takes a sample within every 3 hours, and GOS, lactose, grape are detected by HPLC
Sugar and galactose concentration.Experimental result is as shown in figure 5, with the extension of purification time, lactose, glucose and galactolipin constantly quilt
Consumption, and GOS is not degraded consumption, after purifying 12h, lactose, glucose and galactolipin are almost totally consumed, and realize GOS
Purifying.1min is centrifuged by 15000rpm, obtains GOS supernatants after purification and the full cell precipitation of saccharomyces cerevisiae, after purification GOS
Supernatant obtains the GOS products that purity is 99% by distillation and concentration, and the full cell precipitation of saccharomyces cerevisiae removes.
Embodiment three
It is prepared by the fermentation of the full cell of Kluyveromyces lactis of the endocellular enzyme containing beta galactosidase
The lactic acid yeast kluyveromyces kind of preservation is seeded on flat-plate solid LB culture mediums, 30 DEG C of culture 72h, is made and lives
Change single bacterium colony.Take the single bacterium colony of activation to be seeded in LB liquid medium, 30 DEG C, 160rpm concussion expansion culture 20h, planted
Sub- nutrient solution.In 7-L fermentation tanks, 200mL seed culture fluids are accessed in 4L fermentation mediums, in initial pH value 8.48, turned
Fast 200rpm, throughput 1vvm, 16h is cultivated under the conditions of 27.6 DEG C of temperature, obtain zymotic fluid, be centrifugally separating to obtain Kluyveromyces Lactis dimension
Whole yeast cells precipitate and fermented supernatant fluid, and the full cell precipitation of Kluyveromyces lactis is used for permeabilized treatment and GOS crude products
Reaction purification, fermented supernatant fluid are used for distillation and concentration.Fermentative medium formula is as follows：Lactose 60g/L, peptone 5g/L, yeast
Cream 12g/L, 121 DEG C of sterilizing 20min.
Utilize the full cell of ethanol solution permeabilized treatment Kluyveromyces lactis
Gained fermented supernatant fluid contains the ethanol that concentration is 30g/L, and 35% is obtained by distillation and concentration (temperature is 80 DEG C)
(v/v) ethanol solution, using 35% (v/v) ethanol solution to concentration be the full cell of 50g/L Kluyveromyces lactis in room temperature
Lower progress permeabilized treatment 2min, centrifuge and obtain Ethanol Treatment liquid and permeable lactic acid saccharomyces Kluyveri cell precipitation, second
Alcohol treatment fluid continues on for permeabilized treatment, repeats 3 times, is precipitated to permeable lactic acid saccharomyces Kluyveri cell and adds 5% (w/
V) concentration that pH value is 8 is the washing of 50mmol/L sodium phosphate buffers, then is centrifuged off supernatant, obtains Permeabilized cells.
GOS is prepared using permeable lactic acid saccharomyces Kluyveri cell catalysing lactose
It is 400g/L to prepare lactose solution to mass concentration, adds 18.87g/L permeable lactic acid saccharomyces Kluyveri cells,
Reaction condition is 40 DEG C, pH 8.0 of temperature, speed of agitator 150rpm, takes a sample within every 20 minutes, and GOS, breast are detected by HPLC
Sugar, glucose and galactose concentration.Experimental result obtains GOS crude products, it contains 35.0% as shown in fig. 6, after reaction 1.5h
GOS, 27.4% residual lactose, 25.2% accessory substance glucose and 12.4% accessory substance galactolipin.Centrifuged by 5000rpm
5min, obtains GOS supernatants and permeable lactic acid saccharomyces Kluyveri cell precipitation, and GOS supernatants are used for reaction purification, permeability
Kluyveromyces lactis cell precipitation continues on for catalysing lactose and prepares GOS, repeatable to recycle, as shown in fig. 7, permeability
Cell is reused 14 times, and the yield of GOS crude products is maintained between 34.8%~35.7%.
Utilize the full cell effect purifying GOS crude products of Kluyveromyces lactis
It is 100g/L that GOS crude products are diluted into total carbohydrates concentration, and it is entirely thin to add 15g/L Kluyveromyces lactis
Born of the same parents, reaction condition be 30 DEG C, pH 8.0, speed of agitator 150rpm, take a sample per hour, by HPLC detect GOS, lactose,
Glucose and galactose concentration.Experimental result is as shown in figure 8, with the extension of purification time, and lactose, glucose and galactolipin are not
It is disconnected to be consumed, and GOS is not degraded consumption, after purifying 15h, lactose, glucose and galactolipin are almost totally consumed, and are realized
GOS purifying.5min is centrifuged by 5000rpm, obtains GOS supernatants after purification and the full cell precipitation of Kluyveromyces lactis,
GOS supernatants obtain the high concentration ethanol that concentration is 35% (v/v) and the high-purity GOS productions concentrated by distillation and concentration after purification
Product, the full cell precipitation of Kluyveromyces lactis continue on for reaction purification, repeatable to recycle, as shown in figure 9, recycling
More than 9 times, GOS purity maintains 95%~99%.
Claims (10)
- A kind of 1. environment-friendly preparation method thereof of high-purity oligomate, it is characterized in that comprising the following steps：1) prepared by the fermentation of the whole yeast cells of the endocellular enzyme containing beta galactosidase；2) ethanol solution permeabilized treatment whole yeast cells are utilized；3) galactooligosaccharide is prepared using permeability yeast cells catalysing lactose；4) high-purity oligomate is obtained using whole yeast cells reaction purification galactooligosaccharide crude product.
- 2. the method as described in claim 1, it is characterized in that the method for the step 1) is：Yeast monoclonal is inoculated into seed Yeast starter liquid is prepared in liquid culture medium, then adds 0.1~10% (v/v) yeast kind to rich lactinated biomass culture medium Sub- liquid, it is 16~37 DEG C in temperature, speed of agitator be 50~500rpm, fermentation preparation under conditions of pH value is 4~10, during fermentation Between be 8~48h, zymotic fluid is the zymotic fluid of 1~20g/L of concentration whole yeast cells.
- 3. method as claimed in claim 2, it is characterized in that described yeast is selected from Pichia pastoris, saccharomyces cerevisiae, Kluyveromyces Lactis Tie up yeast or Kluyveromyces fragilis；Described seed liquid culture medium is to contain 1~30g yeast extracts in every liter of culture medium, 1~ 30g peptones and 1~10g sodium chloride；Described rich lactinated biomass is selected from lactose, whey or whey powder；Described training Foster base is containing the rich lactinated biomass of 1~100g, 1~30g yeast extracts and 1~30g peptones in every liter of culture medium；It is described Whole yeast cells contain beta galactosidase endocellular enzyme；Also containing the ethanol that concentration is 5~40g/L in described zymotic fluid.
- 4. the method as described in claim 1, it is characterized in that the method for the step 2) is：Zymotic fluid described above is carried out Centrifuge, obtain whole yeast cells precipitation and fermented supernatant fluid, whole yeast cells are precipitated for permeabilized treatment and oligomeric half The reaction purification of lactose crude product；Use concentration for 20%~50% (v/v) ethanol solution permeabilized treatment concentration be 1~ 100g/L whole yeast cells, permeabilized treatment temperature are 0~37 DEG C, and the permeabilized treatment time is 5s~5min, are centrifuged Ethanol Treatment liquid and permeability yeast cells are obtained, it is 4~10 to add 1%~10% (w/v) pH value to permeability yeast cells The washing of 50mM sodium phosphate buffers, then be centrifuged off supernatant, it is every gram dry thin to obtain beta galactosidase endocellular enzyme enzyme activity Born of the same parents 1 × 103~4 × 104U permeability yeast cells.
- 5. the method as described in claim 1, it is characterized in that the method for the step 3) is：It is in permeability barm cell concentration 5~40g/L, temperature is 20~50 DEG C, under conditions of pH value is 4~10 and speed of agitator is 50~500rpm, using permeability Yeast cells enzyme law catalysis concentration is that 100~500g/L lactose prepares galactooligosaccharide, after 0.25~18h of catalytic reaction, is obtained Concentration is 1~150g/L galactooligosaccharide crude product, while it is 20 to produce glucose that concentration is 50~150g/L and concentration ~100g/L galactolipin accessory substance, catalytic reaction also remain after terminating containing the lactose that concentration is 10~200g/L；Will be oligomeric Galactolipin crude product is centrifuged, acquisition galactooligosaccharide supernatant and permeability yeast cell pellets, on galactooligosaccharide Clear liquid is used for the reaction purification of high-purity oligomate.
- 6. the method as described in claim 1, it is characterized in that the method for the step 4) is：By galactooligosaccharide described above Supernatant carries out 1 to 10 times of dilution, adds the whole yeast cells that concentration is 1~100g/L, is 15~40 DEG C in temperature, pH is 4~10 and speed of agitator be 10~500rpm under conditions of carry out fermented and cultured, consumption lactose, glucose and galactolipin are to purify Galactooligosaccharide supernatant, purification time are 1~30h, obtain galactooligosaccharide solution after purification, pure by centrifuging acquisition Galactooligosaccharide supernatant and whole yeast cells precipitation after change, galactooligosaccharide supernatant obtain purity by distillation and concentration and are 65%~99% galactooligosaccharide product.
- 7. the method as described in claim 4,5 or 6, it is characterized in that described centrifugation bar be centrifugal rotational speed 1000~ 15000rpm, 1~20min of centrifugation time.
- 8. method as claimed in claim 4, it is characterized in that described fermented supernatant fluid by distill obtain concentration be 20~ 50% (v/v) ethanol solution, for whole yeast cells permeabilized treatment；Described vapo(u)rizing temperature is 60~100 DEG C；Described Ethanol Treatment liquid whole yeast cells in step 2) precipitate permeabilized treatment repeated recycling utilize.
- 9. method as claimed in claim 5, it is characterized in that described permeability yeast cell pellets catalysis breast in step 3) Sugar prepares galactooligosaccharide repeated recycling utilize.
- 10. method as claimed in claim 6, it is characterized in that the described solution of galactooligosaccharide after purification contains ethanol；It is described The supernatant of galactooligosaccharide after purification by distillation and concentration obtain 20%~50% (v/v) ethanol solution, it is entirely thin for yeast Born of the same parents' permeabilized treatment；Described whole yeast cells are deposited in reaction purification galactooligosaccharide crude product repetitive cycling profit in step 4) With.
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| CN111334541A (en) * | 2020-02-24 | 2020-06-26 | 天津大学 | Method for preparing high-purity galactooligosaccharide by using β -galactosidase |
| CN111378636A (en) * | 2020-02-24 | 2020-07-07 | 天津大学 | Integrated method for co-production of beta-galactosidase enzyme preparation and ethanol product from lactose-rich biomass |
| WO2023161321A1 (en) | 2022-02-25 | 2023-08-31 | Frieslandcampina Nederland B.V. | Process for preparing high purity galacto-oligosaccharide |
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| CN111378636A (en) * | 2020-02-24 | 2020-07-07 | 天津大学 | Integrated method for co-production of beta-galactosidase enzyme preparation and ethanol product from lactose-rich biomass |
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