CN107099566B - Production method of high-optical-purity alpha-arbutin and high-adhesiveness glucan - Google Patents
Production method of high-optical-purity alpha-arbutin and high-adhesiveness glucan Download PDFInfo
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Abstract
A method for co-producing high optical purity alpha-arbutin and high-adhesiveness glucan by leuconostoc mesenteroides is characterized in that sucrose and hydroquinone are used as substrates, and alpha-arbutin and glucan are obtained by one-step fermentation production in a weakly acidic environment, and specifically comprises the following steps: 1) first-order seed culture: measuring a leuconostoc pseudomesenteroides bacterial solution by using 0.1-0.2% of inoculation amount, inoculating the leuconostoc pseudomesenteroides bacterial solution into a seed culture medium, and culturing at 37 ℃ and 250rpm for 10-12 h; 2) secondary seed culture: transferring the primary seed solution into a seed culture medium by an inoculation amount of 1-2%, and culturing at 37 ℃ and 250rpm for 10-12 h; 3) fermentation culture: inoculating the secondary seed liquid into a fermentation culture medium in an inoculation amount of 5-8%, adjusting the pH value to 6.0-6.8 in the fermentation process, introducing air in the whole fermentation process, controlling the air flow to be 0.1-0.3 vvm, and culturing for 12-16 h at 37 ℃ and 250-300 rpm. The method can directly obtain high optical purity alpha-arbutin and high-adhesiveness glucan by strain fermentation, has high substrate utilization rate, simple and rapid reaction, mild conditions and low energy consumption, and is suitable for industrial production.
Description
Technical Field
The invention relates to the technical field of biological fermentation, in particular to a production method for producing high-optical-purity alpha-arbutin and high-adhesiveness glucan by leuconostoc pseudomesenteroides.
Background
Arbutin has two isomers: alpha-arbutin and beta-arbutin, both epimers, with chemical names of 4-hydroxyphenyl-alpha-D-glucopyranoside and 4-hydroxyphenyl-beta-D-glucopyranoside, the two oxygen glycosidic bonds being in opposite directions in space. Research shows that the intensity and safety of the alpha-arbutin for inhibiting tyrosinase are far greater than those of beta-arbutin, and the whitening effect is 9-10 times of that of the beta-arbutin. Therefore, α -arbutin, as a highly effective cosmetic whitening agent, is currently being used by a large number of worldwide brands: in 2002, new active skin whitening agent containing alpha-arbutin is introduced by Peutaharm, and series cosmetics containing alpha-arbutin are also introduced by brands such as Japan senkyo, DHC and the like. Researches show that the chemical property of the alpha-arbutin is more stable than that of the beta-arbutin, the alpha-arbutin can be conveniently added into various whitening and skin-brightening cosmetics, the alpha-arbutin is most stable when the pH value is 3.5-6.5, the recommended addition amount is 0.2-5%, and the alpha-arbutin can be used in all formulas.
The sources of alpha-arbutin and beta-arbutin are different. The beta-arbutin can be obtained by plant extraction, plant cell culture, artificial synthesis and glycosyl transfer reaction or reverse hydrolysis reaction of glycosyl transferase or glycosidase. The alpha-arbutin can be obtained only by transglycosylation reaction of enzymes of different microorganisms, plant extraction or direct transformation of microorganisms. Kitao et al, who is a Japanese scholarler, use sucrose phosphorylase, add 2g hydroquinone to HEPES buffer solution to react with 30g sucrose at 40 ℃ for 14 hours to obtain 2.3g of alpha-arbutin (JP 06153976). Liuchuqiao and the like are fermented by using Xanthomonas BT2112, alpha-arbutin is synthesized by the hydroquinone through biocatalysis, 12.7g/L of arbutin is obtained through reaction, but the cell fermentation time is long, and the tolerance of the cell to the hydroquinone is relatively low (Liuchuqiao and the like, catalytic bulletin, 2006,27:361 and 364). Fanayama et al, cultured with Bacillus subtilis IFO14140 at 30 ℃ for 4 hours under shaking, aerobically cultured at 37 ℃ for 120 hours, then purified to give a culture broth, and reacted with hydroquinone, sucrose and the culture broth at 40 ℃ for 90 hours in an acetic acid buffer (pH 5.3) to prepare alpha-arbutin (JP 06284896).
Dextran (glucan) is microbial homotype exopolysaccharide synthesized by leuconostoc, belongs to a dextro-glucopyranose polymer, is a microbial metabolic gum with safety and excellent physicochemical properties, can be used as an emulsifier, a suspending agent, a gel, a film-forming agent, a lubricant and the like to be widely applied to the fields of food, petroleum, chemical industry, pharmacy and the like, and has more advantages in the application aspect of biosynthetic gum due to the excellent gelling performance compared with common dextran without high bonding performance.
At present, the preparation method of glucan mainly comprises a fermentation method and an enzyme method, the large-scale application of the glucan produced by the enzyme method in the industry is difficult, further research is needed, the method for industrially producing the glucan is mainly a microbial fermentation method, but the molecular weight of the glucan directly obtained by the fermentation method is generally more than 100KDa, and the bonding property is poor.
Disclosure of Invention
Aiming at the problems, the invention simultaneously prepares the alpha-arbutin with high optical purity and the high-adhesiveness glucan by utilizing the leuconostoc pseudomesenteroides G123 obtained by self-screening through the change of raw materials and the control of fermentation conditions.
The invention aims to provide a method for producing high-optical purity alpha-arbutin and high-adhesiveness glucan, which utilizes a leuconostoc pseudomesenteroides strain to ferment, and directly obtains two products of alpha-arbutin and glucan by one-step fermentation through the control of fermentation conditions and the change of substrates.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
the invention utilizes leuconostoc pseudomesenteroides G123 with a preservation number of CCTCC NO: M2014115 as a substrate, regulates and controls the fermentation pH within a partial acid range of 6.0-6.8, and can produce high-optical purity alpha-arbutin and high-adhesiveness glucan, wherein the optical purity of the obtained high-optical purity alpha-arbutin is 100%, NO arbutin with other configurations is generated, and the molecular weight of the high-adhesiveness glucan is 700-900 Da.
The leuconostoc pseudomesenteroides is inoculated into a fermentation culture medium for fermentation culture after the seed culture step, and the method comprises the following steps:
1) first-order seed culture: measuring a leuconostoc pseudomesenteroides bacterial solution by using 0.1-0.2% of inoculation amount, inoculating the leuconostoc pseudomesenteroides bacterial solution into a seed culture medium, and culturing at 37 ℃ and 250rpm for 10-12 h;
2) secondary seed culture: transferring the primary seed solution into a seed culture medium with the inoculation amount of 1-2%, and culturing at 37 ℃ and 250rpm for 10-12 h;
3) fermentation culture: inoculating the secondary seed liquid into a fermentation culture medium by an inoculation amount of 5-8%, adjusting the initial pH to 6.5-6.8, controlling the pH value to be not lower than 6.0 in the fermentation process by using 20% (wt) NaOH, introducing air in the whole fermentation process, controlling the air flow to be 0.1-0.3 vvm, adding 0.1-0.3% of defoaming agent, culturing at 37 ℃ and 250-300 rpm for 12-16 h.
The seed isThe seed culture medium is: 3-10 g/L of peptone, 3-10 g/L of yeast powder, 3-10 g/L of sodium acetate, and 3-5 ml/L, C of tween-800.56H5O7(NH4)31～5g/L、K2HPO4·7H2O1-5 g/L, nicotinic acid 0.5-2 g/L, MgSO4·7H2O 0.1～1g/L、MnSO4·H2O 0.01～1g/L、CaCl20.01～1g/L、FeCl20.01-1 g/L, NaCl 0.01, 0.01-1 g/L and 5-20 g/L sucrose solution are independently sterilized and supplemented.
The seed culture medium is preferably: 4-8 g/L of peptone, 4-8 g/L of yeast powder, 4-8 g/L of sodium acetate and 801-2 ml/L, C of Tween6H5O7(NH4)32～3g/L、K2HPO4·7H2O2-3 g/L, nicotinic acid 1-2 g/L, MgSO4·7H2O 0.2～0.4g/L、MnSO4·H2O 0.05～0.08g/L、CaCl20.02～0.06g/L、FeCl20.01-0.02 g/L, NaCl 0.01.01-0.02 g/L and 10-12 g/L of sucrose solution are independently sterilized and supplemented.
The seed culture medium is optimally as follows: peptone 5.0g/L, yeast powder 5.0g/L, sodium acetate 5.0g/L, Tween-801 ml/L, C6H5O7(NH4)32.0g/L、K2HPO4·7H2O2.0 g/L, nicotinic acid 1.0g/L, MgSO4·7H2O 0.2g/L、MnSO4·H2O 0.05g/L、CaCl20.02g/L、FeCl20.01g/L, NaCl 0.01.01 g/L and 10g/L sucrose solution are separately sterilized and supplemented.
The fermentation medium is as follows: 3-10 g/L of peptone, 3-10 g/L of yeast powder, 3-10 g/L of sodium acetate, and 3-5 ml/L, C of tween-800.56H5O7(NH4)31～5g/L、K2HPO4·7H2O1-5 g/L, nicotinic acid 0.5-5 g/L, MgSO4·7H2O 0.1～1g/L、MnSO4·H2O 0.01～1g/L、CaCl20.01～1g/L、FeCl20.005-1 g/L, NaCl 0.005-1 g/L, Glu 0.1-3 g/L, Val 0.1-0.3 g/L, 5-40 g/L hydroquinone sterilized separately 1And (3) adding the sucrose for 0 time, independently sterilizing 30-180 g/L of sucrose and simultaneously adding hydroquinone for 10 times.
The fermentation medium is preferably: 4-8 g/L of peptone, 4-8 g/L of yeast powder, 4-8 g/L of sodium acetate and 801-2 ml/L, C of Tween6H5O7(NH4)32～3g/L、K2HPO4·7H2O2-3 g/L, nicotinic acid 1-2 g/L, MgSO4·7H2O 0.2～0.4g/L、MnSO4·H2O 0.05～0.08g/L、CaCl20.02～0.05g/L、FeCl20.01-0.05 g/L, NaCl 0.01.01-0.05 g/L, Glu 0.1.1-1 g/L, Val 0.1-0.2 g/L and 10-35 g/L of hydroquinone are independently sterilized and supplemented for 10 times, and 80-160 g/L of sucrose is independently sterilized and is supplemented with hydroquinone simultaneously for 10 times.
The optimal fermentation medium is peptone 5.0g/L, yeast powder 5.0g/L, sodium acetate 5.0g/L, and Tween-801 ml/L, C6H5O7(NH4)32.0g/L、K2HPO4·7H2O2.0 g/L, nicotinic acid 1.0g/L, MgSO4·7H2O 0.2g/L、MnSO4·H2O 0.05g/L、Cacl20.02g/L、FeCl2Independently sterilizing hydroquinone of 0.01g/L, NaCl 0.01.01 g/L, Glu 0.35.35 g/L, Val 0.115.115 g/L and 15-30 g/L for 10 times, and independently sterilizing sucrose of 90-150 g/L and simultaneously replenishing hydroquinone for 10 times.
Has the advantages that: the high-optical-purity alpha-arbutin and high-adhesiveness glucan are directly obtained through strain fermentation, and are not required to be obtained through enzyme preparation and then enzyme conversion or enzyme decomposition, the substrate utilization rate is high in the production process, the yield of alpha-arbutin relative to hydroquinone is 60-80%, the yield of glucan is about 40% relative to the mass of sucrose, the glucan solution is high in adhesiveness and has high gelling performance, if the glucan is used as a gel, the glucan can form a colloid at the concentration of 0.05-0.3%, the addition amount of the glucan in food is saved, the glucan is a microbial metabolic gel with excellent performance, and the glucan solution has a wide application prospect.
Drawings
FIG. 1 shows the result of fermentation of Leuconostoc pseudomesenteroides G123 expressed by the flight profile of dextran; wherein the fermentation conditions are as follows: controlling pH to be not less than 6.0 in the initial pH of 6.5 and the whole fermentation process, independently sterilizing 15g/L hydroquinone and supplementing 10 times, independently sterilizing 90g/L sucrose and simultaneously supplementing hydroquinone, and respectively supplementing 10 times in a 3L fermentation tank at 37 ℃.
FIG. 2 is a graph showing the plate impact experiment of Leuconostoc pseudomesenteroides G123 on hydroquinone.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as described in the claims.
Leuconostoc pseudomesenteroides (Leuconostoc pseudomesenteroides) strains used in the present invention: the strain is preserved by the patent program of China type culture Collection acknowledged by the China patent office or the International patent organization in the United laboratories, and the preservation numbers of the strain are as follows: CCTCC NO: and M2014115.
Example 1
The method comprises the following steps of (1) fermenting leuconostoc pseudomesenteroides G123 by adopting a 3L fermentation tank, wherein the liquid loading amount is 2L, the fermentation condition is initial pH6.5, the pH is controlled to be 6.0-6.8 in the whole fermentation process, 15G/L hydroquinone is independently sterilized and supplemented for 10 times, 90G/L sucrose is independently sterilized and is supplemented with hydroquinone simultaneously, and the method is divided into 10 times, and specifically comprises the following steps:
(1) seed liquid culture:
first-order seed culture: inoculating the leuconostoc pseudomesenteroides bacterial liquid in a glycerol storage tube by 0.1-0.2% of inoculation amount into a seed culture medium, and culturing at 37 ℃ and 250rpm for 11 h;
secondary seed culture: transferring the primary seed solution into a seed culture medium with the inoculation amount of 1-2%, and culturing at 37 ℃ and 250rpm for 11 h;
the seed culture medium is as follows: peptone 5.0g/L, yeast powder 5.0g/L, sodium acetate 5.0g/L, Tween-801 ml/L, C6H5O7(NH4)32.0g/L、K2HPO4·7H2O2.0 g/L, nicotinic acid 1.0g/L, MgSO4·7H2O 0.2g/L、MnSO4·H2O 0.05g/L、CaCl20.02g/L、FeCl20.01g/L, NaCl 0.01.01 g/L and 10g/L sucrose solution are separately sterilized and supplemented.
(2) Culturing in a fermentation tank:
inoculating the secondary seed solution into a fermentation culture medium by an inoculation amount of 5-8%, adjusting the initial pH to 6.5-6.8, controlling the pH to 6.0-6.8 in the fermentation process by using 20% (wt) NaOH, introducing air in the whole fermentation process, controlling the air flow to 0.1-0.3 vvm, adding 0.1-0.3% of a defoaming agent, independently sterilizing 15g/L of hydroquinone at 37 ℃ and 250-300 rpm for 10 times, and simultaneously sterilizing 90g/L of sucrose and simultaneously replenishing hydroquinone for 10 times.
The fermentation medium is as follows: peptone 5.0g/L, yeast powder 5.0g/L, sodium acetate 5.0g/L, Tween-801 ml/L, C6H5O7(NH4)32.0g/L、K2HPO4·7H2O2.0 g/L, nicotinic acid 1.0g/L, MgSO4·7H2O 0.2g/L、MnSO4·H2O 0.05g/L、CaCl20.02g/L、FeCl20.01g/L, NaCl 0.01, 0.01g/L, Glu 0.35, 0.35g/L, Val 0.115, 0.115g/L and 15g/L of hydroquinone are independently sterilized and supplemented for 10 times, and 90g/L of sucrose is independently sterilized and supplemented with hydroquinone simultaneously and 10 times.
After fermenting for 12h, the contents of sucrose, hydroquinone and alpha-arbutin in the fermentation liquor are detected by HPLC, the content of glucan in the fermentation liquor in each time period is detected by a constant weight method, and the fermentation results are shown in Table 1.
In this example, most of the hydroquinone was converted to arbutin and part of the sucrose was converted to glucan at 12h of fermentation. The molecular weight of the glucan is analyzed by mass spectrometry, and is 700-900 Da as shown in figure 1.
TABLE 1 fermentation results of Leuconostoc pseudomesenteroides G123 using sucrose and hydroquinone as raw materials
Example 2
The leuconostoc pseudomesenteroides G123 is fermented by a 3L fermentation tank, the liquid loading amount is 2L, the fermentation condition is initial pH6.5, the pH is controlled to be 6.0-6.8 in the whole fermentation process, 30G/L hydroquinone is independently sterilized and supplemented for 10 times, 150G/L sucrose is independently sterilized and is supplemented with hydroquinone simultaneously for 10 times, and the method specifically comprises the following steps:
(1) seed liquid culture:
first-order seed culture: inoculating the leuconostoc pseudomesenteroides bacterial liquid in a glycerol storage tube by 0.1-0.2% of inoculation amount into a seed culture medium, and culturing at 37 ℃ and 250rpm for 10 h;
secondary seed culture: transferring the primary seed solution into a seed culture medium with the inoculation amount of 1-2%, and culturing at 37 ℃ and 250rpm for 10 h;
the seed culture medium is as follows: peptone 4.0g/L, yeast powder 4.0g/L, sodium acetate 4.0g/L, Tween-802 ml/L, C6H5O7(NH4)33.0g/L、K2HPO4·7H2O3.0 g/L, nicotinic acid 2.0g/L, MgSO4·7H2O 0.4g/L、MnSO4·H2O 0.8g/L、CaCl20.06g/L、FeCl20.02g/L, NaCl 0.02.02 g/L and 12g/L sucrose solution are separately sterilized and supplemented.
(2) Culturing in a fermentation tank:
inoculating the secondary seed solution into a fermentation culture medium by an inoculation amount of 5-8%, adjusting the initial pH to 6.5-6.8, controlling the pH to 6.0-6.8 in the fermentation process by 10% (wt) NaOH, introducing air in the whole fermentation process, controlling the air flow to 0.1-0.3 vvm, adding 0.1-0.3% of a defoaming agent, independently sterilizing 30g/L of hydroquinone at 37 ℃ and 250-300 rpm, replenishing by 10 times, independently sterilizing 150g/L of sucrose and simultaneously replenishing hydroquinone by 10 times.
The fermentation medium is as follows: peptone 4.0g/L, yeast powder 4.0g/L, sodium acetate 4.0g/L, Tween-802 ml/L, C6H5O7(NH4)33.0g/L、K2HPO4·7H2O3.0 g/L, nicotinic acid 2.0g/L, MgSO4·7H2O 0.4g/L、MnSO4·H2O 0.08g/L、CaCl20.05g/L、FeCl20.05g/L, NaCl 0.05.05 g/L, Glu 1g/L, Val 0.2.2 g/L and 30g/L of hydroquinone are independently sterilized and supplemented for 10 times, and 150g/L of sucrose is independently sterilized and supplemented with hydroquinone simultaneously and 10 times. Fermenting for 16 h.
Example 3
The leuconostoc pseudomesenteroides G123 is fermented by a 3L fermentation tank, the liquid loading amount is 2L, the fermentation condition is initial pH6.5, the pH is controlled to be 6.0-6.8 in the whole fermentation process, 10G/L hydroquinone is independently sterilized and supplemented for 10 times, 80G/L sucrose is independently sterilized and is supplemented with hydroquinone simultaneously for 10 times, and the method specifically comprises the following steps:
(1) seed liquid culture:
first-order seed culture: inoculating the leuconostoc pseudomesenteroides bacterial liquid in a glycerol storage tube by 0.1-0.2% of inoculation amount into a seed culture medium, and culturing at 37 ℃ and 250rpm for 12 h;
secondary seed culture: transferring the primary seed solution into a seed culture medium by an inoculation amount of 1-2%, and culturing at 37 ℃ and 250rpm for 12 h;
the seed culture medium is as follows: peptone 8.0g/L, yeast powder 8.0g/L, sodium acetate 8.0g/L, Tween-805 ml/L, C6H5O7(NH4)35.0g/L、K2HPO4·7H2O5.0 g/L, nicotinic acid 1.5g/L, MgSO4·7H2O 1g/L、MnSO4·H2O 1g/L、CaCl21g/L、FeCl2Independently sterilizing and supplementing 1g/L, NaCl 1g/L and 20g/L sucrose solution.
(2) Culturing in a fermentation tank:
inoculating the secondary seed liquid into a fermentation medium in an inoculation amount of 5-8%, adjusting the initial pH to 6.5-6.8, and adding 20% (wt) Na2CO3Controlling the pH value to be 6.0-6.8 in the fermentation process, introducing air in the whole fermentation process, controlling the air flow to be 0.1-0.3 vvm, adding 0.1-0.3% of defoaming agent, sterilizing 10g/L of hydroquinone independently at 37 ℃ and 250-300 rpm, supplementing 10 times, sterilizing 80g/L of sucrose independently and supplementing hydroquinone simultaneously, and supplementing 10 times.
The fermentation medium is as follows: peptone 8.0g/L, yeast powder 8.0g/L, sodium acetate 8.0g/L, Tween-805 ml/L, C6H5O7(NH4)35.0g/L、K2HPO4·7H2O5.0 g/L, nicotinic acid 5.0g/L, MgSO4·7H2O 1g/L、MnSO4·H2O 1g/L、CaCl21g/L、FeCl21g/L, NaCl 1g/L, Glu 3g/L, Val 0.1.1 g/L and 10g/L of hydroquinone are independently sterilized and supplemented for 10 times, and 80g/L of sucrose is independently sterilized and supplemented with hydroquinone at the same time and 10 times. Fermenting for 13 h.
Example 4
The leuconostoc pseudomesenteroides G123 is fermented by a 3L fermentation tank, the liquid loading amount is 2L, the fermentation condition is the initial pH value of 6.5, the pH value is controlled to be 6.0-6.8 in the whole fermentation process, 40G/L hydroquinone is independently sterilized and supplemented for 10 times, 160G/L sucrose is independently sterilized and is simultaneously supplemented with hydroquinone for 10 times, and the method specifically comprises the following steps:
(1) seed liquid culture:
first-order seed culture: inoculating the leuconostoc pseudomesenteroides bacterial liquid in a glycerol storage tube by 0.1-0.2% of inoculation amount into a seed culture medium, and culturing at 37 ℃ and 250rpm for 12 h;
secondary seed culture: transferring the primary seed solution into a seed culture medium by an inoculation amount of 1-2%, and culturing at 37 ℃ and 250rpm for 12 h;
the seed culture medium is as follows: peptone 3.0g/L, yeast powder 3.0g/L, sodium acetate 3.0g/L, Tween-800.5 ml/L, C6H5O7(NH4)31.0g/L、K2HPO4·7H2O1.0 g/L, nicotinic acid 0.5g/L, MgSO4·7H2O 0.1g/L、MnSO4·H2O 0.01g/L、CaCl20.01g/L、FeCl20.01g/L, NaCl 0.01.01 g/L and 5g/L sucrose solution are separately sterilized and supplemented.
(2) Culturing in a fermentation tank:
inoculating the secondary seed solution into a fermentation culture medium by an inoculation amount of 5-8%, adjusting the initial pH to 6.5-6.8, controlling the pH to 6.0-6.8 in the fermentation process by 10% (wt) NaOH, introducing air in the whole fermentation process, controlling the air flow to 0.1-0.3 vvm, adding 0.1-0.3% of a defoaming agent, independently sterilizing 40g/L of hydroquinone at 37 ℃ and 250-300 rpm, replenishing the hydroquinone for 10 times, independently sterilizing 160g/L of sucrose and replenishing the hydroquinone for 10 times.
The fermentation medium is as follows: peptone 3.0g/L, yeast powder 3.0g/L, sodium acetate 3.0g/L, Tween-800.5 ml/L, C6H5O7(NH4)31.0g/L、K2HPO4·7H2O1.0 g/L, nicotinic acid 0.5g/L, MgSO4·7H2O 0.1g/L、MnSO4·H2O 0.01g/L、CaCl20.01g/L、FeCl20.005g/L, NaCl 0.005, 0.005g/L, Glu 0.1, 0.1g/L, Val 0.1, 0.1g/L and 40g/L of hydroquinone are independently sterilized and supplemented by 10 times, and 160g/L of sucrose is independently sterilized and supplemented by 10 times together with hydroquinone. Fermenting for 14 h.
Example 5
The leuconostoc pseudomesenteroides G123 is fermented by a 3L fermentation tank, the liquid loading amount is 2L, the fermentation condition is the initial pH value of 6.5, the pH value is controlled to be 6.0-6.8 in the whole fermentation process, 35G/L of hydroquinone is independently sterilized and supplemented for 10 times, 30G/L of cane sugar is independently sterilized and supplemented with hydroquinone simultaneously and for 10 times, and the method specifically comprises the following steps:
(1) seed liquid culture:
first-order seed culture: inoculating the leuconostoc pseudomesenteroides bacterial liquid in a glycerol storage tube by 0.1-0.2% of inoculation amount into a seed culture medium, and culturing at 37 ℃ and 250rpm for 12 h;
secondary seed culture: transferring the primary seed solution into a seed culture medium by an inoculation amount of 1-2%, and culturing at 37 ℃ and 250rpm for 12 h;
the seed culture medium is as follows: 10.0g/L of peptone, 10.0g/L of yeast powder, 10.0g/L of sodium acetate and 801ml/L, C of Tween6H5O7(NH4)32.0g/L、K2HPO4·7H2O2.0 g/L, nicotinic acid 1.0g/L, MgSO4·7H2O 0.3g/L、MnSO4·H2O 0.1g/L、CaCl20.1g/L、FeCl20.1g/L, NaCl 0.1.1 g/L and 8g/L sucrose solution are independently sterilized and supplemented.
(2) Culturing in a fermentation tank:
inoculating the secondary seed solution into a fermentation culture medium by an inoculation amount of 5-8%, adjusting the initial pH to 6.5-6.8, controlling the pH to 6.0-6.8 in the fermentation process by using 20% (wt) NaOH, introducing air in the whole fermentation process, controlling the air flow to 0.1-0.3 vvm, adding 0.1-0.3% of a defoaming agent, independently sterilizing 35g/L of hydroquinone at 37 ℃ and 250-300 rpm for 10 times, and simultaneously sterilizing 30g/L of sucrose and simultaneously replenishing hydroquinone for 10 times.
The fermentation medium is as follows: 10.0g/L of peptone, 10.0g/L of yeast powder, 10.0g/L of sodium acetate and 801ml/L, C of Tween6H5O7(NH4)32.0g/L、K2HPO4·7H2O2.0 g/L, nicotinic acid 1.0g/L, MgSO4·7H2O 0.2g/L、MnSO4·H2O 0.05g/L、CaCl20.02g/L、FeCl20.01g/L, NaCl 0.01.01 g/L, Glu 0.5.5 g/L, Val 0.3.3 g/L and 35g/L of hydroquinone are independently sterilized and supplemented for 10 times, and 30g/L of sucrose is independently sterilized and supplemented with hydroquinone at the same time and 10 times. Fermenting for 15 h.
Example 6
The leuconostoc pseudomesenteroides G123 is fermented by a 3L fermentation tank, the liquid loading amount is 2L, the fermentation condition is initial pH6.5, the pH is controlled to be 6.0-6.8 in the whole fermentation process, 5G/L hydroquinone is independently sterilized and supplemented for 10 times, 180G/L sucrose is independently sterilized and is supplemented with hydroquinone simultaneously for 10 times, and the method specifically comprises the following steps:
(1) seed liquid culture:
first-order seed culture: inoculating the leuconostoc pseudomesenteroides bacterial liquid in a glycerol storage tube by 0.1-0.2% of inoculation amount into a seed culture medium, and culturing at 37 ℃ and 250rpm for 12 h;
secondary seed culture: transferring the primary seed solution into a seed culture medium by an inoculation amount of 1-2%, and culturing at 37 ℃ and 250rpm for 12 h;
the seed culture medium is as follows: peptone 5.0g/L, yeast powder 5.0g/L, sodium acetate 5.0g/L, Tween-801 ml/L, C6H5O7(NH4)32.0g/L、K2HPO4·7H2O2.0 g/L, nicotinic acid 1.0g/L, MgSO4·7H2O 0.2g/L、MnSO4·H2O 0.05g/L、CaCl20.02g/L、FeCl20.01g/L, NaCl 0.01.01 g/L and 10g/L sucrose solution are separately sterilized and supplemented.
(2) Culturing in a fermentation tank:
inoculating the secondary seed solution into a fermentation culture medium by an inoculation amount of 5-8%, adjusting the initial pH to 6.5-6.8, controlling the pH to 6.0-6.8 in the fermentation process by using 20% (wt) NaOH, introducing air in the whole fermentation process, controlling the air flow to 0.1-0.3 vvm, adding 0.1-0.3% of a defoaming agent, independently sterilizing 5g/L of hydroquinone at 37 ℃ and 250-300 rpm for 10 times, and independently sterilizing 180g/L of sucrose and simultaneously replenishing hydroquinone for 10 times.
The fermentation medium is as follows: peptone 5.0g/L, yeast powder 5.0g/L, sodium acetate 5.0g/L, Tween-801 ml/L, C6H5O7(NH4)32.0g/L、K2HPO4·7H2O2.0 g/L, nicotinic acid 1.0g/L, MgSO4·7H2O 0.2g/L、MnSO4·H2O 0.05g/L、CaCl20.02g/L、FeCl20.01g/L, NaCl 0.01.01 g/L, Glu 0.35.35 g/L, Val 0.115.115 g/L and 5g/L of hydroquinone are independently sterilized and supplemented for 10 times, and 180g/L of sucrose is independently sterilized and supplemented with hydroquinone simultaneously and 10 times. Fermenting for 16 h.
Example 7
This example illustrates the results of fermentation of Leuconostoc pseudomesenteroides G123 with α -arbutin and dextran in different pH environments, using a 3L fermentor for fermentation, with a liquid loading of 2L, at an initial pH of 7.0, with pH values of 6.0, 7.0 and 8.0 being controlled throughout the fermentation, with 15G/L hydroquinone sterilized separately and supplemented 10 times, 90G/L sucrose sterilized separately and hydroquinone supplemented simultaneously, 10 times, with seed broth culture and fermentor culture conditions the same as in example 1, and with the results of fermentation for 12h shown in Table 2.
In this example, as pH of leuconostoc pseudomesenteroides G123 increases, the residue of hydroquinone and sucrose increases, the amount of arbutin synthesized decreases, and the change in glucan synthesis is small, so that the optimum condition is a slightly acidic environment.
TABLE 2 Effect of different fermentation pH conditions on the Synthesis of alpha-arbutin and Glucan
Example 8
This example illustrates the hydroquinone resistance of Leuconostoc pseudomesenteroides G123, and the results are shown in FIG. 2.
In the embodiment, after the leuconostoc pseudomesenteroides G123 is impacted by hydroquinone with different concentrations, the cell growth amount is obviously reduced along with the increase of the hydroquinone concentration, and the inhibition effect is obvious when the hydroquinone concentration exceeds 5/L, so the hydroquinone concentration is controlled within 1G/L to eliminate the toxic effect of the hydroquinone on the cells.
Claims (7)
1. A method for co-producing alpha-arbutin with high optical purity and glucan with high adhesiveness is characterized in that leuconostoc pseudomesenteroides is used for producing alpha-arbutin and glucan by one-step fermentation in a weakly acidic environment by taking sucrose and hydroquinone as substrates, wherein the leuconostoc pseudomesenteroides is leuconostoc pseudomesenteroides (Leuconostoc pseudomesenteroides)Leuconostoc pseudomesenteroides) G123, the preservation number is CCTCC NO: M2014115; the weak acid environment is a solution with the pH value of 6.0-6.8;
the leuconostoc pseudomesenteroides is inoculated into a fermentation culture medium for fermentation culture after the seed culture step, and the method specifically comprises the following steps:
1) first-order seed culture: measuring a leuconostoc pseudomesenteroides bacterial solution by using 0.1-0.2% of inoculation amount, inoculating the leuconostoc pseudomesenteroides bacterial solution into a seed culture medium, and culturing at 37 ℃ and 250rpm for 10-12 h;
2) secondary seed culture: transferring the primary seed solution into a seed culture medium by an inoculation amount of 1-2%, and culturing at 37 ℃ and 250rpm for 10-12 h;
3) fermentation culture: inoculating the secondary seed liquid into a fermentation culture medium in an inoculation amount of 5-8%, and adopting NaOH or Na2CO3Adjusting the pH value to 6.0-6.8 in the fermentation process, introducing air in the whole fermentation process, controlling the air flow to be 0.1-0.3 vvm, and culturing for 12-16 h at 37 ℃ and 250-300 rpm;
the seed culture medium is as follows: 3-10 g/L of peptone, 3-10 g/L of yeast powder, 3-10 g/L of sodium acetate, and 3-5 ml/L, C of tween-800.56H5O7(NH4)3 1～5g/L、K2HPO4•7H2O1-5 g/L, nicotinic acid 0.5-2 g/L, MgSO4•7H2O 0.1～1g/L、MnSO4•H2O 0.01～1g/L、CaCl2 0.01～1g/L、FeCl2Independently sterilizing and supplementing 0.01-1 g/L and 5-20 g/L sucrose solution with the concentration of 0.01-1 g/L, NaCl 0.01;
the fermentation medium is as follows: 3-10 g/L of peptone, 3-10 g/L of yeast powder, 3-10 g/L of sodium acetate, and 3-5 ml/L, C of tween-800.56H5O7(NH4)3 1～5g/L、K2HPO4•7H2O1-5 g/L, nicotinic acid 0.5-5 g/L, MgSO4•7H2O 0.1～1g/L、MnSO4•H2O 0.01～1g/L、CaCl2 0.01～1g/L、FeCl20.005-1 g/L, NaCl 0.005, 0.005-1 g/L, Glu 0.1, 0.1-3 g/L, Val 0.1, 0.1-0.3 g/L and 15g/L of hydroquinone are independently sterilized and supplemented for 10 times, and 90g/L of sucrose is independently sterilized and is simultaneously supplemented with hydroquinone for 10 times.
2. The method of claim 1, wherein the NaOH or Na2CO3The mass fraction of the solution is 10-20%.
3. The method of claim 1, wherein the seed medium is: 4-8 g/L of peptone, 4-8 g/L of yeast powder, 4-8 g/L of sodium acetate and 801-2 ml/L, C of Tween6H5O7(NH4)3 2～3g/L、K2HPO4•7H2O2-3 g/L, nicotinic acid 1-2 g/L, MgSO4•7H2O 0.2～0.4g/L、MnSO4•H2O 0.05～0.08g/L、CaCl2 0.02～0.06g/L、FeCl20.01-0.02 g/L, NaCl 0.01.01-0.02 g/L and 10-12 g/L of sucrose solution are independently sterilized and supplemented.
4. The method of claim 3, wherein the seed medium is: peptone 5.0g/L, yeast powder 5.0g/L, sodium acetate 5.0g/L, Tween-801 ml/L, C6H5O7(NH4)3 2.0g/L、K2HPO4•7H2O2.0 g/L, nicotinic acid 1.0g/L, MgSO4•7H2O 0.2g/L、MnSO4•H2O 0.05g/L、CaCl20.02g/L、FeCl20.01g/L, NaCl 0.01.01 g/L and 10g/L sucrose solution are separately sterilized and supplemented.
5. The method of claim 1, wherein the fermentation medium is: 4-8 g/L of peptone, 4-8 g/L of yeast powder, 4-8 g/L of sodium acetate and 801-2 ml/L, C of Tween6H5O7(NH4)3 2～3g/L、K2HPO4•7H2O2-3 g/L, nicotinic acid 1-2 g/L, MgSO4•7H2O 0.2～0.4g/L、MnSO4•H2O 0.05～0.08g/L、CaCl20.02～0.05g/L、FeCl20.01-0.05 g/L, NaCl 0.01.01-0.05 g/L, Glu 0.1.1-1 g/L, Val 0.1-0.2 g/L and 15g/L of hydroquinone are independently sterilized and supplemented for 10 times, and 90g/L of sucrose is independently sterilized and is supplemented with hydroquinone simultaneously for 10 times.
6. The method of claim 5, wherein the fermentation medium is peptone 5.0g/L, yeast powder 5.0g/L, sodium acetate 5.0g/L, Tween-801 ml/L, C6H5O7(NH4)3 2.0g/L、K2HPO4•7H2O2.0 g/L, nicotinic acid 1.0g/L, MgSO4•7H2O 0.2g/L、MnSO4•H2O 0.05g/L、CaCl20.02g/L、FeCl20.01g/L, NaCl 0.01, 0.01g/L, Glu 0.35, 0.35g/L, Val 0.115, 0.115g/L and 15g/L of hydroquinone are independently sterilized and supplemented for 10 times, and 90g/L of sucrose is independently sterilized and supplemented with hydroquinone simultaneously and 10 times.
7. A method according to any one of claims 1 to 6, comprising the steps of:
1) first-order seed culture: measuring a leuconostoc pseudomesenteroides bacterial solution by using 0.1-0.2% of inoculation amount, inoculating the leuconostoc pseudomesenteroides bacterial solution into a seed culture medium, and culturing at 37 ℃ and 250rpm for 10-12 h;
2) secondary seed culture: transferring the primary seed solution into a seed culture medium by an inoculation amount of 1-2%, and culturing at 37 ℃ and 250rpm for 10-12 h;
3) fermentation culture: inoculating the secondary seed liquid into a fermentation culture medium in an inoculation amount of 5-8%, and adopting NaOH or Na with the mass fraction of 10-20% (wt)2CO3Adjusting the pH value of the solution to 6.0-6.8 in the fermentation process, introducing air in the whole fermentation process, controlling the air flow to be 0.1-0.3 vvm, and culturing for 12-16 h at 37 ℃ and 250-300 rpm;
the leuconostoc pseudomesenteroides is leuconostoc pseudomesenteroides G123 (Leuconostocpseudomesenteroides) The preservation number is CCTCC NO: M2014115;
the seed culture medium is as follows: peptone 5.0g/L, yeast powder 5.0g/L, sodium acetate 5.0g/L, Tween-801 ml/L, C6H5O7(NH4)3 2.0g/L、K2HPO4•7H2O2.0 g/L, nicotinic acid 1.0g/L, MgSO4•7H2O 0.2g/L、MnSO4•H2O 0.05g/L、CaCl20.02g/L、FeCl2Independently sterilizing and supplementing 0.01g/L, NaCl 0.01.01 g/L and 10g/L sucrose solution;
the fermentation medium is peptone 5.0g/L, yeast powder 5.0g/L, sodium acetate 5.0g/L, Tween-801 ml/L, C6H5O7(NH4)3 2.0g/L、K2HPO4•7H2O2.0 g/L, nicotinic acid 1.0g/L, MgSO4•7H2O 0.2g/L、MnSO4•H2O 0.05g/L、CaCl20.02g/L、FeCl20.01g/L, NaCl 0.01, 0.01g/L, Glu 0.35, 0.35g/L, Val 0.115, 0.115g/L and 15g/L of hydroquinone are independently sterilized and supplemented for 10 times, and 90g/L of sucrose is independently sterilized and supplemented with hydroquinone simultaneously and 10 times.
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