CN103805649A - Method for preparing tagatose - Google Patents
Method for preparing tagatose Download PDFInfo
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- CN103805649A CN103805649A CN201210458165.4A CN201210458165A CN103805649A CN 103805649 A CN103805649 A CN 103805649A CN 201210458165 A CN201210458165 A CN 201210458165A CN 103805649 A CN103805649 A CN 103805649A
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Abstract
The invention relates to a method for preparing tagatose, and in particular to a method for preparing D-tagatose. The method comprises the following step of: (1) catalyzing galactose with lactobacillus acidophilus CCTCC NO:M98004 to obtain D-tagatose.
Description
Technical field
The present invention relates to the preparation method of D-Tag, concrete utilization relates to the method for utilizing Lactobacterium acidophilum to transform the synthetic D-Tag of semi-lactosi.
Background technology
Along with the raising of modern life level, the sugared intake of human body is also in continuous increase, and the health problem of bringing thus also highlights day by day, especially, take obesity, diabetes and carious tooth as main, therefore research and develop sweeting agent low in calories and causing people's attention to substitute sucrose.D-Tag is a small amount of rare sugar existing of a kind of occurring in nature, is the isomer of fructose and D-semi-lactosi.D-Tag physico-chemical property is stable, without any undesired off taste, sugariness is about 92% of sucrose, and heat is only 1/3 left and right (the sucrose 4kcal/g of sucrose, D-Tag 1.5kcal/g), pharmacology and toxicologic study prove that D-Tag has the intestinal microflora of improvement, reduces blood sugar, prevents the multiple prebiotic effects such as carious tooth simultaneously, for human-body safety, nontoxic.In April calendar year 2001, U.S. FDA determines that D-Tag is generally recognized as safe food (GRAS), and subsequently, Korea S, Australia, New Zealand, European Union all ratify D-Tag for food.Food and Argriculture OrganizationFAO (FAO) and the combination food additive council of the World Health Organization (WHO) (JECFA) 57 meetings in 2004 approval D-Tag is as foodstuff additive.In addition, D-Tag is used for the treatment of type II diabetes by Spherix company of the U.S., and three phases of carrying out are at present clinical.
The production method of D-Tag comprises natural separation, chemical catalysis and bio-transformation.Because the content of D-Tag is comparatively rare at the content of occurring in nature, therefore natural separation method is infeasible for industrial-scale production tagatose.The synthetic D-Tag of chemical catalysis exists that side reaction is many, impurity is many, be difficult to separation, the not high problems of security.The synthetic D-Tag of bio-transformation is at present mainly at the heat-resisting L-arabinose isomerase of e. coli expression by genetic engineering means, then enzyme purification immobilization are carried out to the problem that the method existence and stability is poor, cost is high with catalysis D-semi-lactosi generation D-Tag.In addition,, because Host Strains adopts intestinal bacteria, when the products production of directly taking for human body, there is certain risk.Produce D-Tag except utilizing recombination bacillus coli, patent CN200610085922.2 discloses a kind of method of utilizing lactobacillus-fermented to produce D-Tag, but fermentation method often exists that impurity is more, content is lower and unsettled problem, the method maximum output is 450mg/100mL.
Therefore, the problem existing for solving above-mentioned present D-Tag production technology, the D-Tag production method of setting up a kind of efficient, economy, environmental protection is very significant.
Summary of the invention
The object of this invention is to provide a kind of preparation method of effective D-Tag.
One aspect of the present invention provides a kind of method of preparing D-Tag, said method comprising the steps of:
(1) obtain D-Tag with Lactobacterium acidophilum CCTCC M98004 catalysis semi-lactosi.
In a preferred embodiment of the present invention, described method also comprises the step with culture medium culturing Lactobacterium acidophilum CCTCC M98004.
In a preferred embodiment of the present invention, described method also comprises the step that Lactobacterium acidophilum CCTCC M98004 is prepared into quiescent condition thalline.
In a preferred embodiment of the present invention, described method also comprises the step that Lactobacterium acidophilum CCTCC M98004 is prepared into immobilized thallus.
In a preferred embodiment of the present invention, said method comprising the steps of:
(1) with culture medium culturing Lactobacterium acidophilum CCTCC M98004;
(2) the Lactobacterium acidophilum CCTCC M98004 after the cultivation of step (1) is prepared into quiescent condition thalline and/or immobilized thallus; And
(3) the quiescent condition thalline obtaining by step (2) and/or immobilized thallus catalysis semi-lactosi obtain D-Tag.
In a preferred embodiment of the present invention, in obtaining the step of D-Tag, catalysis semi-lactosi can add boric acid or borate.
In a preferred embodiment of the present invention, described borate is selected from the borate of boric acid base metal-salt and divalent metal.
In a preferred embodiment of the present invention, temperature when catalysis semi-lactosi obtains D-Tag is 40-90 ℃, preferably 50-80 ℃, and more preferably 60-70 ℃, is preferably 60-65 ℃.
In a preferred embodiment of the present invention, time when catalysis semi-lactosi obtains D-Tag is 1-100 hour, preferably 10-80 hour, and more preferably 24-72 hour, is preferably 48 hours.
The present invention provides Lactobacterium acidophilum CCTCC M98004 in the purposes of preparing in D-Tag on the other hand.
Method of the present invention can be prepared D-Tag effectively.
Accompanying drawing explanation
Fig. 1 represents to detect D-Tag content by HPLC, and (A is D-semi-lactosi and D-Tag standard substance figure; B is that quiescent condition thalline shown in embodiment 1 detects tagatose result).
Fig. 2 represents that temperature of reaction shown in embodiment 2 and time catalyze and synthesize the impact of D-Tag on immobilized thallus.
Fig. 3 represents that continuous batch of immobilized thallus catalyzes and synthesizes D-Tag.
Embodiment
In the present invention, if not special explanation, percentage ratio (%) or part all refer to weight percentage or the weight part with respect to composition.
In the present invention, if not special explanation, related each component or its preferred ingredient can be combined to form new technical scheme mutually.
In the present invention, if not special explanation, all embodiments mentioned in this article and preferred implementation can be combined to form new technical scheme mutually.
In the present invention, if not special explanation, all technical characterictics mentioned in this article and preferred feature can be combined to form new technical scheme mutually.
In the present invention, if there is no contrary explanation, in composition, the content sum of each component is 100%.
In the present invention, if there is no contrary explanation, in composition, the umber sum of each component can be 100 weight parts.
In the present invention, unless there are other explanations, numerical range " a-b " represents that the breviary that a closes to the arbitrary real array between b represents, wherein a and b are real numbers.For example numerical range " 0-5 " represents all to have listed the whole real numbers between " 0-5 " herein, and " 0-5 " just the breviary of these combinations of values represents.
In the present invention, unless there are other explanations, integer numerical range " a-b " represents that a represents to the breviary of the arbitrary integer combination between b, and wherein a and b are integers.For example integer numerical range " 1-N " represents 1,2 ... N, wherein N is integer.
In the present invention, unless there are other explanations, " its combination " represents the multicomponent mixture of described each element, for example two kinds, three kinds, four kinds and until the multicomponent mixture of maximum possible.
If do not particularly not pointed out, this specification sheets term " one " used refers to " at least one ".
If do not particularly not pointed out, the benchmark of percentage ratio of the present invention (comprising weight percentage) is all the gross weight of described composition.
" scope " disclosed herein is with the form of lower limit and the upper limit.Can be respectively one or more lower limits, and one or more upper limit.Given range limits by a selected lower limit and a upper limit.Selected lower limit and the upper limit define the border of special scope.All scopes that can limit by this way comprise with capable of being combined, and any lower limit can be combined to form a scope with any upper limit.For example, list the scope of 60-120 and 80-110 for special parameter, be interpreted as that the scope of 60-110 and 80-120 also expects.In addition, if the minimum extent value of listing 1 and 2, and if listed maximum range value 3,4 and 5, scope below can all expect: 1-3,1-4,1-5,2-3,2-4 and 2-5.
In this article, except as otherwise noted, the ratio of each component or weight all refer to dry weight.
In this article, except as otherwise noted, each reaction is all carried out at normal temperatures and pressures.
In this article, except as otherwise noted, each reactions steps can sequentially be carried out, and also can not carry out in order.For example, between each reactions steps, can comprise other steps, and also can exchange order between reactions steps.Preferably, reaction method is herein in sequence.
One aspect of the present invention provides a kind of method of preparing D-Tag, said method comprising the steps of:
(1) obtain D-Tag with Lactobacterium acidophilum CCTCC M98004 catalysis semi-lactosi.
The Lactobacterium acidophilum (Lactobacillus acidophilus Lactobacillus acidophilus YIT2004(L) that the present invention is used) be preserved in Chinese Typical Representative culture collection center on February 23rd, 1998, preserving number is CCTCC NO:M98004.Depositary institution address: Wuhan, China Wuhan University.
In a preferred embodiment of the present invention, described Lactobacterium acidophilum CCTCC M98004 passes through culture medium culturing.Therefore,, in a preferred embodiment of the present invention, described method also comprises the step with culture medium culturing Lactobacterium acidophilum CCTCC M98004.
In the present invention, described substratum is conventional, and which substratum those of ordinary skill in the art can direct derivation goes out in conjunction with prior art again and can be used for the present invention reading specification sheets of the present invention.In a preferred embodiment of the present invention, described substratum comprises nitrogenous source, carbon source and special composition.In another preferred embodiment of the present invention, described substratum is blister meat pipe substratum, and described blister meat pipe substratum comprises the trypton(Tryptones of 5 weight parts), the glucose of yeast extract 5,5 weight parts of 5 weight parts, the Zulkovsky starch of 2 weight parts, the extractum carnis of 3 weight parts, the KH2PO4 (pH 7.6) of 2 weight parts.In another preferred embodiment of the present invention, described substratum is fermention medium, and described fermention medium comprises the trypton(Tryptones of 20 weight parts), the yeast extract of 5 weight parts, the glucose of 15 weight parts, the Polypepton(peptone of 5 weight parts), the lactose of 5 weight parts, the K2HPO4 of 5 weight parts, the magnesium sulfate of 1 weight part, the manganous sulfate of 0.15 weight part, the calcium carbonate of 5 weight parts, the pH of Tomato juice 7.0 of 200mL/L).In another preferred embodiment of the present invention, described substratum is blister meat pipe substratum and/or fermention medium.
In a preferred embodiment of the present invention, described Lactobacterium acidophilum CCTCC M98004 is preferably prepared into quiescent condition thalline.Therefore,, in a preferred embodiment of the present invention, described method also comprises the step that Lactobacterium acidophilum CCTCC M98004 is prepared into quiescent condition thalline.
In the present invention, the step that Lactobacterium acidophilum CCTCC M98004 is preferably prepared into quiescent condition thalline is conventional.Those of ordinary skill in the art can be used for Lactobacterium acidophilum CCTCC M98004 to be preferably prepared into quiescent condition thalline in conjunction with can direct derivation in prior art going out which method according to description of the invention again.
In a preferred embodiment of the present invention, the described method that Lactobacterium acidophilum CCTCC M98004 is preferably prepared into quiescent condition thalline is carried out as follows:
First freeze-dried vaccine powder is added sterilized water dilution spread on MRS solid medium, to activate growth.Picking colonies typical on MRS solid medium, inoculate into first order seed substratum (same to fermention medium), 37 ℃ of anaerobism leave standstill cultivation to the logarithmic phase later stage, then are inoculated in second order fermentation substratum according to 10% ratio, and 37 ℃ leave standstill cultivation to the termination of stabilizer later stage.After cultivation finishes, after 4 ℃ of centrifugal phosphoric acid buffer (pH7.0) rinsings that obtain bacterium mud use precooling, centrifugal 4 ℃ of the phosphoric acid buffers that are resuspended in obtain quiescent condition thalline.
In a preferred embodiment of the present invention, described Lactobacterium acidophilum CCTCC M98004 is also preferably prepared into immobilized thallus.Therefore,, in a preferred embodiment of the present invention, described method also comprises the step that Lactobacterium acidophilum CCTCC M98004 is prepared into immobilized thallus.
In the present invention, the step that Lactobacterium acidophilum CCTCC M98004 is preferably prepared into immobilized thallus is conventional.Those of ordinary skill in the art can be used for Lactobacterium acidophilum CCTCC M98004 to be preferably prepared into immobilized thallus in conjunction with can direct derivation in prior art going out which method according to description of the invention again.
In a preferred embodiment of the present invention, the described method that Lactobacterium acidophilum CCTCC M98004 is preferably prepared into immobilized thallus is carried out as follows:
First cryodesiccated Lactobacterium acidophilum is carried out to activation culture.Be inoculated on MRS solid medium, 37 ℃ of anaerobism are cultivated about 2 days, after colonies typical forms, be inoculated into first order seed substratum (same to fermention medium), 37 ℃ of anaerobism leave standstill cultivates the logarithmic phase later stage, be inoculated in second order fermentation substratum according to 10% ratio, 37 ℃ leave standstill and cultivate the stabilizer later stage and stop, being fixed thalline after centrifugal and rinsing again.
Therefore,, in a preferred embodiment of the present invention, the present invention also provides a kind of method of preparing D-Tag, said method comprising the steps of:
(1) with culture medium culturing Lactobacterium acidophilum CCTCC M98004;
(2) the Lactobacterium acidophilum CCTCC M98004 after the cultivation of step (1) is prepared into quiescent condition thalline and/or immobilized thallus; And
(3) the quiescent condition thalline obtaining by step (2) and/or immobilized thallus catalysis semi-lactosi obtain D-Tag.
In a preferred embodiment of the present invention, in order to improve the transformation efficiency of D-Tag, with Lactobacterium acidophilum CCTCC M98004(or quiescent condition thalline and/or immobilized thallus) catalysis semi-lactosi obtains can adding boric acid or borate, for example borate of boric acid base metal-salt and divalent metal in the step of D-Tag.Described divalent metal is preferably Co2+, Mn2+ and combination thereof.
In the present invention, with Lactobacterium acidophilum CCTCC M98004(or quiescent condition thalline and/or immobilized thallus) condition that obtains in the step of D-Tag of catalysis semi-lactosi is conventional.Those of ordinary skill in the art can directly obtain its actual conditions in conjunction with prior art again according to description of the invention.In a preferred embodiment of the present invention, temperature is 40-90 ℃, preferably 50-80 ℃, and more preferably 60-70 ℃, is preferably 60-65 ℃.In a preferred embodiment of the present invention, the time is 1-100 hour, preferably 10-80 hour, and more preferably 24-72 hour, is preferably 48 hours.
Describe bright the present invention in detail below in conjunction with embodiment, these embodiment are presented for purposes of illustration, do not limit the scope of the invention.
Embodiment
Embodiment 1: the preparation of blister meat pipe substratum
Blister meat pipe substratum (1L): trypton 5g, yeast extract 5g, glucose 5g, Zulkovsky starch 2g, extractum carnis 3g, KH2PO42g (pH 7.6).Packing test tube 7mL/ pipe, adds beef slag 0.4g.115 ℃ of sterilizings in 30 minutes are for subsequent use.
Embodiment 2: the preparation of fermention medium
Fermention medium (1L): trypton 20g, yeast extract 5g, glucose 15g, Polypepton5g, lactose 5g, K2HPO45g, magnesium sulfate 1g, manganous sulfate 0.15g, calcium carbonate 5g, the 200mL/L(pH of Tomato juice 7.0).115 ℃ of sterilizings in 30 minutes are for subsequent use.
Embodiment 3: the enlarged culturing of Lactobacterium acidophilum
Get the Lactobacterium acidophilum CCTCC M98004 that lyophilize is preserved, resuspended with the sterilized water of sterilizing after, be diluted to after suitable concn, be coated directly onto MRS solid plate.37 ℃ of anaerobic environments are cultivated 48 hours.The typical Lactobacterium acidophilum bacterium colony of picking, be inoculated in a blister meat pipe (comprising the blister meat pipe substratum of embodiment 1), 37 ℃ of anaerobic environments are cultivated after 48 hours and this blister meat pipe are inoculated into respectively with cultivating in a collection of blister meat pipe, and to obtain, a collection of source is identical, consistent Lactobacterium acidophilum grows.A blister meat pipe is inoculated in first order seed substratum (with the fermention medium of embodiment 2), keep micro-oxygen environment with micro-aerobic aerogenesis bag, 37 ℃ of standing cultivations the logarithmic phase later stage (about 11 hours), then be inoculated into second order fermentation substratum (with the fermention medium of embodiment 2) cultivation to later stage stationary phase (about 13 hours) termination cultivation according to 10% ratio.
Embodiment 4: quiescent condition Bacterium lacticum catalyzes and synthesizes D-Tag
The Lactobacterium acidophilum that enlarged culturing is obtained is placed in freezing on ice, after 4 ℃ of centrifugal phosphoric acid buffer (pH7.0) rinsings that obtain bacterium mud use precooling, the centrifugal bacterium mud obtaining mixes with the D-galactose solution of 100g/L, is placed in 65 ℃ of water-baths and reacts 48 hours, detects the content of D-Tag.The results are shown in Figure 1.The content that integral and calculating can obtain D-Tag reaches 17g/L, and transformation efficiency reaches 17%.Be compared to patent CN200610085922.2 result (450mg/100mL) and have 3 times of raisings.
Embodiment 5: immobilization Lactobacterium acidophilum catalyzes and synthesizes D-Tag
The Lactobacterium acidophilum that enlarged culturing is obtained is placed in freezing on ice, after 4 ℃ of centrifugal phosphoric acid buffer (pH7.0) rinsings that obtain bacterium mud use precooling, after obtaining the sodium alginate soln of milk-acid bacteria thalline 1.5-2% concentration and milk-acid bacteria thalline and fully mixing, splashed in the calcium chloride solution of 1% concentration, room temperature was placed after for some time, centrifugal and by rinsed with sterile water, add the glutaraldehyde solution of 0.2% concentration, be placed on magnetic stirring apparatus, room temperature is placed 1 hour, after pure water rinsing, be resuspended in phosphate buffered saline buffer 4 ℃ for subsequent use.Immobilized thallus is mixed with the D-galactose solution of 100g/L, add boric acid to make its concentration reach 0.3-0.5M, adding Co2+, Mn2+ concentration is (0.1%) again, controls reaction pH between 7.5-8, be placed in 60-65 ℃ of water-bath and react 48 hours, detect the content of D-Tag.In the present embodiment, temperature (50 ℃, 60 ℃, 65 ℃, 70 ℃) and the impact of time (12,24,36,48 hours) on D-Tag resultant quantity have been investigated respectively.The results are shown in Figure 2, show 65 ℃ of left and right of temperature of reaction, about 48 hours time, output is the highest, transformation efficiency nearly 40%.Visible use immobilized thallus, the resultant quantity of adding a certain amount of boron acid and specific divalent metal ion simultaneously and can improve D-Tag.
Embodiment 6: catalysis is batch on the impact synthetic on D-Tag
For the stability of checking immobilized cell catalytic capability, after a catalyzed reaction completes as described in Example 5, after the centrifugal 10min of 10000rpm, obtain immobilized thallus bead, with after the abundant rinsing of phosphate buffered saline buffer, proceed the catalyzed reaction of next batch, have or not change to test its catalytic capability.Result (Fig. 3) shows that the catalytic capability of continuous 7 batches of reaction immobilized thallus has kept good stability.
Claims (10)
1. a method of preparing D-Tag, said method comprising the steps of:
(1) obtain D-Tag with Lactobacterium acidophilum CCTCC M98004 catalysis semi-lactosi.
2. the method for claim 1, is characterized in that, described method also comprises the step with culture medium culturing Lactobacterium acidophilum CCTCC M98004.
3. the method for claim 1, is characterized in that, described method also comprises the step that Lactobacterium acidophilum CCTCC M98004 is prepared into quiescent condition thalline.
4. the method for claim 1, is characterized in that, described method also comprises the step that Lactobacterium acidophilum CCTCC M98004 is prepared into immobilized thallus.
5. the method for claim 1, is characterized in that, said method comprising the steps of:
(1) with culture medium culturing Lactobacterium acidophilum CCTCC M98004;
(2) the Lactobacterium acidophilum CCTCC M98004 after the cultivation of step (1) is prepared into quiescent condition thalline and/or immobilized thallus; And
(3) the quiescent condition thalline obtaining by step (2) and/or immobilized thallus catalysis semi-lactosi obtain D-Tag.
6. the method as described in claim 1 or 5, is characterized in that, in catalysis semi-lactosi obtains the step of D-Tag, can add boric acid or borate.
7. method as claimed in claim 6, is characterized in that, described borate is selected from the borate of boric acid base metal-salt and divalent metal.
8. the method for claim 1, is characterized in that, temperature when catalysis semi-lactosi obtains D-Tag is 40-90 ℃, preferably 50-80 ℃, and more preferably 60-70 ℃, is preferably 60-65 ℃.
9. the method for claim 1, is characterized in that, time when catalysis semi-lactosi obtains D-Tag is 1-100 hour, preferably 10-80 hour, and more preferably 24-72 hour, is preferably 48 hours.
10. Lactobacterium acidophilum CCTCC M98004 is in the purposes of preparing in D-Tag.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105055438A (en) * | 2015-07-21 | 2015-11-18 | 珲春市神怡菌业生物科技开发有限公司 | Lentinan prebiotic composition with gastrointestinal tract function improving effect |
| CN108374031A (en) * | 2015-10-02 | 2018-08-07 | 博努莫斯生化有限责任公司 | The enzymatic production of D-Tag |
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| CN1948500A (en) * | 2006-05-26 | 2007-04-18 | 江南大学 | Preparation method of functional sweetener D-tatai sugar |
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| CN1948500A (en) * | 2006-05-26 | 2007-04-18 | 江南大学 | Preparation method of functional sweetener D-tatai sugar |
| CN101558077A (en) * | 2006-12-11 | 2009-10-14 | Cj第一制糖株式会社 | Manufacturing method of tagatose using galactose isomerization of high yield |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105055438A (en) * | 2015-07-21 | 2015-11-18 | 珲春市神怡菌业生物科技开发有限公司 | Lentinan prebiotic composition with gastrointestinal tract function improving effect |
| CN108374031A (en) * | 2015-10-02 | 2018-08-07 | 博努莫斯生化有限责任公司 | The enzymatic production of D-Tag |
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Application publication date: 20140521 |