CN1948500A - Preparation method of functional sweetener D-tatai sugar - Google Patents
Preparation method of functional sweetener D-tatai sugar Download PDFInfo
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Abstract
一种功能性甜味剂D－塔格糖的制备方法，具体涉及微生物发酵生物转化制备D－塔格糖的一种方法，属于食品生物技术领域。用乳酸菌作为菌种，以半乳糖为转化底物，添加碳源、氮源及无机盐组成发酵培养基，发酵法生物转化制备D－塔格糖。所用乳酸菌在MRS培养基中，25－40℃培养，生长良好，作为种子培养液，发酵培养基碳源为葡萄糖、乳糖、麦芽糖或可溶性淀粉，氮源选用蛋白胨、酵母膏、玉米浆、大豆粕中的一种或几种混合使用，发酵后经检测，在发酵液中D－塔格糖浓度达250－450mg/100ml。本发明所得产品安全可靠，具有很高的生理功能。The invention discloses a preparation method of functional sweetener D-tagatose, specifically relates to a method for preparing D-tagatose through microbial fermentation and biotransformation, and belongs to the field of food biotechnology. Lactic acid bacteria are used as strains, galactose is used as conversion substrate, carbon source, nitrogen source and inorganic salt are added to form fermentation medium, and D-tagatose is prepared by fermentation biotransformation. The lactic acid bacteria used are cultured in MRS medium at 25-40°C and grow well. They are used as seed culture medium. The carbon source of the fermentation medium is glucose, lactose, maltose or soluble starch, and the nitrogen source is peptone, yeast extract, corn steep liquor, and soybean meal. One or more of them are used in combination. After fermentation, the concentration of D-tagatose in the fermentation broth reaches 250-450mg/100ml. The product obtained by the invention is safe and reliable, and has high physiological functions.
Description
Technical field
The present invention relates to a kind of preparation method of functional sweetener D-tatai sugar, be specifically related to a kind of method that the microbial fermentation bio-transformation prepares the D-tagatose, belong to technical field of food biotechnology.
Background technology
Tagatose is a kind of at the comparatively rare natural tagatose of occurring in nature, belongs to a kind of of rare sugar.International rare sugared association (ISRS) is defined as " existing but a few class monose and the derivative thereof of content at nature " to rare sugar.Tagatose (tagatose) is " epimer " of fructose, extensively be present in occurring in nature, numerous food product, as the cow's milk of sterilizing, ultra-high-temperature sterilized milk, milk powder, hot cocoa, various cheese, the yogurt of some kind, infant formula, and all have a certain amount of tagatose in the certain plants.Its sweet taste is similar to sucrose, and caloric value has only 1.5kcal/g, therefore is called as low-yield sweeting agent.The D-tagatose also has good functional characteristics simultaneously, does not increase glucose level in the body after eating as the normal people, can suppress and slow down the rising of blood sugar behind the diabetes patient, can not cause carious tooth; In heating and bake process Maillard reaction taking place, produce good colour, can be used as the substitute or and the composite use of other sweeting agent of sucrose in food-processing.The production of tagatose at present has that natural extract is separated, chemical catalysis is synthetic and method such as bio-transformation.Because tagatose is a kind of comparatively rare monose, it is extremely low at natural content, natural extract separation costs height, be not suitable for the requirement of industrialized production, and chemical catalysis has many adverse factors, for example under basic reaction conditions, can form many by products and chemical pollutant, and the numerous by products that form make purification step become complicated.Recent study personnel produce tagatose in the method for seeking bio-transformation.Once report used Mycobacterium smegmatis and Arthrobacter globformis bacterium dehydrogenase oxidoreductase melampyrum and hexan-hexol to generate tagatose, but because raw materials cost price height is not suitable for suitability for industrialized production.The L-arabinose isomerase that the somebody utilizes Lactobacillus gayonii metabolism to produce successfully transforms semi-lactosi and generates tagatose, has proposed to utilize milk-acid bacteria to produce the imagination of tagatose.At present, mainly by gene recombination, the L-arabinose isomerase that produces at e. coli expression carries out then to the bio-transformation of tagatose.Mainly be that thermophilric bacteria L-arabinose isomerase gene is passed through the intestinal bacteria transgene expression,, all have shortcoming substrate semi-lactosi specificity difference although increased the thermotolerance of enzyme.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of functional sweetener D-tatai sugar, particular content relates to a kind of method of utilizing the lactobacillus-fermented bio-transformation to prepare the D-tagatose.The L-arabinose isomerase that the milk-acid bacteria that the present invention adopts produces is obtaining result preferably aspect thermotolerance and the Substratspezifitaet, obtained unusual effect by the fermentation conversion, for the bio-transformation of tagatose provides a new way.
Technical scheme of the present invention: with milk-acid bacteria (Lactobacillus plantarum, Lactobacilluspentose) as bacterial classification, with the semi-lactosi is conversion of substrate, adopts carbon source, nitrogenous source and inductor and inorganic salt to form fermention medium, and the fermentation method bio-transformation prepares the D-tagatose.
(1) bacterial classification:
Lactobacillus plantarum AS1.550；Lactobacillus plantarum AS1.557；
Lactobacillus plantarum CICC6003；Lactobacillus plantarum CICC6014；
Lactobacillus pentose BCRC 11053；Lactobacillus pentose BCRC 15317。
When transforming as strain bio, select for use Lactobacillus plantarum AS1.557 or Lactobacillus pentose BCRC 15317 effects better with milk-acid bacteria.
(2) substratum, in g/100ml:
Storage medium: glucose 1.5, yeast extract paste 1, lime carbonate 1.5, agar 1.5, pH 6.5-7.0.
Seed culture medium: glucose 1, peptone 1, yeast extract paste 1, anhydrous sodium acetate 1, potassiumphosphate 0.02, sal epsom 0.02, manganous sulfate 0.005, pH 6.5-7.0.
(3) preparation technology:
Culture presevation: used culture presevation on storage medium, 4 ℃ of preservations, two week switchings once.
Seed culture: change seed culture medium over to from storage medium, cultivate and activated for 2 generations, cultivate 12h at every turn.
Fermentation: the mass concentration of substrate semi-lactosi in substratum is 1%, seed culture fluid is with the inoculum size of 1%-3%, insert and add different carbon sources, in the fermention medium that nitrogenous source and inductor and inorganic salt are formed, 250ml triangular flask liquid amount is 50ml, carbon source interpolation level is the 0.5%-3% of fermention medium quality, nitrogenous source interpolation level is the 0.5%-3.5% of fermention medium quality, the fermention medium composition is counted with g/100ml: semi-lactosi 0.5-2.0, carbon source 0.5-3.0, nitrogenous source 0.5-3.5, L-arabinose 0.15, anhydrous sodium acetate 1, potassiumphosphate 0.02, sal epsom 0.02, manganous sulfate 0.005, pH 7.0, and 25-40 ℃ leaves standstill the fermented liquid that cultivation 12-20h obtains containing the D-tagatose.Used carbon source is glucose, lactose, maltose or Zulkovsky starch, and used nitrogenous source is that in peptone, yeast extract paste, corn steep liquor, the soybean meal one or more are used with.
(4) separation and purification:
The fermented liquid that will contain the D-tagatose carries out ion-exchange with D001 type strongly acidic styrene resin, and deionized water wash again through the resin dedicated purifying of U20, concentrates, crystallization, and the D-tagatose gets product after the drying.
Preferred fermention medium is in g/100ml: semi-lactosi 1, and glucose 0.5, peptone 1, yeast extract paste 1, anhydrous sodium acetate 1, potassiumphosphate 0.02, sal epsom 0.02, manganous sulfate 0.005, L-arabinose 0.15, pH 7.0 is better.
The method that detects D-tagatose content is as follows:
It is centrifugal to get fermented liquid, supernatant liquor filtering with microporous membrane (0.22 μ m), and HPLC analyzes on the filtrate.HPLC condition: Waters2410 chromatographic column: Sugarpak 1.65mmid * 300mm, moving phase: pure water, detector: differential refraction detector, column temperature: 85 ℃, flow velocity: 0.4ml/min, sample size: 10 μ l, tagatose standard specimen concentration: 0.5%.
Beneficial effect of the present invention: the invention provides a kind of method of utilizing the lactobacillus-fermented bio-transformation to prepare the D-tagatose, show that the output of 12hD-tagatose can reach 359-450mg/100ml by the method for fermentation.The L-arabinose isomerase that adopts milk-acid bacteria to produce is obtaining result preferably aspect thermotolerance and the Substratspezifitaet, obtained unusual effect by the fermentation conversion, for the bio-transformation of D-tagatose provides a new way.
The biological material source explanation
Used bacterial classification is Lactobacillus plantarum AS1.550; Lactobacillus plantarumAS1.557; Lactobacillus plantarum CICC6003; Lactobacillus plantarum CICC6014; Lactobacillus pentose BCRC11053; Lactobacillus pentose BCRC15317, above bacterial classification is all own open, the expression of AS numbering (China General Microbiological Cuture CollectionCenter.CGMCC.Institute ofMicribiology, Chinese Academy of Science: Chinese microorganism strain council common micro-organisms center).CICC numbering expression (China National Institute ofFoodand Fermentation Industries: Chinese culture presevation council Research for Industrial Microbial Germ preservation administrative center).BCRC numbering expression (Bioresource Collection and Research Center: Biological resources preservation research centre, Taiwan).Thereafter numbering is represented deposit number, specifically checks each bacterial classification database webpage.
Embodiment
By experiment, obtained the principal element of influence fermentation conversion D-tagatose productive rate:
Embodiment 1 different strain is to the influence of D-tagatose productive rate
In fermention medium, add 1% semi-lactosi (analytical pure), the above-mentioned preferred substratum of by specification carries out fermenting experiment to some milk-acid bacterias of primary election, behind the fermentation 12h fermented liquid is detected, the result is as shown in table 1, Lactobacillus plantarum AS 1.557, the D-tagatose productive rate of Lactobacillus pentose BCRC15317 is higher, and different strain has difference comparatively significantly to the generation of D-tagatose.
Table 1 different strain is to producing the influence of D-tagatose
| Bacterial classification | Tagatose (mg/100ml) |
| Lactobacillus plantarum AS1.550 Lactobacillus plantarum AS1.557 Lactobacillus plantarum CICC6003 Lactobacillus plantarum CICC6014 Lactobacillus pentose BCRC11053 Lactobacillus pentose BCRC15317 | 250 450 359 320 281 370 |
Embodiment 2 fermentation times are to producing the influence of D-tagatose
The described fermentation condition of by specification, operational condition is with embodiment 1, by Lactobacillusplantarum AS1.557 is produced the detection of D-tagatose in the different fermentations time, D-tagatose content is the highest after finding fermentation 12h, along with fermentation time prolongs, D-tagatose content descends, and shows that bacterial strain has the ability of utilizing the D-tagatose.So behind the fermentation 12h, stop fermentation immediately.Fermentation time is as shown in table 2 to the influence of producing the D-tagatose.
Table 2 fermentation time is to producing the influence of D-tagatose
| Time (h) | 6 | 12 | 24 | 36 |
| Tagatose (mg/100ml) | 185 | 450 | 375 | 164 |
Claims (3)
1. the preparation method of a D-tagatose, it is characterized in that with milk-acid bacteria (Lactobacillus plantarum, Lactobacillus pentose) as bacterial classification, with the semi-lactosi is conversion of substrate, adopt carbon source, nitrogenous source and inductor and inorganic salt to form fermention medium, the fermentation method bio-transformation prepares the D-tagatose;
(1) bacterial classification: adopt milk-acid bacteria Lactobacillus plantarum AS1.550; Lactobacillusplantarum AS1.557; Lactobacillus plantarum CICC6003; Lactobacillus plantarumCICC6014; Lactobacillus pentose BCRC11053 or Lactobacillus pentose BCRC15317;
(2) substratum, in g/100ml:
Storage medium: glucose 1.5, yeast extract paste 1, lime carbonate 1.5, agar 1.5, pH 6.5-7.0;
Seed culture medium: glucose 1, peptone 1, yeast extract paste 1, anhydrous sodium acetate 1, potassiumphosphate 0.02, sal epsom 0.02, manganous sulfate 0.005, pH 6.5-7.0;
(3) preparation technology:
Culture presevation: used culture presevation on storage medium, 4 ℃ of preservations, two week switchings once;
Seed culture: change seed culture medium over to from storage medium, cultivate and activated for 2 generations, cultivate 12h at every turn;
Fermentation: the mass concentration of substrate semi-lactosi in substratum is 1%, seed culture fluid is with the inoculum size of 1%-3%, insert and add different carbon sources, in the fermention medium that nitrogenous source and inductor and inorganic salt are formed, 250ml triangular flask liquid amount is 50ml, carbon source interpolation level is the 0.5%-3% of fermention medium quality, nitrogenous source interpolation level is the 0.5%-3.5% of fermention medium quality, the fermention medium composition is counted with g/100ml: semi-lactosi 0.5-2.0, carbon source 0.5-3.0, nitrogenous source 0.5-3.5, L-arabinose 0.15, anhydrous sodium acetate 1, potassiumphosphate 0.02, sal epsom 0.02, manganous sulfate 0.005, pH 7.0, and 25-40 ℃ leaves standstill cultivation 12-20h, obtains containing the fermented liquid of D-tagatose, used carbon source is a glucose, lactose, maltose or Zulkovsky starch, used nitrogenous source are peptone, yeast extract paste, corn steep liquor, in the soybean meal one or more are used with;
(4) separation and purification:
The fermented liquid that will contain the D-tagatose carries out ion-exchange with D001 type strongly acidic styrene resin, and deionized water wash again through the resin dedicated purifying of U20, concentrates, crystallization, and the D-tagatose gets product after the drying.
2. the preparation method of D-tagatose according to claim 1 is characterized in that fermention medium is in g/100ml: semi-lactosi 1, glucose 0.5, peptone 1, yeast extract paste 1, anhydrous sodium acetate 1, potassiumphosphate 0.02, sal epsom 0.02, manganous sulfate 0.005, L-arabinose 0.15, pH 7.0.
3. the preparation method of D-tagatose according to claim 1 is characterized in that bacterial classification selects Lactobacillus plantarum AS1.557 or Lactobacillus pentose BCRC15317 for use.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101691597B (en) * | 2009-09-17 | 2012-09-05 | 上海交通大学 | Methods for preparing L-tagatose |
| CN102876752A (en) * | 2012-09-07 | 2013-01-16 | 山东绿健生物技术有限公司 | Production method for crystalline tagatose |
| CN103805649A (en) * | 2012-11-14 | 2014-05-21 | 上海信谊药厂有限公司 | Method for preparing tagatose |
| CN104561188A (en) * | 2014-12-26 | 2015-04-29 | 山东富欣生物科技股份有限公司 | Process for preparing tagatose through cottonseed meal |
| CN116789718A (en) * | 2022-03-18 | 2023-09-22 | 中国科学院天津工业生物技术研究所 | High-purity crystalline D-tagatose, compositions containing the same, and preparation methods and uses |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6057135A (en) * | 1992-01-16 | 2000-05-02 | Kraft Foods, Inc. | Process for manufacturing D-tagatose |
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- 2006-05-26 CN CNB2006100859222A patent/CN100526470C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101691597B (en) * | 2009-09-17 | 2012-09-05 | 上海交通大学 | Methods for preparing L-tagatose |
| CN102876752A (en) * | 2012-09-07 | 2013-01-16 | 山东绿健生物技术有限公司 | Production method for crystalline tagatose |
| CN103805649A (en) * | 2012-11-14 | 2014-05-21 | 上海信谊药厂有限公司 | Method for preparing tagatose |
| CN104561188A (en) * | 2014-12-26 | 2015-04-29 | 山东富欣生物科技股份有限公司 | Process for preparing tagatose through cottonseed meal |
| CN116789718A (en) * | 2022-03-18 | 2023-09-22 | 中国科学院天津工业生物技术研究所 | High-purity crystalline D-tagatose, compositions containing the same, and preparation methods and uses |
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| CN100526470C (en) | 2009-08-12 |
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