CN100526470C - Preparation method of functional sweetener D-tatai sugar - Google Patents
Preparation method of functional sweetener D-tatai sugar Download PDFInfo
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- CN100526470C CN100526470C CNB2006100859222A CN200610085922A CN100526470C CN 100526470 C CN100526470 C CN 100526470C CN B2006100859222 A CNB2006100859222 A CN B2006100859222A CN 200610085922 A CN200610085922 A CN 200610085922A CN 100526470 C CN100526470 C CN 100526470C
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Abstract
This invention discloses a preparation of functional edulcorant D-tagatose, concretely, relating to a preparation of producing D-tagatose by fermenting microbes and bioconversion, belonging to food biotechnology field. D-tagatose is prepared by fermentation method bioconversion, using lactobacillus as strain, galactose as transformation substrate, fermentation medium composed of by adding carbon source, nitrogen source and inorganic salt. Lactobacillus used is in MRS nutrient medium, cultivating at 25-40DEG C,growing well, as seed culture solution, glucose, lactose, maltose or starch soluble as carbon source of fermentation medium, using one or several of peptone, yeast extract, corn steep liquor, soybean meal to mix up as nitrogen source, detecting after fermenting, concentration of D-tagatose in the broth reaching to 250-450mg/100ml. The product attained in this invention is safe and reliable, having a high physiologic function.
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 globiformis 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 plant lactobacillus (Lactobacillus plantarum), Lactobacillus pentosus (Lactobacillus pentose) is 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:
Plant lactobacillus AS1.550; Plant lactobacillus AS1.557;
Lactobacillus pentosus BCRC11053; Lactobacillus pentosus BCRC15317.
When transforming as strain bio, select for use plant lactobacillus AS1.557 or Lactobacillus pentosus BCRC15317 effect 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: plant lactobacillus AS1.550; Plant lactobacillus AS1.557;
Lactobacillus pentosus BCRC11053; Lactobacillus pentosus BCRC15317, above bacterial classification is all open, the expression of AS numbering (China General Microbiological Cuture CollectionCenter.CGMCC.Institute of Micribiology, Chinese Academy of Science: Chinese microorganism strain council common micro-organisms center).BCRC numbering expression (Bioresource Collection andResearch 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, plant lactobacillus AS1.557, the D-tagatose productive rate of Lactobacillus pentosus 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
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 plant lactobacillus 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
Claims (3)
1. the preparation method of a D-tagatose, it is characterized in that with plant lactobacillus (Lactobacillusplantarum), Lactobacillus pentosus (Lactobacillus pentose) is 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 plant lactobacillus AS1.550; Plant lactobacillus AS1.557; Lactobacillus pentosus BCRC11053 or Lactobacillus pentosus BCRC 15317;
(2) substratum, in g/100ml:
Storage medium: glucose 1.5, yeast extract paste 1, lime carbonate 1.5, agar 1.5, pH6.5-7.0;
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, pH6.5-7.0;
(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, pH7.0,25-40 ℃ 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, pH7.0.
3. the preparation method of D-tagatose according to claim 1 is characterized in that bacterial classification selects plant lactobacillus AS1.557 or Lactobacillus pentosus BCRC15317 for use.
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| CN100526470C true CN100526470C (en) | 2009-08-12 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
Citations (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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6057135A (en) * | 1992-01-16 | 2000-05-02 | Kraft Foods, Inc. | Process for manufacturing D-tagatose |
Non-Patent Citations (2)
| Title |
|---|
| L-***糖异构酶蹙眉性质及其生物合成塔格糖的研究. 翁玮慧，张华，江波.食品工业科技，第27卷第1期. 2006 |
| L-***糖异构酶蹙眉性质及其生物合成塔格糖的研究. 翁玮慧，张华，江波.食品工业科技，第27卷第1期. 2006 * |
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