CN109393426A - A kind of composite sweetener - Google Patents
A kind of composite sweetener Download PDFInfo
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- CN109393426A CN109393426A CN201811148626.1A CN201811148626A CN109393426A CN 109393426 A CN109393426 A CN 109393426A CN 201811148626 A CN201811148626 A CN 201811148626A CN 109393426 A CN109393426 A CN 109393426A
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- China
- Prior art keywords
- concentration
- sucrose
- recombinant microorganism
- sample
- rebaudioside
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/30—Artificial sweetening agents
- A23L27/33—Artificial sweetening agents containing sugars or derivatives
- A23L27/36—Terpene glycosides
Abstract
The present invention relates to a kind of composite sweeteners, belong to technical field of food additives.The technical problem to be solved by the present invention is to provide composite sweeteners in good taste.The sweetener is synthesized using whole-cell catalytic, and using rebaudioside A as substrate, using recombinant microorganism as catalyst, in the presence of sucrose, zinc chloride and trisodium citrate, catalysis substrate is reacted, and obtains sweetener composition.The present invention is converted using whole-cell catalytic, and method is simple, is controlled the composition of the Rebaudiodside A D and Rebaudiodside A M of the available specific proportion of conversion condition, can be reduced production cost.Composite sweetener of the present invention, it is similar to cane-sugar taste, any artificial synthetic ingredient is not introduced, and pure natural, noenergy is water-soluble also preferable.
Description
Technical field
The present invention relates to a kind of composite sweeteners, belong to technical field of food additives.
Background technique
Sweetener refers to the substance that can assign soft drink sweet taste.Currently, the most commonly used natural sweetener of people is sucrose,
Sucrose can provide sweet taste for people, and mouthfeel is also generally received by people, and still, the heat in sucrose is higher, patient of diabetes
Person and obese people are required to carry out strict control in the diet, and therefore, it is necessary to empty calory or replacement sucrose low in calories
Sweetener.
Stevioside (steviol glycosides) is that separation is extracted from STEVIA REBAUDIANA (Stevia rehaudiana) blade
Obtained a kind of stevioside glycosides compound, have high sugariness (for 250~300 times of sucrose), it is low in calories (be the 1/ of sucrose
300) it, has no toxic side effect, the advantages that no carcinogen, edible safety, receives the extensive of the multiple fields such as scientific circles, industrial circle
Pay attention to, becomes the third natural sucrose substitute praised highly with Development volue and health except cane suger, beet sugar, quilt
It is described as " third place in the world sugar source " in the world.
Stevioside glycoside compound component is numerous, all has tetracyclic diterpene parent nucleus and has different degrees of glycosylation modified
And different degrees of sweet taste mouthfeel, currently, identified steviol glycoside compound mainly have stevioside (Stevioside.ST),
Rebaudioside A (RA), dulcoside B (RC), Rebaudiodside A D (RD), Rebaudiodside A M (RM) etc., wherein only stevioside and Lai Bao
Enlightening glycosides A is commercialized application, is widely used in the food processing fields such as beverage, food, flavoring agent, drinks, dairy products.
Although stevioside and rebaudioside A have low in calories, Gao Tiandu, there is also taste after the hardship in addition to sweet taste,
Its taste flavor can not be suitable with sucrose.And although Rebaudiodside A D is similar to sucrose taste, its dissolubility is bad.And mesh
Before, although there is some sweetener compositions, preparation method is to compound to obtain using purer raw material, and this method is to raw material
Purity requirement it is high, need to compound again after feedstock purification, higher cost.
Therefore, it is necessary to develop a kind of composite sweetener novel, production cost is low, in good taste, can reach
Taste flavor similar with sucrose, is received by the majority of consumers.
Summary of the invention
The technical problem to be solved by the present invention is to provide a kind of composite sweeteners in good taste.
Composite sweetener of the present invention, is prepared by following methods: using rebaudioside A as substrate, being with recombinant microorganism
Catalyst, in the presence of sucrose, zinc chloride and trisodium citrate, catalysis substrate is reacted, and obtains sweetener composition,
In, the cell concentration OD600 of recombinant microorganism is 80~120, and rebaudioside A concentration is 1~80g/L；Trisodium citrate concentration
For 50~80mmol/L, zinc oxide concentration is 0.5~2mmol/L, and sucrose concentration is 30~50% (W/V), pH value is 7.5~
8.5, the recombinant microorganism contains EUGT11 encoding gene and UGT76G1 encoding gene.
Preferably, reaction temperature is 35~40 DEG C, and the reaction time is 20~60h.
Preferably, the cell concentration OD600 of recombinant microorganism is 100, and rebaudioside A concentration is 30g/L；Lemon
Sour three na concns are 60mmol/L, and zinc oxide concentration 1mmol/L, sucrose concentration is 40% (W/V), pH value 8.0.
Preferably, reaction temperature is 37 DEG C, and the reaction time is for 24 hours.
Preferably, the recombinant microorganism is recombination bacillus coli, recombinant yeast, recombined bacillus subtilis, recombination
Corynebacterium glutamicum or recombination streptomycete.
Compared with prior art, the invention has the following advantages that
1) composite sweetener of the present invention, is transformed using whole-cell catalytic, and method is simple, and control conversion condition can obtain
To the composition of the Rebaudiodside A D and Rebaudiodside A M of specific proportion, production cost can be reduced.
2) composite sweetener of the present invention, it is similar to cane-sugar taste, any artificial synthetic ingredient is not introduced, it is pure natural, it is incompetent
Amount, it is water-soluble also preferable.
Specific embodiment
Composite sweetener of the present invention, is synthesized using whole-cell catalytic, using rebaudioside A as substrate, is with recombinant microorganism
Catalyst, in the presence of sucrose, zinc chloride and trisodium citrate, catalysis substrate is reacted, and obtains sweetener composition,
In, the cell concentration OD600 of recombinant microorganism is 80~120, and rebaudioside A concentration is 1~80g/L；Trisodium citrate concentration
For 50~80mmol/L, zinc oxide concentration is 0.5~2mmol/L, and sucrose concentration is 30~50% (W/V), pH value is 7.5~
8.5, the recombinant microorganism contains EUGT11 encoding gene and UGT76G1 encoding gene.In this way, RA can be converted
At the mixture of RD and RM.
In the method for the present invention, sucrose concentration is w/v, i.e. sucrose weight in reaction system and entire reactant
The ratio between volume of system, i.e. W/V.
Preferably, reaction temperature is 35~40 DEG C, and the reaction time is 20~60h.
Preferably, the cell concentration OD600 of recombinant microorganism is 100, and rebaudioside A concentration is 5g/L；Lemon
Sour three na concns are 60mmol/L, and zinc oxide concentration 1mmol/L, sucrose concentration is 40% (W/V), pH value 8.0, reaction temperature
Degree is 37 DEG C, and the reaction time is for 24 hours.In this way, RA can be converted to the mixture of RD and RM, examined through HPLC sample introduction
Survey, Rebaudiodside A D in product: the weight ratio of Rebaudiodside A M is 3:1.
Using composite sweetener of the invention, not only high water solubility, also has mouthfeel similar with sucrose.
Wherein, the EUGT11 encoding gene that recombinant microorganism contains be it is existing, can be found in GenBank database
(Genbank code:AK121682).UGT76G1 is UDP-glycosyltransferase 76G1, amino acid sequence number
For AAR06912.1, UGT76G1 encoding gene is as shown in sequence 1.
The recombinant microorganism can be obtained using existing gene engineering method, for example, by rice EUGT11 encoding gene,
Stevia rebaudianum UGT76G1 encoding gene connect construction recombination plasmid with carrier, is then transformed into microorganism, obtains recombinant microorganism.
In order to improve the conversion ratio of RA, it is preferred that the recombinant microorganism is recombination bacillus coli, recombinant yeast, again
Group bacillus subtilis, recombination corynebacterium glutamicum or recombination streptomycete.
A specific embodiment of the invention is further described below with reference to embodiment, is not therefore limited the present invention
System is among the embodiment described range.
Embodiment 1
1, the preparation of recombinant microorganism:
It extracts rice (Oryzasativa) leaf total serum IgE and rice cDNA is obtained by reverse transcription.According to GeneBank data
EUGT11 gene order (AccessionNo.AK121682) in library, designs PCR amplification primer, and upstream and downstream primer introduces respectively
BamH I, the site Hind III, PCR amplification obtain EUGT11 encoding gene.
It extracts STEVIA REBAUDIANA total serum IgE and rice cDNA is obtained by reverse transcription.According to the EUGT11 in GeneBank database
Gene order (AccessionNo.AK121682), designs PCR amplification primer, and upstream and downstream primer introduces Nde I, Xho respectively
The site III, PCR amplification obtain UGT76G1 encoding gene.
EUGT11 segment and expression vector pETDuet are used into BamH I and Hind III double digestion respectively, recycle purpose piece
It is attached after section with ligase, obtains pETDuet-EUGT11.
UGT76G1 segment and pETDuet-EUGT11 are used into Nde I and Xho III double digestion respectively, recycle target fragment
It is attached afterwards with ligase, obtains pETDuet-EUGT11-UGT76G1.
Recombinant plasmid pETDuet-EUGT11-UGT76G1 is converted into competent cell E.coliBL21 (DE3), is led to
The screening of ammonia benzyl chloramphenicol resistance is crossed, recombinant microorganism is obtained.
2, thallus culture and protein expression:
It chooses monoclonal to be inoculated in LB culture medium of the 2mL containing Amp (ammonia benzyl mycin, 100 μ g/mL) (20mL small test tube), 37
DEG C culture 4h, then 1% be inoculated in 100mLM9 culture medium (500mL triangular flask), 37 DEG C, 250r/m CMC model 2h
(OD600~0.6), is impregnated with tap water, and cooling 10min adds IPTG (working concentration 100mM), is placed in 22 DEG C, 180r/m condition
Lower inducing expression 20h receives bacterium, ice bath.
M9 nutrient media components such as table 1:
Table 1
| M9 nutrient media components | 1L dosage | Remarks |
| 5 × M9 salt | 200mL | Combinable sterilizing (121 DEG C of 20min) |
| Glycerol | 4mL | |
| 0.1M MgSO4(0.6g constant volume 50mL) | 20mL | Individually sterilizing (121 DEG C of 20min) |
| 0.02M CACl2(0.11g constant volume 50mL) | 5mL | Individually sterilizing (121 DEG C of 20min) |
5 × M9 salt component are as follows: Na2HPO4·12H2O 8.55g/100mL, KH2PO41.5g/100mL, NaCl 0.25g/
100mL, NH4Cl 0.5g/100mL。
3, conversion of resting cells
The OD600 for measuring bacterium solution is then centrifuged for (4 DEG C, 3000g, be centrifuged 10min) and abandons supernatant collection thallus, thin with tranquillization
Dysuria with lower abdominal colic reaction buffer (i.e. sodium phosphate buffer pH8.0, added substrate, sucrose, zinc chloride and trisodium citrate etc.) is resuspended
Thallus is to OD600=100.37 DEG C of incubators are stood for 24 hours, and then 12000r/m room temperature is centrifuged 10min, and supernatant is taken to cross 0.22 μm of filter
Film obtains product.Wherein, substrate rebaudioside A concentration be 5g/L, sucrose concentration be 40% (w/v), zinc oxide concentration 1mM,
Trisodium citrate concentration is 60mM.
HPLC sample detection, tests the amount of the RD and RM in product, and the weight ratio of discovery RD and RM is 3:1.
Embodiment 2~4
Using the method for embodiment 1, only changes partial parameters when conversion, obtain product, wherein the parameter of change such as table
Shown in 2.
Table 2
| Response parameter | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 |
| OD600 | 100 | 80 | 120 | 110 |
| Rebaudioside A concentration (g/L) | 5 | 1 | 3 | 10 |
| Trisodium citrate concentration (mmol/L) | 60 | 50 | 70 | 80 |
| Zinc oxide concentration (mmol/L) | 1 | 0.5 | 1.5 | 2 |
| Sucrose concentration (%, w/v) | 40 | 30 | 40 | 50 |
| PH value | 8.0 | 8.5 | 7.5 | 8.0 |
| Reaction temperature (DEG C) | 37 | 35 | 38 | 40 |
| Reaction time (h) | 24 | 20 | 28 | 30 |
The amount for measuring the RD and RM in product prepared by embodiment 2~4, records the weight ratio of RD and RM, the results are shown in Table 3.
Table 3
| Embodiment number | The weight ratio of RD and RM |
| Embodiment 1 | 3:1 |
| Embodiment 2 | 1.5:1 |
| Embodiment 3 | 4:1 |
| Embodiment 4 | 9:1 |
1 organoleptic analysis of test example
Adopt by reference 12311 sensory testing methods (three of 4120 sensory testing methods of BS ISO (triangle test) and GB
Point is examined) test evaluation analysis is carried out to sweetener composition, specific analytical method is as follows:
Method And Principle: at the same to valuation officer provide one group of three sample, wherein two be it is identical, valuation officer chooses
Single sample.
Equipment: responsible person is examined to select equipment according to product property and sample size etc..The equipment used should not influence to examine
Test result.The preferential standardized equipment needed using inspection is met.
Sampling: it is sampled by the sampling standard of examined product.If incomplete without such standard or sampling standard
Where applicable then negotiates the agreed methods of sampling by each side concerned.
Environment: condition needed for meeting GB 10220.
Valuation officer's condition: condition as defined in Ying Fuhe GB 10220, all valuation officers should have same qualification and inspection
Test ability.
Valuation officer's quantity: valuation officer's number is depending on examining purpose and the level of signifiance.Usually 6 or more experts；Or
15 or more optimizing evaluation persons；Or 25 or more primary assessors.7 or more experts are needed in 0.1% level of signifiance.
It examines responsible person: examining responsible person that should not generally participate in inspection, if participated in, also not it should be recognized that sample number into spectrum.Inspection
The preliminary introduction of evaluation can not be influenced with regard to relevant issues and properties of samples by testing responsible person, when being related to examining taint object, be answered
Prepare a non-taint object sample and a taint object sample compareed therewith.
The preparation of test sample: providing the sample A and B of sufficient amount, and every three sample surveys are one group.
Combine by following six kinds: ABB, AAB, ABA, BAA, BBA, BAB prepare sample equal in number from laboratory sample
Product group.
Examination requirements: valuation officer cannot be made to draw a conclusion from the mode that sample provides to the property of sample.It should be with same
Mode (identical equipment, same containers, identical quantity product and aligned identical form (triangle, straight line etc.)) prepares various inspections
Sample sets.In any sample sets, the temperature of sample survey is identical, such as possible, every other sample in the inspection series provided
The temperature of product group also Ying Xiangtong.The container for containing sample survey should number, and usually randomly select three digits.It examines, compiles every time
Number Ying Butong.
Inspection technology: telling valuation officer to examine purpose, and degree should not make their conclusion generate bias.By the several of preparation
Group sample is randomly assigned to valuation officer.Valuation officer checks each group sample survey in defined order, and order is in a series of inspections
It answers identical.When evaluating same group of three test sample, valuation officer should have the chance of duplicate test to every kind of test sample.It examines
The sample size and volume that responsible person can tell valuation officer to provide if necessary.When the multiple of the number deficiency 6 of valuation officer,
Following two ways can be taken.
A. give up redundant sample group；
B. 6 groups of samples are provided for each valuation officer and does duplicate test.
When valuation officer cannot identify its difference, allow to answer " indifference ".
As a result expression:
" variant " or " indifference " answer number is counted, when valuation officer's number n value is greater than 100, in the different levels of signifiance
The minimal number X of answer needed for upper determining three point test difference is calculated as follows, takes immediate integer value.Its
Middle α is significance, is a desired value.
The value of Z changes according to level of significance α in formula:
α≤0.05Z=1.64
α≤0.01Z=2.33
α≤0.001Z=3.10
In assumed statistical inspection, it is recognized that the probability value of small probability event be referred to as the conspicuousness water of assumed statistical inspection
It is flat, it is denoted as α.The value of α is smaller, and the significance of this hypothesis testing is higher.For example α is set as 0.05, it means that sampling point
95% sample in cloth can be regarded as normality sample, and the sample at both ends 5% can be regarded as extreme sample.If a sample falls into 95%
In normality sample, then it is just seen as from this totality, the difference of it and other samples in totality is only in other words
Accidental error caused by sampling, without statistically significant difference.One sample will be fallen into 5% extreme sample, so that it may
Refuse it from this overall judgement, and think that it is overall from others, in other words other samples of it and this totality
Difference be not sampling error, have statistically significant difference.
In the present embodiment,
" 0.1% level of signifiance (α≤0.001) is variant " correspondence " dissmilarity "
" 5% level of signifiance (α≤0.05) is indifference " correspondence " similar "
" 1% level of signifiance (α≤0.01) is indifference " correspondence " closely similar "
" 0.1% level of signifiance (α≤0.001) is indifference " correspondence " indifference "
When " indifference " answer occupies biggish ratio, illustrate the difference of two samples lower than valuation officer's detection threshold.
The above evaluation analysis method is applied to the product of analysis Examples 1 to 4, contrast sample is sucrose, by sample and
Contrast sample is tested after being made into the identical aqueous solution of sugariness respectively by the above sensory testing methods requirement, effective evaluation people
Member: 135 primary assessors allow to answer " indifference ".Its mouthfeel comparative test the results are shown in Table 4.
Table 4
In table 4, DM 1:1 mixture, DM 1:4 mixture are all made of RD (> 95%) and RM (> 95%) is mixed to get.It is logical
Cross table 4 statistics indicate that: the product that production method of the invention obtains, mouthfeel are similar to sucrose.
Claims (5)
1. a kind of composite sweetener, it is characterised in that: be prepared by following methods: micro- to recombinate using rebaudioside A as substrate
Biology is catalyst, and in the presence of sucrose, zinc chloride and trisodium citrate, catalysis substrate is reacted, and obtains sweetener group
Close object, wherein the cell concentration OD600 of recombinant microorganism is 80~120, and rebaudioside A concentration is 1~80g/L；Citric acid three
Na concn is 50~80mmol/L, and zinc oxide concentration is 0.5~2mmol/L, and sucrose concentration is 30~50% (W/V), and pH value is
7.5~8.5, the recombinant microorganism contains EUGT11 encoding gene and UGT76G1 encoding gene.
2. composite sweetener according to claim 1, it is characterised in that: reaction temperature is 35~40 DEG C, and the reaction time is
20~60h.
3. composite sweetener according to claim 1, it is characterised in that: the cell concentration OD600 of recombinant microorganism is
100, rebaudioside A concentration is 30g/L；Trisodium citrate concentration is 60mmol/L, zinc oxide concentration 1mmol/L, and sucrose is dense
Degree is 40% (W/V), pH value 8.0.
4. composite sweetener according to claim 2 or 3, it is characterised in that: reaction temperature is 37 DEG C, and the reaction time is
24h。
5. composite sweetener according to claim 1, it is characterised in that: the recombinant microorganism be recombination bacillus coli,
Recombinant yeast, recombined bacillus subtilis, recombination corynebacterium glutamicum or recombination streptomycete.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811148626.1A CN109393426A (en) | 2018-09-29 | 2018-09-29 | A kind of composite sweetener |
| PCT/CN2018/113715 WO2020062437A1 (en) | 2018-09-29 | 2018-11-02 | Composite sweetening agent and production method therefor |
| US16/588,427 US11274328B2 (en) | 2018-09-29 | 2019-09-30 | Methods for producing rebaudioside D and rebaudioside M and compositions thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811148626.1A CN109393426A (en) | 2018-09-29 | 2018-09-29 | A kind of composite sweetener |
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| Publication Number | Publication Date |
|---|---|
| CN109393426A true CN109393426A (en) | 2019-03-01 |
Family
ID=65466620
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| CN201811148626.1A Pending CN109393426A (en) | 2018-09-29 | 2018-09-29 | A kind of composite sweetener |
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Citations (7)
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|---|---|---|---|---|
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| CN103397064A (en) * | 2013-08-14 | 2013-11-20 | 苏州汉酶生物技术有限公司 | Method for preparing rebaudioside M through enzyme method |
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| WO2013022989A2 (en) * | 2011-08-08 | 2013-02-14 | Evolva Sa | Recombinant production of steviol glycosides |
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| US20160186225A1 (en) * | 2013-02-06 | 2016-06-30 | Evolva Sa | Methods for improved production of rebaudioside d and rebaudioside m |
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Application publication date: 20190301 |