WO2020062437A1 - 复合甜味剂及其生产方法 - Google Patents

复合甜味剂及其生产方法 Download PDF

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Publication number
WO2020062437A1
WO2020062437A1 PCT/CN2018/113715 CN2018113715W WO2020062437A1 WO 2020062437 A1 WO2020062437 A1 WO 2020062437A1 CN 2018113715 W CN2018113715 W CN 2018113715W WO 2020062437 A1 WO2020062437 A1 WO 2020062437A1
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Prior art keywords
rebaudioside
sweetener composition
concentration
recombinant
sucrose
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PCT/CN2018/113715
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English (en)
French (fr)
Inventor
王勇
吕华军
孙雨伟
李建华
裴亮
华君
Original Assignee
四川盈嘉合生科技有限公司
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Priority claimed from CN201811148647.3A external-priority patent/CN109234341A/zh
Priority claimed from CN201811148626.1A external-priority patent/CN109393426A/zh
Priority claimed from CN201811148627.6A external-priority patent/CN109349596A/zh
Application filed by 四川盈嘉合生科技有限公司 filed Critical 四川盈嘉合生科技有限公司
Publication of WO2020062437A1 publication Critical patent/WO2020062437A1/zh

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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin

Definitions

  • the invention relates to a composite sweetener and a production method thereof, and belongs to the technical field of food additives.
  • a sweetener is a substance that imparts a sweet taste to a soft drink.
  • sucrose the most commonly used natural sweetener is sucrose.
  • Sucrose can provide people with a sweet taste and its taste is generally accepted.
  • the calories in sucrose are high. Diabetics and obese people need to be strict in their diet. Control, therefore, requires no or low-calorie sweeteners instead of sucrose.
  • Steviol sugarsides are a class of steviol glycoside compounds extracted and isolated from the leaves of Stevia rehaudiana. They have high sweetness (250 to 300 times that of sucrose) and low calories (1/300 of that of sucrose). ), No toxic side effects, no carcinogens, edible safety and other advantages, has been widely valued by the scientific community, industry and many other fields, becoming the third development and health respected after sugarcane and beet sugar.
  • the natural sucrose substitute is internationally known as "the third sugar source in the world”.
  • Stevioside compounds have many components, all of which have a tetracyclic diterpene mother nucleus and have varying degrees of glycosylation modification and varying degrees of sweet taste.
  • the major stevia compounds identified are stevioside (Stevioside.ST) Rebaudioside A (RA), Rebaudioside C (RC), Rebaudioside D (RD), Rebaudioside M (RM), etc., of which only stevioside and rebaudioside A are Commercial applications, widely used in food processing fields such as beverages, foods, flavoring agents, alcohol, dairy products.
  • stevioside and rebaudioside A have low calories and high sweetness, there are also bitter aftertastes other than sweetness, and their taste and flavor cannot be comparable to sucrose.
  • Rebaudioside D although similar in taste to sucrose, has poor solubility. Therefore, there is a need to develop a novel sweetener composition with good mouthfeel so that it can achieve a mouthfeel similar to sucrose, which is accepted by consumers.
  • the current sweetener compositions are prepared by using relatively pure raw materials. This method requires high purity of the raw materials. The raw materials need to be purified and then mixed, which has a high cost. Therefore, there is a need for a method for producing a sweetener composition with a lower production cost.
  • the first technical problem solved by the present invention is to provide a sweetener composition with good taste.
  • the present invention also provides another sweetener composition, namely, sweetener composition II.
  • the second technical problem solved by the present invention is that the technical problem solved by the present invention is to provide a method for producing a sweetener composition with low production cost.
  • the production method of the sweetener composition of the present invention uses Rebaudioside A as a substrate and a recombinant microorganism as a catalyst, and catalyzes the reaction of the substrate in the presence of sucrose, zinc chloride and trisodium citrate to obtain sweetness.
  • Flavor composition wherein the microbial cell concentration OD600 of the recombinant microorganism is 80 to 120, the rebaudioside A concentration is 1 to 80 g / L, the concentration of trisodium citrate is 50 to 80 mmol / L, and the concentration of zinc chloride is 0.5 ⁇ 2mmol / L, 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.
  • the reaction temperature is 35-40 ° C, and the reaction time is 20-60 hours.
  • the microbial cell concentration of the recombinant microorganism is 100
  • the concentration of rebaudioside A is 5 g / L
  • the concentration of trisodium citrate is 60 mmol / L
  • the concentration of zinc chloride is 1 mmol / L
  • the concentration of sucrose is 40% (W / V)
  • pH was 8.0.
  • the reaction temperature is 37 ° C. and the reaction time is 24 h.
  • the recombinant microorganism is a recombinant E. coli, a recombinant yeast, a recombinant Bacillus subtilis, a recombinant Corynebacterium glutamicum, or a recombinant Streptomyces.
  • the invention also provides a sweetener composition prepared by the above preparation method.
  • the present invention has the following advantages:
  • the invention can effectively solve the taste and flavor problems of sweeteners, making it similar to sucrose, without introducing any artificially synthesized ingredients, maintaining the pure natural characteristics of rebaudioside A, D, and M, and without introducing any energy ingredients,
  • the obtained sweetener has the characteristic of being energy-free.
  • the present invention can effectively solve the water solubility problem of rebaudioside D and improve the water solubility of sweeteners.
  • the present invention adopts whole-cell catalytic conversion, the method is simple, and the conversion conditions can be controlled to obtain a combination of rebaudioside D and rebaudioside M, which does not need to be purified and then recombined, which can reduce production costs.
  • a sweetener composition similar to the taste of sucrose can be obtained, without introducing any artificially synthesized ingredients, pure natural, no energy, and good water solubility.
  • RD and RM can be mixed to increase water solubility.
  • RD and RM are mixed in a ratio of 3: 1, their taste is similar to sucrose.
  • RD and RM are mixed in a ratio of 9: 1, the taste is very similar to sucrose.
  • the RD and RM in the present invention are both highly pure, and their purity is greater than 95% (w / w).
  • the present invention also provides another sweetener composition, namely, the sweetener composition II, which is a mixture of the above-mentioned sweetener composition I and rebaudioside A, and is sweetened by weight.
  • the sweetener composition of the present invention can also be made by mixing the sweetener composition I with other sugar alcohol sweeteners.
  • it can be mixed with erythritol to suppress bitterness and reduce sweetness. multiple. It can also be mixed with xylitol and the like.
  • the RA purity in the present invention is greater than 97% (w / w).
  • the sweetener composition of the present invention can be prepared by a simple mixing method. For example, rebaudioside D (> 95%, w / w) and rebaudioside M (> 95%, w / w), just mix in proportion.
  • the production method of the sweetener composition of the present invention adopts whole-cell catalytic synthesis, uses rebaudioside A as a substrate, and a recombinant microorganism as a catalyst, and catalyzes the base in the presence of sucrose, zinc chloride and trisodium citrate.
  • the reaction was carried out to obtain a sweetener composition, wherein the concentration of the recombinant microorganism OD600 was 80 to 120, the concentration of rebaudioside A was 1 to 80 g / L, and the concentration of trisodium citrate was 50 to 80 mmol / L.
  • the zinc concentration is 0.5 to 2 mmol / L
  • the sucrose concentration is 30 to 50% (W / V)
  • the pH value is 7.5 to 8.5.
  • the recombinant microorganism contains an EUGT11 encoding gene and a UGT76G1 encoding gene. By this method, RA can be converted into a mixture of RD and RM.
  • the reaction temperature is 35-40 ° C, and the reaction time is 20-60 hours.
  • the microbial concentration of the recombinant microorganism is OD600 of 100, rebaudioside A concentration is 5g / L; trisodium citrate concentration is 60mmol / L, zinc chloride concentration is 1mmol / L, and sucrose concentration is 40% W / V (ie mass-to-volume ratio), pH value is 8.0, reaction temperature is 37 ° C, and reaction time is 24h.
  • RA can be converted into a mixture of RD and RM.
  • the weight ratio of rebaudioside D: rebaudioside M in the product is 3: 1.
  • RA can be successfully converted into a mixture of RD and RM.
  • the method is simple, and the obtained product has not only high water solubility but also a mouthfeel similar to sucrose.
  • the EUGT11 encoding gene contained in the recombinant microorganism is an existing gene and can be found in the GenBank database (Accession No. AK121682.1).
  • UGT76G1 is UDP-glycosyltransferase 76G1, and its amino acid sequence number is AAR06912.1.
  • the gene encoding UGT76G1 is shown in Sequence 1.
  • the recombinant microorganism can be obtained by using existing genetic engineering methods. For example, a rice EUGT11 encoding gene and a stevia UGT76G1 encoding gene are connected to a vector to construct a recombinant plasmid, and then transformed into the microorganism to obtain a recombinant microorganism.
  • the recombinant microorganism is preferably a recombinant E. coli, a recombinant yeast, a recombinant Bacillus subtilis, a recombinant Corynebacterium glutamicum, or a recombinant Streptomyces.
  • Rice (Oryzasativa) leaf total RNA was extracted and rice cDNA was obtained by reverse transcription.
  • EUGT11 gene sequence accesion No. AK121682
  • PCR amplification primers were designed, and the upstream and downstream primers were introduced into the BamH I and Hind III sites, respectively.
  • the EUGT11 coding gene was obtained by PCR amplification.
  • EUGT11 gene sequence accesion No. AK121682
  • GeneBank database PCR amplification primers were designed. The upstream and downstream primers were introduced into the Nde I and Xho III sites, respectively, and the UGT76G1 encoding gene was obtained by PCR amplification.
  • the EUGT11 fragment and the expression vector pETDuet were digested with BamH I and Hind III, respectively, and the target fragment was recovered and ligated with ligase to obtain pETDuet-EUGT11.
  • the UGT76G1 fragment and pETDuet-EUGT11 were double-digested with Nde I and Xho III, respectively, and the target fragment was recovered and ligated with ligase to obtain pETDuet-EUGT11-UGT76G1.
  • the recombinant plasmid pETDuet-EUGT11-UGT76G1 was transformed into competent cells E. coli BL21 (DE3), and the recombinant microorganism was obtained by ampicillin resistance screening.
  • Recombinant microorganisms were picked and inoculated in 2 mL of LB medium (20 mL small test tube) containing Amp (ampicillin, 100 ⁇ g / mL), cultured at 37 ° C for 4 h, and then 1% inoculated in 100 ml of LM9 medium (500 mL Erlenmeyer flask), 37 ° C. Incubate at 250r / m for 2h (OD600 ⁇ 0.6), soak in tap water, cool for 10min, add IPTG (working concentration 100mM), place at 22 ° C, induce expression for 20h at 180r / m, collect bacteria, and ice bath.
  • LB medium 20 mL small test tube
  • Amp ampicillin, 100 ⁇ g / mL
  • LM9 medium 500 mL Erlenmeyer flask
  • M9 medium The components of M9 medium are shown in Table 1:
  • M9 medium components 1L dosage Note 5 ⁇ M9 salt 200mL Can be combined sterilized (121 °C 20min) glycerin 4mL Zh 0.1M MgSO 4 (0.6g 50mL) 20mL Individual sterilization (121 °C 20min) 0.02M CACl 2 (0.11g constant volume 50mL) 5mL Individual sterilization (121 °C 20min)
  • the 5 ⁇ M9 salt components are: Na 2 HPO 4 ⁇ 12H 2 O 8.55 g / 100 mL, KH 2 PO 4 1.5 g / 100 mL, NaCl 0.25 g / 100 mL, and NH 4 Cl 0.5 g / 100 mL.
  • the substrate rebaudioside A had a concentration of 5 g / L, a sucrose concentration of 40% (w / v), a zinc chloride concentration of 1 mM, and a trisodium citrate concentration of 60 mM.
  • Example 2 The method of Example 1 was used to change only a part of the parameters during the conversion to obtain a product, wherein the changed parameters are shown in Table 2.
  • Example 1 Example 2 Example 3 Example 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 chloride 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 (°C) 37 35 38 40 Response time (h) twenty four 20 28 30
  • Test example 1 sensory analysis
  • a group of three samples is provided to the evaluator at the same time, two of which are identical, and the evaluator picks out a single sample.
  • Equipment The person in charge of inspection selects equipment according to the nature of the product and the number of samples. The equipment used shall not affect the inspection results. Preferential use of standardized equipment that meets inspection needs.
  • Sampling shall be carried out according to the sampling standards of the tested products. If there is no such standard or the sampling standard is not completely applicable, the sampling method shall be agreed upon by the parties concerned.
  • Evaluator conditions The conditions specified in GB 10220 should be met, and all evaluators should have equal qualifications and inspection capabilities.
  • Number of evaluators The number of evaluators is based on the purpose and significance of the test. Usually more than 6 experts; or more than 15 preferred evaluators; or more than 25 junior evaluators. More than 7 experts are required at a significant level of 0.1%.
  • the person in charge of inspection should generally not participate in the inspection, and if they do, they should not know the sample number.
  • the person in charge of the inspection can make a preliminary introduction on the relevant issues and the nature of the sample without affecting the evaluation.
  • a non-dye sample and a contrasted dye sample should be prepared.
  • Preparation of test samples Provide a sufficient amount of samples A and B, with each three test samples as a group.
  • ABB, AAB, ABA, BAA, BBA, BAB an equal number of sample groups are prepared from laboratory samples.
  • test sample groups shall be prepared in the same way (same equipment, same container, same quantity of product, and same arrangement (triangle, line, etc.)).
  • the temperature of the test sample is the same in any sample group, and if possible, the temperature of all other sample groups in the test series provided should also be the same.
  • Containers for inspection samples should be numbered, and generally three digits are randomly selected. The number should be different for each inspection.
  • Testing techniques Tell the evaluators that the purpose of the test is to such an extent that they do not bias their conclusions.
  • the prepared groups of samples were randomly assigned to the evaluators.
  • the evaluator checks the test samples of each group in the prescribed order, and the order should be the same in the same series of tests. When evaluating three test samples in the same group, the evaluator shall have the opportunity to repeat the test for each test sample.
  • the inspection leader can tell the evaluator the number and volume of samples provided if necessary. When the number of evaluators is less than a multiple of 6, the following two methods can be adopted.
  • the minimum number of answers required to determine the significant difference in the three-point test at different significance levels X is calculated according to the following formula, which is the closest Integer value. Where ⁇ is the significance level and is an expected value.
  • the probability value of the accepted small probability event is called the significance level of the statistical hypothesis test, and is recorded as ⁇ .
  • ⁇ is set to 0.05, which means that 95% of the samples in the sampling distribution are counted as normal samples, and 5% of the samples at both ends are counted as extreme samples. If a sample falls into 95% of the normal samples, then it is regarded as coming from this population, or the difference between it and other samples in the population is only the accidental error caused by sampling, and there is no statistically significant difference. If a sample falls into the 5% extreme sample, it can reject the assertion that it comes from this population, and think that it comes from another population, or that the difference between it and other samples of this population is not a sampling error, and has statistical significance. Significant difference.
  • the above evaluation and analysis method is applied to analyze different compositions.
  • the comparison sample is sucrose.
  • the sample and the comparison sample are respectively formulated into the same sweetness aqueous solution.
  • the experiments are performed according to the above sensory analysis method requirements. Effective reviewers: 135 junior reviewers , Allowing to answer "no difference”.
  • the results of the taste comparison test are shown in Table 4.
  • DM 1: 9 mixture, DM 1: 1 mixture, and DM 1: 4 mixture are all obtained by mixing RD (> 95%) and RM (> 95%).
  • DM3: 1 mixture, DM 1.5: 1 mixture, The DM 4: 1 mixture and the DM 9: 1 mixture were prepared in Examples 1 to 4 of the present invention.
  • the data in Table 4 indicate that the product obtained by the production method of the present invention has a taste similar to sucrose. And when the ratio of high-purity rebaudioside D (> 95%) to high-purity rebaudioside M (> 95%) (w / w) is 1.5-9: 1, the taste is similar to sucrose.
  • a mixture of rebaudioside D and rebaudioside M in a weight ratio of 3: 1 (DM 3: 1 mixture) and rebaudioside A (RA) are mixed in a certain weight ratio to obtain a sweetener composition.
  • Rebaudioside A (> 97%)
  • DM 3 1 mixture
  • DM 3 1 mixture-Rebaudioside A (1: 9, w / w)
  • DM 3 1 mixture-Rebaudioside A (9: 1, w / w)
  • DM 3 1 mixture-Rebaudioside A (3: 7, w / w)
  • the comparison sample was sucrose.
  • the sample and the comparison sample were respectively prepared into an aqueous solution with the same sweetness, and the experiments were performed according to the above sensory analysis method requirements.
  • Effective evaluators 135 junior evaluators, allowed to answer "no difference”.
  • the results of the taste comparison test are shown in Table 5.
  • a mixture of rebaudioside D and rebaudioside M in a weight ratio of 3: 1 (DM 3: 1 mixture) and rebaudioside A (RA) are mixed in a certain weight ratio to obtain a sweetener composition.
  • Rebaudioside A (> 97%)
  • DM 3 1 mixture
  • DM 3 1 mixture-Rebaudioside A (3: 7, w / w)
  • DM 3 1 mixture-Rebaudioside A (7: 3, w / w)
  • DM 3 1 mixture-Rebaudioside A (4: 6, w / w)
  • the comparison sample was sucrose.
  • the sample and the comparison sample were respectively prepared into an aqueous solution with the same sweetness, and the experiment was performed according to the above sensory analysis method requirements.
  • Effective evaluators 125 junior evaluators, allowed to answer "no difference”.
  • the results of the taste comparison test are shown in Table 6.
  • a mixture of rebaudioside D and rebaudioside M in a weight ratio of 3: 1 (DM 3: 1 mixture) and rebaudioside A (RA) are mixed in a certain weight ratio to obtain a sweetener composition.
  • Rebaudioside A (> 97%)
  • DM 3 1 mixture
  • DM 3 1 mixture-Rebaudioside A (4: 6, w / w)
  • DM 3 1 mixture-Rebaudioside A (6: 4, w / w)
  • DM 3 1 mixture-Rebaudioside A (5: 5, w / w)
  • the comparison sample was sucrose.
  • the sample and the comparison sample were respectively prepared into an aqueous solution with the same sweetness, and the experiment was performed according to the above sensory analysis method requirements.
  • Effective evaluators 128 junior evaluators, allowed to answer "no difference”.
  • the results of the taste comparison test are shown in Table 7.

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Abstract

一种口感好的甜味剂组合物，由莱鲍迪苷D和莱鲍迪苷M组成，且按重量比，莱鲍迪苷D:莱鲍迪苷M＝1.5～9:1。该甜味剂采用全细胞催化合成，以莱鲍迪苷A为底物，以重组微生物为催化剂，在蔗糖、氯化锌和柠檬酸三钠的存在下，催化底物进行反应得到，所述重组微生物含有EUGT11编码基因和UGT76G1编码基因。

Description

复合甜味剂及其生产方法 技术领域
本发明涉及一种复合甜味剂及其生产方法，属于食品添加剂技术领域。
背景技术
甜味剂是指能赋予软饮料甜味的物质。目前，人们使用最多的天然甜味剂为蔗糖，蔗糖能够为人们提供甜味，其口感也被人们普遍接受，但是，蔗糖中的热量较高，糖尿病患者以及肥胖人群均需要在饮食中进行严格控制，因此，需要无热量或者低热量的代替蔗糖的甜味剂。
甜菊糖(steviol glycosides)是从甜叶菊(Stevia rehaudiana)叶片中提取分离得到的一类甜菊糖苷类化合物，具有高甜度(为蔗糖的250～300倍)、低热量(为蔗糖的1/300)，无毒副作用，无致癌物质，食用安全等优点，受到了科学界、产业界等多个领域的广泛重视，成为继甘蔗糖、甜菜糖之外的第三种具有开发价值和健康推崇的天然蔗糖替代品，被国际上誉为“世界第三糖源”。
甜菊糖苷类化合物组分众多，均具有四环二萜母核并且有不同程度的糖基化修饰及不同程度的甜味口感，目前，被识别的甜菊糖苷化合物主要有甜菊苷(Stevioside.ST)、莱鲍迪苷A(RA)、莱鲍迪苷C(RC)、莱鲍迪苷D(RD)、莱鲍迪苷M(RM)等，其中，仅甜菊苷和莱鲍迪苷A被商业化应用，广泛用于饮料、食品、调味剂、酒类、乳制品等食品加工领域。
虽然甜菊苷和莱鲍迪苷A具有低热量、高甜度，但是还存在除甜味之外的苦后味，其口感风味无法与蔗糖相当。而莱鲍迪苷D虽然与蔗糖口味相似，但是其溶解性不好。因此，需要开发一种新型的口感好的甜味剂组合物，使其能够达到与蔗糖相似的口感风味，被广大消费者所接受。
此外，目前的甜味剂组合物，其制备方法均是采用较纯的原料复配得到，该方法对原料的纯度要求高，需要将原料提纯后再进行复配，成本较高。因此，需要一种生产成本较低的甜味剂组合物的生产方法。
发明内容
本发明解决的第一个技术问题是提供一种口感好的甜味剂组合物。
本发明甜味剂组合物Ⅰ，由莱鲍迪苷D和莱鲍迪苷M组成，且按重量比，莱鲍迪苷D:莱鲍迪苷M＝1.5～9:1。
优选的，按重量比，莱鲍迪苷D:莱鲍迪苷M＝3～9:1。
作为优选方案，按重量比，莱鲍迪苷D:莱鲍迪苷M＝3:1。
本发明还提供另一种甜味剂组合物，即甜味剂组合物Ⅱ。
本发明甜味剂组合物Ⅱ，由上述的甜味剂组合物Ⅰ与莱鲍迪苷A混合而成，且按重量比，甜味剂组合物Ⅰ:莱鲍迪苷A＝1～9:1～9。
优选的，按重量比，甜味剂组合物Ⅰ:莱鲍迪苷A＝3～7:3～7。
作为优选方案，按重量比，甜味剂组合物Ⅰ:莱鲍迪苷A＝4～6:4～6。
本发明解决的第二个技术问题是本发明解决的技术问题是提供一种生产成本低的甜味剂组合物的生产方法。
本发明甜味剂组合物的生产方法，以莱鲍迪苷A为底物，以重组微生物为催化剂，在蔗糖、氯化锌和柠檬酸三钠的存在下，催化底物进行反应，得到甜味剂组合物，其中，重组微生物的菌体浓度OD600为80～120，莱鲍迪苷A浓度为1～80g/L；柠檬酸三钠浓度为50～80mmol/L，氯化锌浓度为0.5～2mmol/L，蔗糖浓度为30～50％(W/V)，pH值为7.5～8.5，所述重组微生物含有EUGT11编码基因和UGT76G1编码基因。
优选的，反应温度为35～40℃，反应时间为20～60h。
作为优选方案，重组微生物菌体浓度OD600为100，莱鲍迪苷A浓度为5g/L；柠檬酸三钠浓度为60mmol/L，氯化锌浓度为1mmol/L，蔗糖浓度为40％(W/V)，pH值为8.0。
作为优选方案，反应温度为37℃，反应时间为24h。
优选的，所述重组微生物为重组大肠杆菌、重组酵母菌、重组枯草芽孢杆菌、重组谷氨酸棒状杆菌或重组链霉菌。
本发明还提供上述制备方法制备得到的甜味剂组合物。
与现有技术相比，本发明具有以下优点：
1)本发明能有效解决甜味剂的口感风味问题，使之与蔗糖相似，不引入任何人工合成成分，保持了莱鲍迪苷A、D和M纯天然的特点，不引入任何能量成分，得到的甜味剂具有无能量的特点。
2)本发明能有效解决莱鲍迪苷D的水溶性问题，提高甜味剂的水溶性。
3)本发明采用全细胞催化转化，方法简单，控制转化条件可以得到特定配比的莱鲍迪苷D和莱鲍迪苷M的组合物，无需提纯后再复配，可以降低生产成本。采用该方 法，可以得到一种与蔗糖口感相似的甜味剂组合物，不引入任何人工合成成分，纯天然，无能量，水溶性也较好。
具体实施方式
本发明甜味剂组合物Ⅰ，由莱鲍迪苷D和莱鲍迪苷M组成，且按重量比，莱鲍迪苷D:莱鲍迪苷M＝1.5～9:1。研究发现，RD和RM以一定比例混合后，能够相辅相成，改善甜味剂的口感，使其能够达到口感与蔗糖相似。此外，RD和RM混合后，还能提高水溶性。
优选的，按重量比，莱鲍迪苷D:莱鲍迪苷M＝3～9:1。
作为优选方案，莱鲍迪苷D:莱鲍迪苷M＝3:1。当RD与RM以3:1的比例混合后，其口感与蔗糖相似。
作为优选方案，莱鲍迪苷D:莱鲍迪苷M＝9:1。当RD与RM以9:1的比例混合后，其口感与蔗糖非常相似。
本发明中的RD和RM均为高纯的，其纯度均大于95％(w/w)。
优选的，本发明还提供另一种甜味剂组合物，即甜味剂组合物Ⅱ，它是由上述甜味剂组合物Ⅰ与莱鲍迪苷A混合而成，且按重量比，甜味剂组合物Ⅰ:莱鲍迪苷A＝1～9:1～9。发明人发现，将RA、RD和RM按一定比例混合后，其口感更佳。
优选的，按重量比，甜味剂组合物Ⅰ:莱鲍迪苷A＝3～7:3～7；采用该比例，得到的组合物其口感与蔗糖非常相似。
更优选的，按重量比，甜味剂组合物Ⅰ:莱鲍迪苷A＝4～6:4～6；采用该比例，得到的组合物其口感与蔗糖无差异。
此外，本发明的甜味剂组合物，还可以由甜味剂组合物Ⅰ与其他糖醇类甜味剂混合而成，比如，可以与赤藓糖醇混合，从而抑制苦味，并且降低甜度倍数。也可与木糖醇等混合使用。
本发明中的RA纯度大于97％(w/w)。
本发明的甜味剂组合物，其制备方法可以采用简单的混合，比如，直接称取莱鲍迪苷D(>95％,w/w)与莱鲍迪苷M(>95％,w/w)，按比例混合即可。
本发明甜味剂组合物的生产方法，采用全细胞催化合成，以莱鲍迪苷A为底物，以重组微生物为催化剂，在蔗糖、氯化锌和柠檬酸三钠的存在下，催化底物进行反应，得到甜味剂组合物，其中，重组微生物菌体浓度OD600为80～120，莱鲍迪苷A浓度 为1～80g/L；柠檬酸三钠浓度为50～80mmol/L，氯化锌浓度为0.5～2mmol/L，蔗糖浓度为30～50％(W/V)，pH值为7.5～8.5，所述重组微生物含有EUGT11编码基因和UGT76G1编码基因。通过该方法，能够将RA转化成RD和RM的混合物。
优选的，反应温度为35～40℃，反应时间为20～60h。
作为优选方案，重组微生物的菌体浓度OD600为100，莱鲍迪苷A浓度为5g/L；柠檬酸三钠浓度为60mmol/L，氯化锌浓度为1mmol/L，蔗糖浓度为40％(W/V，即质量体积比)，pH值为8.0，反应温度为37℃，反应时间为24h。通过该方法，能够将RA转化成RD和RM的混合物，经HPLC进样检测，产物中莱鲍迪苷D:莱鲍迪苷M的重量比为3:1。
采用本发明方法，能够成功将RA转化成RD和RM的混合物，其方法简单，得到的产物不仅水溶性高，还具有与蔗糖相似的口感。
本发明方法中，重组微生物含有的EUGT11编码基因为现有的，在GenBank数据库中可以查到(Accession No.AK121682.1)。UGT76G1为UDP-glycosyltransferase 76G1，其氨基酸序列号为AAR06912.1，UGT76G1编码基因如序列1所示。
该重组微生物可以采用现有的基因工程方法得到，比如，将水稻EUGT11编码基因、甜菊UGT76G1编码基因与载体连接构建重组质粒，然后转化到微生物中，得到重组微生物。
为了提高RA的转化率，优选的，所述重组微生物为重组大肠杆菌、重组酵母菌、重组枯草芽孢杆菌、重组谷氨酸棒状杆菌或重组链霉菌。
下面结合实施例对本发明的具体实施方式做进一步的描述，并不因此将本发明限制在所述的实施例范围之中。
实施例1
1、重组微生物的制备：
提取水稻(Oryzasativa)叶总RNA并通过反转录获得水稻cDNA。根据GeneBank数据库中的EUGT11基因序列(AccessionNo.AK121682)，设计PCR扩增引物，上下游引物分别引入BamH I、Hind III位点，PCR扩增得到EUGT11编码基因。
提取甜叶菊总RNA并通过反转录获得水稻cDNA。根据GeneBank数据库中的EUGT11基因序列(AccessionNo.AK121682)，设计PCR扩增引物，上下游引物分别引入Nde I、Xho III位点，PCR扩增得到UGT76G1编码基因。
将EUGT11片段与表达载体pETDuet分别用BamH I和Hind III双酶切，回收目的片段后以连接酶进行连接，得到pETDuet-EUGT11。
将UGT76G1片段与pETDuet-EUGT11分别用Nde I和Xho III双酶切，回收目的片段后以连接酶进行连接，得到pETDuet-EUGT11-UGT76G1。
将重组质粒pETDuet-EUGT11-UGT76G1转化至感受态细胞E.coliBL21(DE3)中，通过氨苄霉素抗性筛选，获得重组微生物。
2、菌体培养及蛋白表达：
挑重组微生物接种于2mL含Amp(氨苄霉素，100μg/mL)的LB培养基中(20mL小试管)，37℃培养4h，然后1％接种于100mLM9培养基中(500mL三角瓶)，37℃，250r/m条件培养2h(OD600～0.6)，用自来水浸泡，冷却10min，加IPTG(工作浓度100mM)，置于22℃，180r/m条件下诱导表达20h，收菌，冰浴。
M9培养基组分如表1：
表1
M9培养基组分 1L用量 备注
5×M9盐 200mL 可合并灭菌(121℃20min)
甘油 4mL  
0.1M MgSO 4(0.6g定容50mL) 20mL 单独灭菌(121℃20min)
0.02M CACl 2(0.11g定容50mL) 5mL 单独灭菌(121℃20min)
5×M9盐组分为：Na 2HPO 4·12H 2O 8.55g/100mL，KH 2PO 4 1.5g/100mL，NaCl 0.25g/100mL，NH 4Cl 0.5g/100mL。
3、静息细胞转化
测定菌液的OD600，然后离心(4℃，3000g，离心10min)弃上清收集菌体，用静息细胞转化反应缓冲液(即磷酸钠缓冲液pH8.0，附加底物、蔗糖、氯化锌和柠檬酸三钠等)重悬菌体至OD600＝100。37℃培养箱静置24h，然后12000r/m室温离心10min，取上清过0.22μm滤膜，得到产物。其中，底物莱鲍迪苷A浓度为5g/L，蔗糖浓度为40％(w/v)，氯化锌浓度为1mM，柠檬酸三钠浓度为60mM。
HPLC进样检测，测试产物中的RD和RM的量，发现RD和RM的重量比为3:1。
实施例2～4
采用实施例1的方法，仅改变转化时的部分参数，得到产物，其中，改变的参数如表2所示。
表2
反应参数 实施例1 实施例2 实施例3 实施例4
OD600 100 80 120 110
莱鲍迪苷A浓度(g/L) 5 1 3 10
柠檬酸三钠浓度(mmol/L) 60 50 70 80
氯化锌浓度(mmol/L) 1 0.5 1.5 2
蔗糖浓度(％，w/v) 40 30 40 50
pH值 8.0 8.5 7.5 8.0
反应温度(℃) 37 35 38 40
反应时间(h) 24 20 28 30
测定实施例2～4制备的产物中的RD和RM的量，记录RD和RM的重量比，结果见表3。
表3
实施例编号 RD和RM的重量比
实施例1 3:1
实施例2 1.5:1
实施例3 4:1
实施例4 9:1
试验例1感官分析
参照采用BS ISO 4120感官分析方法学(三角试验)和GB 12311感官分析方法(三点检验)对甜味剂组合物进行测试评价分析，具体分析方法如下：
方法原理：同时向评价员提供一组三个样品，其中二个是完全相同的，评价员挑出单个的样品。
设备：检验负责人根据产品性质和样品数量等选择设备。使用的设备不应影响检验结果。优先使用符合检验需要的标准化设备。
抽样：按被检产品的抽样标准进行抽样。如果没有这样的标准或抽样标准不完全适用时，则由有关各方协商议定抽样方法。
环境：满足GB 10220所需条件。
评价员条件：应符合GB 10220规定的条件，所有评价员应该具有同等的资格和检验能力。
评价员数量：评价员数是根据检验目的与显著水平而定。通常是6个以上专家；或15个以上优选评价员；或25个以上初级评价员。在0.1％显著水平上需7个以上专家。
检验负责人：检验负责人一般不应参加检验，如果参加，也不应知道样品编号。检验负责人可就有关问题和样品性质进行不影响评价的初步介绍，当涉及检验玷染物时，应准备一个非玷染物样品和一个与之对照的玷染物样品。
被检样品的制备：提供足够量的样品A和B，每三个检验样品为一组。
按下述六种组合：ABB、AAB、ABA、BAA、BBA、BAB，从实验室样品中制备数目相等的样品组。
检验要求：不能使评价员从样品提供的方式中对样品的性质作出结论。应以同一方式〔相同设备、相同容器、相同数量产品和相同排列形式(三角形，直线等)〕制备各种检验样品组。任一样品组中，检验样品的温度是相同的，如可能，提供的检验系列中所有其他样品组的温度也应相同。盛装检验样品的容器应编号，一般是随机选取三位数。每次检验，编号应不同。
检验技术：告诉评价员检验目的，其程度应不使他们的结论产生偏倚。将制备的几组样品随机分配给评价员。评价员按规定次序检查各组检验样品，次序在同一系列检验中应相同。在评价同一组三个被检样品时，评价员对每种被检样品应有重复检验的机会。检验负责人在必要时可以告诉评价员提供的样品数量和体积。当评价员的数目不足6的倍数时，可采取下述两种方式。
a.舍弃多余样品组；
b.为每个评价员提供6组样品做重复检验。
当评价员不能鉴别其差异时，允许回答“无差异”。
结果的表达：
统计“有差异”或“无差异”答案数，当评价员人数n值大于100时，在不同显著水平上确定三点试验显著性差别所需答案的最少数目X按以下公式计算，取最接近的整数值。其中α是显著性水平，为一个预期值。
Figure PCTCN2018113715-appb-000001
式中Z的值根据显著性水平α而变化：
α≤0.05   Z＝1.64
α≤0.01   Z＝2.33
α≤0.001  Z＝3.10
在统计假设检验中，公认的小概率事件的概率值被称为统计假设检验的显著性水平，记为α。α的取值越小，此假设检验的显著性水平越高。比如α设为0.05，这意味着抽样分布中的95％的样本算作常态样本，两端5％的样本算作极端样本。一个样本要是落入95％的常态样本中，那么它就被看做来自这一总体，或者说它和总体中的其他样本的差异仅仅是抽样造成的偶然误差，没有统计上显著的差异。一个样本要落入5％的极端样本中，就可以拒绝它来自这一总体的论断，而认为它来自其它的总体，或者说它和这一总体的其它样本的差异不是抽样误差，具有统计上显著的差异。
在本实施例中，
“0.1％显著水平(α≤0.001)为有差异”对应“不相似”
“5％显著水平(α≤0.05)为无差异”对应“相似”
“1％显著水平(α≤0.01)为无差异”对应“非常相似”
“0.1％显著水平(α≤0.001)为无差异”对应“无差异”
“无差异”答案占有较大的比例时，说明两个样品的差异低于评价员觉察阈。
将以上评价分析方法应用于分析不同的组合物，对比样品为蔗糖，将样品和对比样品分别配成甜度相同的水溶液后按以上感官分析方法要求进行实验，有效评价人员：135个初级评价员，允许回答“无差异”。其口感比较试验结果见表4。
表4
Figure PCTCN2018113715-appb-000002
Figure PCTCN2018113715-appb-000003
表4中，DM 1:9混合物、DM 1:1混合物、DM 1:4混合物均采用RD(>95％)和RM(>95％)混合得到，DM3:1混合物、DM 1.5:1混合物、DM 4:1混合物和DM 9:1混合物为本发明实施例1～4中制备得到的。通过表4的数据表明：本发明的生产方法得到的产物，其口感与蔗糖相似。且当高纯莱鲍迪苷D(>95％)与高纯莱鲍迪苷M(>95％)配比范围(w/w)在1.5～9:1时，其口感与蔗糖相似。
试验例2
将莱鲍迪苷D和莱鲍迪苷M重量比3:1的混合物(DM 3:1混合物)，和莱鲍迪苷A(RA)按一定的重量比混合，得到甜味剂组合物。
按照实施例1中记载的评价分析方法，分析莱鲍迪苷A(>97％)、DM 3:1混合物、DM 3:1混合物-莱鲍迪苷A(1:9,w/w)、DM 3:1混合物-莱鲍迪苷A(9:1,w/w)、DM 3:1混合物-莱鲍迪苷A(3:7,w/w)，对比样品为蔗糖。将样品和对比样品分别配成甜度相同的水溶液后按以上感官分析方法要求进行实验，有效评价人员：135个初级评价员，允许回答“无差异”。其口感比较试验结果见表5。
表5
Figure PCTCN2018113715-appb-000004
通过上述数据表明：当莱鲍迪苷D-莱鲍迪苷M 3:1(w/w)的混合物与高纯莱鲍迪苷A(>97％)配比范围(w/w)在1:9～9:1时，其口感与蔗糖相似。
试验例3
将莱鲍迪苷D和莱鲍迪苷M重量比3:1的混合物(DM 3:1混合物)，和莱鲍迪苷A(RA)按一定的重量比混合，得到甜味剂组合物。
按照实施例1中记载的评价分析方法，分析莱鲍迪苷A(>97％)、DM 3:1混合物、DM 3:1混合物-莱鲍迪苷A(3:7,w/w)、DM 3:1混合物-莱鲍迪苷A(7:3,w/w)、DM 3:1混合物-莱鲍迪苷A(4:6,w/w)，对比样品为蔗糖。将样品和对比样品分别配成甜度相同的水溶液后按以上感官分析方法要求进行实验，有效评价人员：125个初级评价员，允许回答“无差异”。其口感比较试验结果见表6。
表6
Figure PCTCN2018113715-appb-000005
通过上述数据表明：当莱鲍迪苷D-莱鲍迪苷M 3:1(w/w)的混合物与高纯莱鲍迪苷A(>97％)配比范围(w/w)在3:7～7:3时，其口感与蔗糖非常相似。
试验例4
将莱鲍迪苷D和莱鲍迪苷M重量比3:1的混合物(DM 3:1混合物)，和莱鲍迪苷A(RA)按一定的重量比混合，得到甜味剂组合物。
按照实施例1中记载的评价分析方法，分析莱鲍迪苷A(>97％)、DM 3:1混合物、DM 3:1混合物-莱鲍迪苷A(4:6,w/w)、DM 3:1混合物-莱鲍迪苷A(6:4,w/w)、DM 3:1混合物-莱鲍迪苷A(5:5,w/w)，对比样品为蔗糖。将样品和对比样品分别配成甜度相同的水溶液后按以上感官分析方法要求进行实验，有效评价人员：128个初级评价员，允许回答“无差异”。其口感比较试验结果见表7。
表7
Figure PCTCN2018113715-appb-000006
通过上述数据表明：当莱鲍迪苷D-莱鲍迪苷M 3:1(w/w)的混合物与高纯莱鲍迪苷A(>97％)配比范围(w/w)在4:6～6:4时，其口感与蔗糖无差异。

Claims (12)

  1. 甜味剂组合物Ⅰ，其特征在于：由莱鲍迪苷D和莱鲍迪苷M组成，且按重量比，莱鲍迪苷D:莱鲍迪苷M＝1.5～9:1。
  2. 根据权利要求1所述的甜味剂组合物Ⅰ，其特征在于：按重量比，莱鲍迪苷D:莱鲍迪苷M＝3～9:1。
  3. 根据权利要求2所述的甜味剂组合物Ⅰ，其特征在于：按重量比，莱鲍迪苷D:莱鲍迪苷M＝3:1。
  4. 甜味剂组合物Ⅱ，其特征在于：由权利要求1或2所述的甜味剂组合物Ⅰ与莱鲍迪苷A混合而成，且按重量比，甜味剂组合物Ⅰ:莱鲍迪苷A＝1～9:1～9。
  5. 根据权利要求4所述的甜味剂组合物Ⅱ，其特征在于：按重量比，甜味剂组合物Ⅰ:莱鲍迪苷A＝3～7:3～7。
  6. 根据权利要求5所述的甜味剂组合物Ⅱ，其特征在于：按重量比，甜味剂组合物Ⅰ:莱鲍迪苷A＝4～6:4～6。
  7. 一种甜味剂组合物的生产方法，其特征在于：以莱鲍迪苷A为底物，以重组微生物为催化剂，在蔗糖、氯化锌和柠檬酸三钠的存在下，催化底物进行反应，得到甜味剂组合物，其中，重组微生物的菌体浓度OD600为80～120，莱鲍迪苷A浓度为1～80g/L；柠檬酸三钠浓度为50～80mmol/L，氯化锌浓度为0.5～2mmol/L，蔗糖浓度为30～50％(W/V)，pH值为7.5～8.5，所述重组微生物含有EUGT11编码基因和UGT76G1编码基因。
  8. 根据权利要求7所述的甜味剂组合物的生产方法，其特征在于：反应温度为35～40℃，反应时间为20～60h。
  9. 根据权利要求7所述的甜味剂组合物的生产方法，其特征在于：重组微生物菌体浓度OD600为100，莱鲍迪苷A浓度为5g/L；柠檬酸三钠浓度为60mmol/L，氯化锌浓度为1mmol/L，蔗糖浓度为40％(W/V)，pH值为8.0。
  10. 根据权利要求8或9所述的甜味剂组合物的生产方法，其特征在于：反应温度为37℃，反应时间为24h。
  11. 根据权利要求7所述的甜味剂组合物的生产方法，其特征在于：所述重组微生物为重组大肠杆菌、重组酵母菌、重组枯草芽孢杆菌、重组谷氨酸棒状杆菌或重组链霉菌。
  12. 权利要求7～11任一项所述的甜味剂组合物的生产方法生产得到的甜味剂组合物。
PCT/CN2018/113715 2018-09-29 2018-11-02 复合甜味剂及其生产方法 WO2020062437A1 (zh)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103974628A (zh) * 2012-05-22 2014-08-06 谱赛科有限责任公司 高纯度的甜菊醇糖苷
CN105051195A (zh) * 2013-02-06 2015-11-11 埃沃尔瓦公司 用于提高莱鲍迪苷d和莱鲍迪苷m之产生的方法
CN107404919A (zh) * 2015-03-11 2017-11-28 守田化学工业株式会社 甜味剂组合物及含有该组合物的食品
CN107949632A (zh) * 2015-05-29 2018-04-20 嘉吉公司 用于使用高pH生产甜菊醇糖苷的发酵方法和由此获得的组合物
WO2018112189A1 (en) * 2016-12-14 2018-06-21 The Coca-Cola Company Preparing novel steviol glycosides by bioconversion

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103974628A (zh) * 2012-05-22 2014-08-06 谱赛科有限责任公司 高纯度的甜菊醇糖苷
CN105051195A (zh) * 2013-02-06 2015-11-11 埃沃尔瓦公司 用于提高莱鲍迪苷d和莱鲍迪苷m之产生的方法
CN107404919A (zh) * 2015-03-11 2017-11-28 守田化学工业株式会社 甜味剂组合物及含有该组合物的食品
CN107949632A (zh) * 2015-05-29 2018-04-20 嘉吉公司 用于使用高pH生产甜菊醇糖苷的发酵方法和由此获得的组合物
WO2018112189A1 (en) * 2016-12-14 2018-06-21 The Coca-Cola Company Preparing novel steviol glycosides by bioconversion

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