Fermentation preparation method of sweetening agent

Abstract

The invention belongs to the technical field of biochemical engineering, and particularly relates to a fermentation preparation method of a sweetening agent. The method is based on a recombinant saccharomyces cerevisiae strain, a compound carbon source is adopted, the content of residual ethanol and the range of RQ are strictly controlled to feed back, regulate and control material supplementation so as to regulate and control a cell metabolic pathway, and meanwhile, a nonionic surfactant is added to realize large-scale stable output of a target sweetening agent. The method disclosed by the invention has the beneficial effects that the utilization rate of a carbon source is increased, the accumulation of intermediate products is reduced, the dynamic stability of cell membranes and the transmembrane transport capacity of cells are enhanced, and the yield of rubusoside and rebaudioside M in the sweetening agent is greatly increased; the problems of low yield, more byproducts, high cost, difficulty in later-stage purification treatment and the like which are difficult to meet industrial production in the prior art are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and in particular relates to a fermentation preparation method of a sweetener. Background Art

[0002] Sweeteners can improve taste and adjust flavor, and are widely used in the food, beverage and pharmaceutical industries. As health issues such as hyperlipidemia, diabetes and obesity are gaining more and more attention, natural non-nutritive sweeteners are becoming the main development trend of sweeteners.

[0003] Stevioside is a class of tetracyclic diterpene glycoside compounds found in stevia. Its sweetness is 114-350 times that of sucrose and its calorific value is only 1 / 300 of that of sucrose. As a natural non-nutritive sweetener that is highly favored in the food and pharmaceutical fields, it has been widely used.

[0004] Steviosides from stevia include more than 60 types such as stevioside, stevioside, and rebaudioside AO. Among them, stevioside and rebaudioside A account for a high proportion in stevia and have been commercialized. Rebaudioside M has the advantages of higher sweetness, better taste, and no aftertaste, and the market demand for it is increasing year by year. In addition, stevioside, which also accounts for a relatively low proportion in stevia, has the effects of anti-cancer, lowering blood sugar, and balancing metabolic disorders caused by a high-sugar, high-fat diet, and has a high medicinal value.

[0005] Therefore, the development and application of rare steviol glycosides, such as stevioside and rebaudioside M, have become a research hotspot in the field of natural non-nutritive sweeteners.

[0006] At present, there are two main ways to obtain rubusoside and rebaudioside M: plant extraction and microbial synthesis. The plant extraction process mainly uses the corresponding plant dry leaves as raw materials, and obtains the product through soaking extraction, filtration and impurity removal, resin adsorption, elution, decolorization, crystallization and other processes. Rubusoside is the most abundant in the dry leaves of Chinese sweet tea, but it only accounts for 5%, and the proportion of rebaudioside M in stevia dry leaves is less than 1%, which makes the plant extraction method have many problems in terms of raw material sources, environmental protection, product safety, etc., and the production cost is high.

[0007] As for the microbial synthesis method, it mainly includes whole-cell catalysis and enzyme catalysis. Generally, recombinant Escherichia coli or recombinant Saccharomyces cerevisiae cells are used, and the corresponding substrates are added to obtain products through cell catalysis or enzyme conversion, and then the final product is obtained through extraction and crystallization. However, the substrate of rubusoside is generally steviol or stevioside, and the substrate of rebaudioside M is generally rebaudioside A, rebaudioside D or rebaudioside E. Due to the limitation of the substrate acquisition method, the microbial synthesis method also faces high production cost pressure and safety and environmental protection pressure.

[0008] The de novo synthesis of steviol glycosides using cell factories has the advantages of lower cost and higher yield. Recombinant microorganisms that can synthesize stevioside and rebaudioside M from scratch have been obtained. For example, the recombinant Saccharomyces cerevisiae strain obtained through synthetic biology strategies can express multiple enzymes involved in regulating the synthesis pathway of steviol glycosides, open up the complete biosynthetic pathway from glucose to stevioside and rebaudioside M, and achieve rebalancing of the metabolic network.

[0009] Although a cell factory for the de novo synthesis of rubusoside and rebaudioside M has been successfully constructed, the current fermentation method has problems such as unstable cell growth, low product yield, and a large number of by-products, making it difficult to meet the requirements of industrial production. Summary of the invention

[0010] In order to solve the above technical problems, the present invention provides a fermentation preparation method of a sweetener.

[0011] The method provided by the present invention is based on a recombinant saccharomyces cerevisiae strain, uses a basic fermentation medium for fermentation, and timely adjusts the addition of a supplementary medium during the fermentation process to strictly control the residual ethanol concentration in the fermentation process within a range of 0.01 to 1.5 g / L, and the cell respiration entropy within a range of 0.6 to 2.0, so that the cell metabolic pathway flows more toward the synthesis of the target product, thereby obtaining a sweetener; The sweeteners include but are not limited to betaine and rebaudioside M; The basic fermentation medium includes a carbon source, a nitrogen source, inorganic salts, trace metal elements, and vitamins; The carbon source includes but is not limited to glucose, sucrose, glycerol, fructose, sugarcane molasses, and ethanol; The vitamin is selected from at least one of thiamine hydrochloride and inositol, and the vitamin concentration is 1 to 4 g / L; In the supplemented culture medium, the weight ratio of glucose: sugarcane molasses: ethanol: water is 0-6:3-4:3-5:1-4.

[0012] Preferably, the sweetener is a combination of betaine and rebaudioside M.

[0013] Preferably, during the fermentation process, the residual ethanol concentration is controlled within the range of 0.01-1.0 g / L, and the cell respiration entropy is within the range of 0.6-1.2.

[0014] More preferably, during the fermentation process, the residual ethanol concentration is controlled within the range of 0.01-0.8 g / L, and the cellular respiration entropy is within the range of 0.6-0.8.

[0015] As further preferred, the components of the basic fermentation medium are as follows: Glucose 0~30 g / L, cane molasses 0~40 g / L, yeast extract powder 8~20 g / L, peptone 10~30 g / L, potassium dihydrogen phosphate 10~15 g / L, ammonium sulfate 8~15 g / L, magnesium sulfate 2~5 g / L, thiamine hydrochloride 1~2 g / L, inositol 1~2 g / L, FeSO4·7H2O 0.1~1.0 g / L, Zn SO4·7 H2O 0.1~1.0 g / L, MnSO4·H2O 0.001~0.1 g / L, defoaming agent 0.05~0.2 g / L.

[0016] As further preferred, if the carbon source is glucose, the concentration of glucose is 10-30 g / L; if the carbon source is sugarcane molasses, the concentration of sugarcane molasses is 20-40 g / L.

[0017] As further preferred, in the supplemented medium, the weight ratio of each component is glucose: sugarcane molasses: ethanol: water = 1:4:3:2; Alternatively, the weight ratio of the components is glucose: sugarcane molasses: ethanol: water = 0:3:5:2.

[0018] As a further preferred embodiment, during the fermentation process, when the OD of the fermentation liquid is 600 When the value is ≥50, add nonionic surfactant to the fermentation system.

[0019] Preferably, the equilibrium value of the nonionic surfactant is 10-15, and the concentration of the nonionic surfactant is 0.1-10 g / L.

[0020] As further preferred, the nonionic surfactant is selected from any one of Tween-100, Tween-80, and fatty alcohol polyoxyethylene ether-9 (AEO-9).

[0021] More preferably, the nonionic surfactant is AEO-9.

[0022] Furthermore, the present invention provides a method for preparing a sweetener by fermentation, which specifically comprises the following steps: (1) After activating the recombinant Saccharomyces cerevisiae strain, inoculate it into the seed culture medium at an inoculum rate of 5-10% based on the volume of the seed culture medium, and culture it at 25-35°C and 200-300 rpm for 24-36 h to obtain a seed solution; (2) inoculating the seed solution obtained by culturing in (1) into the basic fermentation medium at an inoculation rate of 10-20% based on the volume of the fermentation medium, and supplementing the medium for batch fermentation. During the entire fermentation process, the temperature is controlled at 25-35° C., the pH is maintained at 5.0-5.8 with aqueous ammonia, the dissolved oxygen is controlled at 20%-50%, and the weight ratio of glucose: sugarcane molasses: ethanol: water in the supplementing medium is 0-6:3-4:3-5:1-4; (3) During the fermentation process, when both the dissolved oxygen and pH suddenly increased, the culture medium was added to control the residual ethanol concentration in the range of 0.01-1.5 g / L and the cell respiration entropy in the range of 0.6-2.0. (4) When the OD of the fermentation liquid 600 When the value is ≥50, a sterilized nonionic surfactant is added to the fermentation system, wherein the equilibrium value of the nonionic surfactant is 10-15 and the concentration of the nonionic surfactant is 0.1-10 g / L; (5) The sweetener content is measured by liquid chromatography-mass spectrometry. When the sweetener content in the fermentation broth no longer increases, the fermentation is stopped.

[0023] The above-mentioned technical scheme of the present invention is used to produce rubusoside and rebaudioside M, which overcomes the problems in the prior art that are difficult to meet industrial production, such as unstable cell growth, low yield, many by-products, high cost, and difficulty in subsequent purification treatment.

[0024] The beneficial effects of the present invention are: (1) The use of a composite carbon source of glucose / sugarcane molasses / ethanol in the supplementary medium and the addition of sugarcane molasses and inositol to the basal fermentation medium can effectively avoid the anti-Pasteur effect, improve energy utilization and increase cell stability, which greatly increases biomass and product yields, especially the yield of rubusoside, which reaches more than 13 g / L. (2) During the fermentation process, the ethanol concentration range and RQ range are strictly limited, and the metabolic pathway is regulated at the cellular level to flow more toward the synthesis of rebaudioside M, which effectively reduces by-products and increases the proportion of rebaudioside M in the product, thereby reducing production costs and increasing product yields; (3) The addition of non-ionic surfactants during the fermentation process enhanced the dynamic stability of the cell membrane, promoted the transmembrane transport of rubusoside, and reduced the accumulation of intermediates. At the end of the fermentation, the yield of rebaudioside M reached 5.15 g / L, which greatly improved the yield and reduced the difficulty of subsequent purification treatment. At the same time, the biomass was reduced, which improved the utilization efficiency of the fermentation raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The graph is a graph showing the yield of the target product under different ratios of carbon sources in Examples 1-4 of the present invention; Figure 2 This is a graph showing the effect of narrowing the range of ethanol and RQ control on the yield and ratio of the target product in Examples 4-6 of the present invention; Figure 3 This is a graph showing the yield of the product after adding a nonionic surfactant in Example 7 of the present invention. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0027] Example 1 A fermentation preparation method of a sweetener (a combination of rubusoside and rebaudioside M) comprises the following steps: (1) The activated recombinant Saccharomyces cerevisiae strain 63SP was inoculated into a 1 L shake flask containing 200 mL of seed culture medium at an inoculation ratio of 5% (V / V) and cultured at 30°C and 220 rpm for 24 h. The composition of the seed culture medium is shown in Table 1 below.

[0028] Table 1 Seed culture medium Components Yeast extract powder Peptone glucose concentration 10 g / L 20 g / L 20 g / L

[0029] (2) The seed liquid obtained by culturing in (1) was inoculated into 4 L of basic fermentation medium at an inoculation ratio of 10% (V / V), and fed-batch fermentation was carried out in a 10 L Shanghai Guoqiang FUS10-1619 fermenter. During the entire fermentation process, the temperature was controlled at 30°C, the pH was maintained at 5.5 with ammonia water, the air flow was always maintained at 1 vvm, the tank pressure was 0.03 MPa, the initial stirring speed was 200 rpm, and the stirring speed was linked to the dissolved oxygen. The dissolved oxygen was controlled at 20%-50% by adjusting the speed.

[0030] The basic fermentation medium used is prepared on the basis of the traditional brewer's yeast culture medium in the art. The components of the basic fermentation medium are shown in Table 2 below.

[0031] Table 2 Basic fermentation medium Components Concentration (g / L) Components Concentration (g / L) glucose 20 Thiamine Hydrochloride 1 Yeast extract powder 10 Inositol 1 Peptone 20 FeSO4·7H2O 0.5 Potassium dihydrogen phosphate 10 ZnSO4·7H2O 0.5 Ammonium sulfate 10 MnSO4·H2O 0.005 Magnesium sulfate 4 Defoaming agent 0.1

[0032] (3) During the fermentation process, when both the dissolved oxygen and pH values ​​suddenly increased, the supplementary culture medium was added. The flow rate of the supplementary culture medium was feedback-controlled based on two parameters: the ethanol concentration during the fermentation process and the cellular respiration entropy (RQ). By adjusting the flow rate of the supplementary culture medium, the ethanol concentration was controlled within the range of 0.01-1.5 g / L, and the RQ was controlled within the range of 0.6-1.8.

[0033] Among them, ethanol concentration was measured using Silman M-100 biosensor analyzer; RQ uses Sunny Optical SHP8400PMS process gas mass spectrometer to measure the oxygen concentration and carbon dioxide concentration in the fermentation tail gas in real time, and then combines the fermentation liquid volume and the air flow rate to calculate the real-time oxygen uptake rate and carbon dioxide release rate and RQ through the built-in program of the tail gas analysis system.

[0034] The supplemented culture medium used was prepared according to Table 3 below.

[0035] Table 3 Supplementary culture medium components Components glucose water Ratio (w / w) 60% 40%

[0036] (4) Two methods were used to evaluate the growth of bacteria during the fermentation process.

[0037] One of them is to measure the absorbance (OD) of the diluted fermentation broth at a wavelength of 600 nm using a spectrophotometer. 600 value) to estimate the concentration of Saccharomyces cerevisiae cells in the fermentation broth; another method is to determine the dry cell weight (DCW), which is to centrifuge the fermentation broth, collect the wet cells, wash the wet cells with deionized water three times, dry them in an oven at 105℃ for 24h, and finally weigh the mass of the obtained dry matter.

[0038] (5) Detect the production of rubusoside and rebaudioside M in the fermentation broth using liquid chromatography-mass spectrometry. The specific operation is as follows: Pretreatment of fermentation broth: First, the fermentation broth was crushed with a high-pressure homogenizer, and the pressure was set to 700 Bar. Then, the crushed fermentation broth was centrifuged, and 4 times the volume of acetonitrile was added to the supernatant to remove impurities, and then the supernatant was centrifuged for later use.

[0039] LC-MS detection method, see Table 4 below.

[0040] Table 4 LC-MS detection method

[0041] At the end of the three experiments, the statistical results of the yield and biomass of the target products rubusoside and rebaudioside M are shown in Table 5 below.

[0042] Table 5 Biomass and yield of target products <![CDATA[OD 600 Value]]> Cell dry weight (g / L) Rubusoside production (mg / L) Rebaudioside M production (mg / L) 1 56 23.5 2874.3 154.1 2 50 21.0 2567 137.6 3 45 18.9 2309.7 123.8

[0043] Example 2 A fermentation preparation method for a sweetener, the specific steps are as follows: (1) The inoculum size used in seed culture was 10%, and the strains, seed culture medium and seed culture conditions used were the same as those in Example 1; (2) The fermentation inoculation ratio was 15%, and the rest of the fermentation temperature, pH, air flow, pressure, initial rotation speed, and dissolved oxygen control method were the same as those in Example 1; The basic fermentation medium was different from that in Example 1, wherein the dosage of thiamine hydrochloride and inositol was 2 g / L, glucose was discarded and 30 g / L of sugarcane molasses was added; (3) During the fermentation process, the ethanol concentration and RQ control ranges were the same as those in Example 1; The supplemented culture medium used was prepared according to Table 6 below.

[0044] Table 6 Supplementary culture medium components Components glucose Sugarcane molasses water Ratio (w / w) 40% 40% 20%

[0045] The method for evaluating the growth status of the bacteria and the method for detecting the yield of the target products rubusoside and rebaudioside M are the same as those in Example 1.

[0046] The statistical results of the yield and biomass of the target products rubusoside and rebaudioside M at the end of the three experiments are shown in Table 7 below.

[0047] Table 7 Biomass and target product yield <![CDATA[OD 600 Value]]> Cell dry weight (g / L) Rubusoside production (mg / L) Rebaudioside M production (mg / L) 1 80 31.5 7846.6 308.2 2 76 29.9 7454.3 292.8 3 81 32.0 7944.7 312.1

[0048] Example 3 A fermentation method for preparing a sweetener, the specific steps of which are different from those of Example 2 in that the supplementary culture medium used in (3) is prepared according to Table 8 below.

[0049] Table 8 Supplementary culture medium components Components glucose Sugarcane molasses Ethanol water Ratio (w / w) 10% 40% 30% 20%

[0050] The detection method for evaluating the bacterial growth status and the yield of the target products rubusoside and rebaudioside M is the same as that in Example 1.

[0051] The statistical results of the target product yield and biomass at the end of the three experiments are shown in Table 9 below.

[0052] Table 9 Biomass and yield of target products <![CDATA[OD 600 Value]]> Cell dry weight (g / L) Rubusoside production (mg / L) Rebaudioside M production (mg / L) 1 83 32.7 11946.3 617.9 2 77 30.3 11082.7 573.3 3 85 33.5 12234.1 632.8

[0053] Example 4 A method for preparing a sweetener by fermentation, wherein the specific operation is different from that of Example 2 in that the supplementary culture medium used in (3) is prepared according to Table 10 below.

[0054] Table 10 Supplementary medium components Components Sugarcane molasses Ethanol water Ratio (w / w) 30% 50% 20%

[0055] The detection method for evaluating the bacterial growth status and the yield of the target products rubusoside and rebaudioside M is the same as that in Example 1.

[0056] The statistical results of the target product yield and biomass at the end of the three experiments are shown in Table 11 below.

[0057] Table 11 Biomass and yield of target products <![CDATA[OD 600 Value]]> Cell dry weight (g / L) Rubusoside production (mg / L) Rebaudioside M production (mg / L) 1 95 35.8 12634.8 1016.8 2 91 34.3 12102.8 974.0 3 97 36.6 12900.8 1038.2

[0058] In Examples 1-4, different types of supplementary culture media were added during the fermentation process. The yields of the target products rubusoside and rebaudioside under different ratios of carbon sources are shown in the attached table. Figure 1 shown.

[0059] Example 5 A fermentation method for preparing a sweetener, which is different from Example 4 in that: In (3), the ethanol concentration is controlled at 0.01-1.0 g / L by adjusting the flow rate of the supplementary medium, and the RQ is controlled at 0.6-1.2 at the same time. The components of the supplementary medium are the same as those in Example 4.

[0060] The detection method for evaluating the bacterial growth status and the yield of the target products rubusoside and rebaudioside M is the same as that in Example 1.

[0061] The statistical results of the target product yield and biomass at the end of the three experiments are shown in Table 12 below.

[0062] Table 12 Biomass and yield of target products <![CDATA[OD 600 Value]]> Cell dry weight (g / L) Rubusoside production (mg / L) Rebaudioside M production (mg / L) 1 96 36.5 13301.9 1840.8 2 91 34.6 12609.1 1745.0 3 98 37.3 13579.0 1879.2

[0063] Example 6 A fermentation method for preparing a sweetener, which is different from Example 4 in that: In (3), the ethanol concentration is controlled at 0.01-0.8 g / L by adjusting the flow rate of the supplementary medium, and the RQ is controlled at 0.6-0.8 at the same time. The components of the supplementary medium are the same as those in Example 4.

[0064] The detection method for evaluating the bacterial growth status and the yield of the target products rubusoside and rebaudioside M is the same as that in Example 1.

[0065] The statistical results of the target product yield and biomass at the end of the three experiments are shown in Table 13 below.

[0066] Table 13 Biomass and target product yield <![CDATA[OD 600 Value]]> Cell dry weight (g / L) Rubusoside production (mg / L) Rebaudioside M production (mg / L) 1 101 38.9 13895.4 2810.7 2 95 36.6 13069.3 2643.7 3 98 37.7 13482.7 2727.2

[0067] In Examples 4-6, the influence of reducing the range of ethanol and RQ control on the target products rubusoside and rebaudioside M is shown in the attached Figure 2 shown.

[0068] Example 7 A fermentation method for preparing a sweetener, which is different from Example 4 in that: (2) The inoculation ratio of the medium fermentation was 20%, and the amount of sugarcane molasses in the basic fermentation medium was 34 g / L; (3) During the fermentation process, the ethanol concentration was controlled between 0.01 and 0.8 g / L by adjusting the flow rate of the supplementary medium, and the RQ was controlled between 0.6 and 0.8. (4) When the OD of the fermentation liquid 600 When the value is ≥50, add the sterilized nonionic surfactant AEO-9 to the fermentation system to make the final concentration of AEO-9 in the fermentation broth ≤10g / L.

[0069] The detection method for evaluating the bacterial growth status and the yield of the target products rubusoside and rebaudioside M is the same as that in Example 1.

[0070] The statistical results of the target product yield and biomass at the end of the three experiments are shown in Table 14 below.

[0071] Table 14 Biomass and yield of target products <![CDATA[OD 600 Value]]> AEO-9 concentration (g / L) Cell dry weight (g / L) Rubusoside production (mg / L) Rebaudioside M production (mg / L) 1 96 1 36.7 13168.5 4201.0 2 87 4 33.9 12698.6 5151.5 3 51 8 23.3 10505.1 1976.5

[0072] In this example, nonionic surfactant AEO-9 was added during the fermentation process. The yield changes of the target products rubusoside and rebaudioside M obtained by fermentation are shown in the attached Figure 3 shown.

[0073] The above experimental results show that after the non-ionic surfactant AEO-9 was added during the fermentation process in this embodiment, the yields of the target products rubusoside and rebaudioside M were increased, especially the yield of rebaudioside M was significantly increased.

Claims

1. A fermentation method for preparing a sweetener, characterized in that: The method is based on a recombinant Saccharomyces cerevisiae strain, using a basic fermentation medium for fermentation, and during the fermentation process, by adding a supplementary medium to strictly control the concentration of residual ethanol within the range of 0.01-1.5 g / L and the cell respiration entropy within the range of 0.6-2.0, to obtain a sweetener; The sweeteners include but are not limited to betaine and rebaudioside M; The basic fermentation medium includes a carbon source, a nitrogen source, inorganic salts, trace metal elements, and vitamins; The carbon source includes but is not limited to glucose, sucrose, glycerol, fructose, sugarcane molasses, and ethanol; The vitamin is selected from at least one of thiamine hydrochloride and inositol, and the vitamin concentration is 1 to 4 g / L; In the supplemented culture medium, the weight ratio of glucose: sugarcane molasses: ethanol: water is 0-6:3-4:3-5:1-4.

2. The fermentation method for preparing a sweetener according to claim 1, characterized in that: The components of the basic fermentation medium are as follows: Glucose 0~30 g / L, cane molasses 0~40 g / L, yeast extract powder 8~20 g / L, peptone 10~30 g / L, potassium dihydrogen phosphate 10~15 g / L, ammonium sulfate 8~15 g / L, magnesium sulfate 2~5 g / L, thiamine hydrochloride 1~2 g / L, inositol 1~2 g / L, FeSO4·7H2O 0.1~1.0 g / L, Zn SO4·7 H2O 0.1~1.0 g / L, MnSO4·H2O 0.001~0.1 g / L, defoaming agent 0.05~0.2 g / L.

3. The fermentation method for preparing a sweetener according to claim 1, characterized in that: During the fermentation process, when the OD 600 When the value is ≥50, add nonionic surfactant to the fermentation system.

4. The fermentation method for preparing a sweetener according to claim 3, characterized in that: The equilibrium value of the nonionic surfactant is 10-15, and the concentration of the nonionic surfactant is 0.1-10 g / L.

5. The fermentation method for preparing a sweetener according to claim 1, characterized in that: The steps include: (1) After activating the recombinant Saccharomyces cerevisiae strain, inoculate it into the seed culture medium at an inoculum rate of 5-10% based on the volume of the seed culture medium, and culture it at 25-35°C and 200-300 rpm for 24-36 h to obtain a seed solution; (2) inoculating the seed solution obtained by culturing in (1) into the basic fermentation medium at an inoculation rate of 10-20% based on the volume of the fermentation medium, and supplementing the medium for batch fermentation. During the entire fermentation process, the temperature is controlled at 25-35° C., the pH is maintained at 5.0-5.8 with aqueous ammonia, the dissolved oxygen is controlled at 20%-50%, and the weight ratio of glucose: sugarcane molasses: ethanol: water in the supplementing medium is 0-6:3-4:3-5:1-4; (3) During the fermentation process, when both the dissolved oxygen and pH suddenly increased, the culture medium was added to control the residual ethanol concentration in the range of 0.01-1.5 g / L and the cell respiration entropy in the range of 0.6-2.

0. (4) When the OD of the fermentation liquid 600 When the value is ≥50, a sterilized nonionic surfactant is added to the fermentation system, wherein the equilibrium value of the nonionic surfactant is 10-15 and the concentration of the nonionic surfactant is 0.1-10 g / L; (5) The sweetener content is measured by liquid chromatography-mass spectrometry. When the sweetener content in the fermentation broth no longer increases, the fermentation is stopped.

Images