A viscosity-reducing and efficiency-increasing fermentation method for high polysaccharide-producing yeast

Abstract

The application provides a viscosity-reducing and synergistic fermentation method of high polysaccharide-producing yeast. The method is as follows: (1) inoculating the high polysaccharide-producing yeast as the main producing strain into a synergistic fermentation medium to start fermentation; (2) inoculating the liamocins-producing Aureobasidium pullulans as the auxiliary strain into the fermentation system to perform synergistic fermentation at 8-18 hours after the start of fermentation; (3) adding a sucrose solution to the fermentation system from 28-44 hours after the start of fermentation, adjusting the pH to 4.8-5.5, and obtaining the fermentation liquor after 60-80 hours of fermentation. The application firstly inoculates the high polysaccharide-producing yeast as the main producing strain and the liamocins-producing Aureobasidium pullulans as the auxiliary strain to perform staggered time inoculation and co-culture, forms the metabolic division of labor of sugar production by the main strain and viscosity reduction by the auxiliary strain. The auxiliary strain synthesizes liamocins by using the metabolic by-products and sucrose of the main producing strain, in-situ reduces the viscosity of the fermentation liquor, does not need to add exogenous chemical reagents, and realizes low addition and self-regulation.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to a method for reducing viscosity and enhancing efficiency of high-polysaccharide-producing yeast fermentation. Background Technology

[0002] Yeast extracellular polysaccharides (EPS) are long-chain polymers secreted by yeast during their growth and metabolism. Among them, β-1,3-glucan, a representative of active polysaccharides, possesses significant biological activities such as immunomodulation and antioxidant activity, and has broad application prospects in food preservation, functional dairy products, and pharmaceuticals. Kluyveromyces martensii, a recognized safe (GRAS) industrial strain, is an important chassis cell for EPS production. However, in the later stages of fermentation, when the EPS yield of a single high-yield yeast reaches 20-30 g / L, the fermentation broth exhibits typical non-Newtonian pseudoplastic fluid characteristics, with the apparent viscosity rising sharply to over 6000 mPa·s. This leads to a decrease in the gas-liquid mass transfer coefficient of over 80%, dissolved oxygen levels falling below the critical oxygen concentration, and the cell metabolism shifting from aerobic respiration to anaerobic fermentation. The activity of polysaccharide synthases is inhibited, and the yield caps at around 30 g / L, unable to break through further.

[0003] To address the aforementioned viscosity bottleneck, existing technologies mainly employ the following strategies: (1) Mechanical enhancement, such as increasing stirring speed and aeration rate, but this significantly increases energy consumption and high shear force may damage the bacterial cells; (2) Exogenous addition of chemical viscosity reducers, such as Tween-80, polyethylene glycol, and other surfactants, but there is a risk of chemical residues, which does not meet the safety standards for food-grade and pharmaceutical-grade polysaccharides, and increases raw material costs by 15% to 20%, with complex subsequent purification processes; (3) Genetically modified strains, which can reduce viscosity by knocking out hyphal morphology genes or heterologously expressing surfactant synthesis genes through gene editing, but these are genetically modified microorganisms, facing dual obstacles of food safety regulatory review and public acceptance, resulting in long industrialization cycles and high approval costs. All of the above methods are passive viscosity reduction strategies and fail to achieve in-situ, self-regulating viscosity during fermentation.

[0004] In addition, high-polysaccharide yeast fermentation produces a large amount of metabolic byproducts such as ethanol, glycerol, and organic acids, which are discharged as waste in traditional processes. This results in a carbon source loss of about 15% to 25% and increases the burden of wastewater treatment, leading to a significant waste of metabolic byproducts.

[0005] Biosurfactants are amphiphilic molecules produced by microbial metabolism, possessing advantages such as low toxicity, biodegradability, and environmental friendliness. Liamocins are a class of polyol ester biosurfactants secreted by *Aureobasidium pullulans*, composed of hydrophilic polyol head groups (such as mannitol) and lipophilic O-acetylated 3,5-dihydroxydecanoic acid chains, exhibiting good surface activity, antibacterial, and emulsifying functions. *Aureobasidium pullulans* can synthesize liamocins using inexpensive carbon sources such as glucose and sucrose, and they exhibit specific inhibitory effects against pathogenic bacteria such as streptococci. However, current research mainly focuses on the fermentation and product extraction of liamocins by single strains, and there are no reports of using liamocins as an auxiliary bacteria for synergistic fermentation with high-polysaccharide-producing yeasts to achieve in-situ viscosity reduction and efficiency enhancement, let alone co-culture strategies that utilize the metabolic byproducts of the main producing bacteria as carbon sources for auxiliary bacteria to achieve metabolite resource utilization.

[0006] Therefore, developing a synergistic fermentation method that eliminates the need for exogenous chemical reagents, utilizes endogenous biosurfactants synthesized by auxiliary bacteria to achieve in-situ viscosity reduction, and simultaneously utilizes metabolic byproducts to improve polysaccharide yield and quality is of great significance for promoting the green and efficient production of yeast extracellular polysaccharides. Summary of the Invention

[0007] Technical Problem to be Solved: To address the aforementioned technical problems, the purpose of this invention is to provide a high-polysaccharide-producing yeast fermentation method for reducing viscosity and enhancing efficiency. This method, for the first time, proposes using a high-polysaccharide-producing yeast as the main producing strain, focusing on extracellular polysaccharide synthesis; and using a naturally occurring liamocins-producing short-stem fungus as an auxiliary strain, focusing on biosurfactant synthesis. The two strains are co-cultured with staggered inoculation times, establishing a metabolic division of labor where the main strain produces sugar and the auxiliary strain reduces viscosity. The auxiliary strain utilizes the metabolic byproducts of the main producing strain—ethanol, glycerol, and organic acids—as auxiliary carbon sources, and supplements this with sucrose as the main carbon source for liamocins synthesis, naturally synthesizing liamocins and reducing the viscosity of the fermentation broth in situ. This achieves low-addition and self-regulating results without the need for any external chemical reagents.

[0008] Technical solution: A method for reducing viscosity and enhancing fermentation efficiency of high-yield polysaccharide yeast, comprising the following steps: (1) Inoculate high-polysaccharide-producing yeast into a co-fermentation medium to start fermentation, wherein the inoculation amount of the main producing yeast is 3% to 8% of the fermentation liquid volume; (2) 8 to 18 hours after the start of fermentation, budding short-stem mold that produces liamocins is inoculated into the fermentation system as an auxiliary bacteria for co-fermentation. The amount of auxiliary bacteria inoculated is 1% to 5% of the fermentation liquid volume. The ratio of viable bacteria of the main producing bacteria to the auxiliary bacteria at the time of inoculation is 2:1 to 6:1. The early stage of fermentation is the polysaccharide synthesis period of the main producing bacteria. The main producing bacteria consume glucose to synthesize extracellular polysaccharides and at the same time produce ethanol, glycerol and organic acid metabolic byproducts. The auxiliary bacteria adapt to the co-culture environment and use the metabolic byproducts to start the initial synthesis of liamocins. The viscosity of the fermentation liquid is allowed to rise naturally to 2000 to 5000 mPa·s as polysaccharides accumulate. (3) The middle and late stages of fermentation are the viscosity reduction period. Starting 28 to 44 hours after the start of fermentation, sucrose solution is added to the fermentation system to adjust the pH to 4.8 to 5.5. Sucrose serves as the exclusive carbon source for the auxiliary bacteria to synthesize liamocins. The auxiliary bacteria use sucrose and residual metabolic byproducts to synthesize liamocins in large quantities. When the concentration of liamocins exceeds the critical micelle concentration, liamocins, as an amphiphilic biosurfactant, reduces the viscosity of the fermentation broth through a triple mechanism: reducing surface tension to improve gas-liquid mass transfer, competitively forming hydrogen bonds with the hydroxyl groups of polysaccharide chains to weaken the intermolecular hydrogen bond network, and the steric hindrance of micelles to prevent entanglement between polysaccharide chains. After 60 to 80 hours of fermentation, the fermentation broth is obtained, and the final apparent viscosity of the fermentation broth is less than 4000 mPa·s.

[0009] Furthermore, the high-polysaccharide-producing yeast mentioned in step (1) is Kluyveromyces marxianus, with accession number CGMCC NO.39233, deposited by the China General Microbiological Culture Collection Center, and deposited on June 15, 2026.

[0010] Furthermore, the inoculation amount of the main producing bacteria in step (1) is 4% to 6% of the fermentation liquid volume.

[0011] Furthermore, the co-fermentation medium described in step (1) comprises: 60-100 g / L glucose, 1.5-3.0 g / L ammonium sulfate, 1.0-2.0 g / L potassium dihydrogen phosphate, 0.3-1.0 g / L magnesium sulfate heptahydrate, 0.1-0.5 g / L calcium chloride, 5-15 mL / L vegetable oil, 2.0-4.0 g / L citric acid, 1.0-3.0 g / L sodium citrate, and an initial pH of 5.8-6.5.

[0012] Furthermore, the fermentation temperature in step (1) is 26~30℃, the pH is 5.8~6.5, the aeration rate is 0.8~1.2vvm, and the stirring speed is 150~250 rpm.

[0013] Furthermore, the inoculation time of the auxiliary bacteria in step (2) is 12 to 18 hours after the start of fermentation, and the inoculation amount is 2% to 3% of the fermentation liquid volume.

[0014] Furthermore, the co-fermentation conditions described in step (2) are: temperature 24~28℃, pH 5.3~6.0, dissolved oxygen maintained at 15%~35% saturation, aeration rate 1.0~1.5 vvm, and stirring speed 180~280 rpm.

[0015] Furthermore, the start time for adding sucrose in step (3) is 30 to 42 hours after the start of fermentation.

[0016] Furthermore, in step (3), during the viscosity reduction period after the addition of sucrose, the temperature is 24~28℃, the dissolved oxygen is maintained at 35%~65% saturation, the aeration rate is 1.2~2.0 vvm, and the stirring speed is 150~220 rpm.

[0017] Furthermore, the total amount of sucrose added in step (3) is 25~50 g / L, and the flow rate is controlled to maintain the sucrose concentration in the fermentation broth at 5~20 g / L.

[0018] Furthermore, in step (3), a citric acid-sodium citrate buffer system is used to maintain the pH.

[0019] Furthermore, it also includes an online viscosity monitoring step: when the apparent viscosity of the fermentation broth exceeds 4500~5500 mPa·s, the sucrose flow acceleration rate is increased in advance.

[0020] Furthermore, it also includes product separation and purification steps: (a) Centrifuge the fermentation broth from step (3) of claim 1 at 6000~10000 rpm and 2~6℃ for 10~20 minutes to remove the cells and take the supernatant; let the supernatant stand for 4~12 hours, collect the liamocins sediment oil layer at the bottom, and remove residual cell debris by microfiltration of the middle supernatant at 0.22~0.45 μm; (b) The supernatant of microfiltration is concentrated under reduced pressure to 1 / 4 to 1 / 2 of the original volume, 2 to 4 times the volume of 90% to 96% ethanol is added and pre-cooled, and the mixture is allowed to stand at 0 to 6°C for 8 to 16 hours. The precipitate is collected by centrifugation to obtain crude polysaccharide and supernatant. (c) The supernatant from step (b) is combined with the liamocins sediment oil layer from step (a), and ethanol is added to a final concentration of 80% to 90%. After standing at low temperature, the precipitate is collected by centrifugation as a mixture of liamocins and low molecular weight oligosaccharides. (d) The crude polysaccharide was washed with anhydrous ethanol 1-3 times and then freeze-dried under vacuum to obtain the polysaccharide component; the mixture of liamocins and low molecular weight oligosaccharides was washed with n-hexane 1-2 times to remove residual vegetable oil, and then washed with anhydrous ethanol 1-2 times and then dried under vacuum to obtain the liamocins component.

[0021] Furthermore, the method for preparing the seed culture of the main producing bacteria is as follows: the main producing bacteria are inoculated into the seed culture medium and cultured at 26-30℃ and 150-220 rpm for 16-26 hours with shaking. The OD of the seed culture is then measured. 600 The pH value reaches 6-15; the seed culture medium contains: glucose 25-40 g / L, yeast extract 3-8 g / L, peptone 3-8 g / L, potassium dihydrogen phosphate 0.5-2.0 g / L, magnesium sulfate heptahydrate 0.2-1.0 g / L, pH 5.8-6.5.

[0022] Furthermore, the preparation method of the auxiliary bacterial seed culture is as follows: the auxiliary bacteria are inoculated into the seed culture medium and cultured at 26~30℃ and 150~250 rpm for 36~60 hours with shaking. The biomass of the seed culture is not less than 6 g / L dry weight, and the proportion of yeast cells is not less than 75%. The seed culture medium contains: glucose 15~30 g / L, tryptone 15~25 g / L, yeast extract 8~15 g / L, pH 5.8~6.5.

[0023] The present invention also provides a fermentation product prepared by the above method, wherein the fermentation product comprises a polysaccharide component mainly composed of β-1,3-glucan and a liamocins component, the polysaccharide yield being not less than 30 g / L and the liamocins concentration being not less than 4 g / L.

[0024] Furthermore, the total polysaccharide content of the fermentation product is not less than 80%; the purity of the liamocins product is not less than 55%, and the emulsification index is not less than 35%.

[0025] The present invention also provides the application of the above-mentioned fermentation products in food preservation or functional dairy products. Beneficial effects

[0026] 1. This invention is the first to propose using a high-polysaccharide-producing yeast as the main producing strain, focusing on extracellular polysaccharide synthesis; and using a naturally occurring liamocins-producing styrax as an auxiliary strain, focusing on biosurfactant synthesis. The two strains are co-cultured with staggered inoculation times, forming a metabolic division of labor where the main strain produces sugar and the auxiliary strain reduces viscosity. The auxiliary strain utilizes the metabolic byproducts of the main producing strain—ethanol, glycerol, and organic acids—as auxiliary carbon sources, and supplements this with sucrose as the main carbon source for liamocins synthesis, naturally synthesizing liamocins and reducing the viscosity of the fermentation broth in situ without the need for any external chemical reagents, achieving low-addition and self-regulating processes.

[0027] 2. This invention innovatively employs a timing strategy of inoculating the main producing bacteria first and then delaying the inoculation of auxiliary bacteria by 12-18 hours. In the early stage, the main producing bacteria are allowed to quickly establish population dominance and consume large amounts of glucose to synthesize polysaccharides. When the viscosity of the fermentation broth rises to 2000-5000 mPa·s and dissolved oxygen begins to be limited, the auxiliary bacteria are introduced. They utilize the accumulated metabolic byproducts and the added sucrose to initiate liamocins synthesis, precisely targeting the viscosity peak and achieving viscosity reduction on demand.

[0028] 3. This invention breaks through the traditional mindset of reducing viscosity throughout the entire process or passively enduring high viscosity, proposing a dynamic viscosity reduction strategy based on the time-series accumulation of liamocins and a two-stage dissolved oxygen regulation strategy. During the synthesis period, from the start of fermentation to the initiation of sucrose feeding, viscosity reduction is not forced, allowing the viscosity to naturally rise to 2000 to 5000 mPa·s as polysaccharides accumulate. Within this viscosity range, the local concentrations of polysaccharide synthase and the substrate UDP-glucose are relatively high, which is conducive to the synthesis reaction. At the same time, the main producing bacteria are facultative anaerobic bacteria, which can maintain metabolic activity even under medium to low dissolved oxygen conditions. The viscosity reduction period spans from the start of sucrose feeding to the end of fermentation. During this time, liamocins accumulates above the critical micelle concentration, acting as an amphiphilic molecule to exert a triple viscosity-reducing effect: First, it reduces the surface tension from approximately 72 mN / m to approximately 35-47 mN / m, significantly improving the gas-liquid interface properties and restoring dissolved oxygen mass transfer efficiency, recovering dissolved oxygen saturation from 15%-35% to 35%-65%. Second, the hydrophilic head groups (mannitol or glycerol groups) of liamocins competitively form hydrogen bonds with the hydroxyl groups of the polysaccharide chains, weakening the hydrogen bond network between polysaccharide molecules and reducing entanglement between triple helical chains. Third, liamocins micelles encapsulate some polysaccharide segments, creating a steric hindrance effect and preventing the re-entanglement of chains. These three synergistic effects achieve a controllable viscosity reduction from 4000-6000 mPa·s to below 3700 mPa·s, while the restoration of dissolved oxygen promotes the continued synthesis and secretion of polysaccharides by the main producing bacteria.

[0029] 4. In this invention, the auxiliary bacteria convert some of the byproducts such as ethanol and glycerol emitted by the main producing bacteria into liamocins, while simultaneously supplementing sucrose as the main carbon source for liamocins synthesis, with a yield of no less than 4 grams per liter. Liamocins not only reduce viscosity in situ but also serve as a natural component of the final product, endowing the composite product with antibacterial and emulsifying functions, thus transforming viscosity reduction costs into product value-added benefits. Compared to single polysaccharide products, polysaccharide-liamocins composite products have broader application prospects in food preservation, functional dairy products, and other fields. Detailed Implementation

[0030] This invention proposes a fermentation method for high-yield polysaccharide yeast that reduces viscosity and enhances fermentation efficiency. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following will provide a more detailed description of the invention in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0031] The high-polysaccharide-producing yeast described below is Kluyveromyces martensii H2, with accession number CGMCC NO.39233, deposited at the China General Microbiological Culture Collection Center, on June 15, 2026. The auxiliary bacteria are budding short-stem mold ( Aureobasidium pullulans Purchased from China General Microbiological Culture Collection Center, catalog number: CGMCC 3.3984.

[0032] The preparation method of Kluyveromyces martensii H2 seed culture is as follows: the main producing strain is inoculated into seed culture medium and cultured at 28℃ and 200 rpm for 24 hours with shaking. The OD of the seed culture is then measured. 600 The seed culture medium contains: 35 g / L glucose, 7 g / L yeast extract, 7 g / L peptone, 1.5 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, and pH 6.0.

[0033] The method for preparing the seed culture of *Brucea buddingis* is as follows: auxiliary bacteria are inoculated into the seed culture medium and cultured at 28℃ and 200 rpm for 50 hours with shaking. The biomass of the seed culture is 6.4 g / L dry weight, and the proportion of yeast cells is 80.4%. The seed culture medium contains: glucose 25 g / L, tryptone 20 g / L, yeast extract 12 g / L, pH 6.2.

[0034] The co-fermentation medium contained: 80 g / L glucose, 2.0 g / L ammonium sulfate, 1.5 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.3 g / L calcium chloride, 10 mL / L vegetable oil, 3.0 g / L citric acid, 2.0 g / L sodium citrate, and an initial pH of 6.0.

[0035] Example 1 A method for reducing viscosity and enhancing fermentation efficiency of high-polysaccharide-producing yeast includes the following steps: (1) Kluyveromyces martensii H2 was inoculated into the co-fermentation medium to start fermentation. The fermentation temperature was 28℃, the pH was 6.2, the aeration rate was 1.0 vvm, the stirring speed was 200 rpm, and the inoculation amount of the main product was 5% of the fermentation liquid volume; (2) Twelve hours after the start of fermentation, budding short-stem mold producing liamocins was inoculated into the fermentation system as an auxiliary bacteria for co-fermentation. The fermentation temperature was 26℃, pH 5.6, dissolved oxygen was maintained at 25% saturation, aeration rate was 1.2 vvm, stirring speed was 220 rpm, the amount of auxiliary bacteria inoculated was 3% of the fermentation liquid volume, and the ratio of viable bacteria between the main producing bacteria and the auxiliary bacteria at the time of inoculation was 3:1. (3) Starting 36 hours after the start of fermentation, sucrose solution was added to the fermentation system. The flow rate was controlled to maintain the sucrose concentration in the fermentation broth at 10 g / L. The viscosity was monitored online. When the apparent viscosity of the fermentation broth exceeded 5000 mPa·s, the sucrose flow rate was increased. The pH was adjusted to 5.2 and maintained using a citric acid-sodium citrate buffer system. The temperature was 26℃, dissolved oxygen was maintained at 50% saturation, the aeration rate was 1.5 vvm, and the stirring speed was 180 rpm. After 72 hours of fermentation, the fermentation broth was obtained. The total amount of sucrose added was 35.4 g / L. The final apparent viscosity of the fermentation broth was measured to be 3202 mPa·s. (4) Centrifuge the fermentation broth at 8000 rpm and 4℃ for 15 minutes to remove the cells and take the supernatant; let the supernatant stand for 6 hours, collect the liamocins sediment oil layer at the bottom, and remove residual cell debris by 0.45 μm microfiltration of the middle supernatant. (5) The supernatant of microfiltration was concentrated under reduced pressure to 1 / 3 of the original volume, 3 times the volume of pre-cooled 95% ethanol was added, and the mixture was allowed to stand at 4°C for 12 hours. The precipitate was collected by centrifugation to obtain crude polysaccharide and supernatant. (6) The supernatant from step (5) is combined with the liamocins sediment oil layer from step (4), and ethanol is added to a final concentration of 85%. After standing at 4°C for 12 hours, the precipitate is collected by centrifugation as a mixture of liamocins and low molecular weight oligosaccharides. (7) The crude polysaccharide was washed twice with anhydrous ethanol and then freeze-dried under vacuum to obtain the polysaccharide component; liamocins and low molecular weight polysaccharides The oligosaccharide mixture was washed once with hexane to remove residual vegetable oil, then washed once with anhydrous ethanol, and finally vacuum dried to obtain the liamocins component.

[0036] Example 2 The difference from Example 1 is that: in step (2), the inoculation time of the auxiliary bacteria is 8 hours after the start of fermentation, and the amount of auxiliary bacteria inoculated is 2% of the fermentation liquid volume; in step (3), the sucrose feeding start time is 30 hours after the start of fermentation, and the total sucrose feeding amount is 39.8 g / L.

[0037] Example 3 The difference from Example 1 is that: in step (2), the inoculation time of the auxiliary bacteria is 18 hours after the start of fermentation, and the amount of auxiliary bacteria inoculated is 2% of the fermentation liquid volume; in step (3), the sucrose feeding start time is 42 hours after the start of fermentation, and the total sucrose feeding amount is 30.1 g / L.

[0038] Example 4 The difference from Example 1 is that: in step (2), the amount of auxiliary bacteria inoculated is 5% of the fermentation liquid volume, and the ratio of the number of live bacteria of the main producing bacteria to the number of auxiliary bacteria is 2:1; in step (3), the total sucrose feed rate is 45.3 g / L and the aeration rate is 1.8 vvm.

[0039] Example 5 The difference from Example 1 is that in step (3), the pH is adjusted to 5.4 and maintained using a citric acid-sodium citrate buffer system, the temperature is 28°C, and the stirring speed is 200 rpm.

[0040] Example 6 The difference from Example 1 is that the fermentation time in step (3) is 78 hours and the total sucrose feed rate is 40.7 g / L.

[0041] Comparative Example 1 (single main producing strain, no auxiliary strains, no viscosity reduction measures) The difference from Example 1 is that no auxiliary bacteria, *Synthia spp.*, were inoculated, and no sucrose was added. All other conditions were the same as in Example 1.

[0042] Comparative Example 2 (Single dominant producing bacteria + exogenous chemical surfactant Tween-80) The difference from Example 1 is that: no auxiliary bacteria, *Bacillus buddingus*, were inoculated, and no sucrose was added; Tween-80 was added at the 12th hour of fermentation to a final concentration of 0.3% (v / v), and Tween-80 was added again at the 36th hour to a final concentration of 0.3% (v / v), for a cumulative addition of 0.6% (v / v). All other conditions were the same as in Example 1.

[0043] Comparative Example 3 (simultaneous inoculation of two bacteria) The difference from Example 1 is that in step (1), the main producing strain, Kluyveromyces martensii H2, and the auxiliary strain, Cladophora buddingis, are inoculated simultaneously at a volume ratio of 5% and 3%, respectively. The remaining conditions are the same as in Example 1.

[0044] Comparative Example 4 (Assistant bacteria were inoculated before the main producing bacteria) The difference from Example 1 is that in step (1), the auxiliary bacteria *Bacillus buddingus* was first inoculated into the fermentation medium at a volume ratio of 3%, and after being cultured at 26°C for 12 hours, the main producing bacteria *Kluyveromyces martensii* H2 was inoculated at a volume ratio of 5%. That is, the auxiliary bacteria were inoculated before the main producing bacteria. The remaining conditions were the same as in Example 1.

[0045] Comparative Example 5 (no sucrose added, relying solely on metabolic byproducts as a carbon source for auxiliary bacteria) The difference from Example 1 is that in step (3), no sucrose solution is added, and the auxiliary bacteria only use the metabolic byproducts (ethanol, glycerol, organic acids) and residual glucose produced by the main producing bacteria as carbon sources to synthesize liamocins. The other conditions are the same as in Example 1.

[0046] Comparative Example 6 (pH drops to 4.0 during viscosity reduction period) The difference from Example 1 is that in step (3), citric acid is used to adjust the pH to 4.0 and maintain it, and the citric acid-sodium citrate buffer system is not used. The other conditions are the same as in Example 1.

[0047] Comparative Example 7 (Sucrose added at a fixed rate without online viscosity monitoring) The difference from Example 1 is that in step (3), viscosity is not monitored online, and the sucrose solution is added at a fixed rate (35 g / L sucrose is added at a constant rate over 36-72 hours), without adjusting the flow rate according to viscosity changes. The other conditions are the same as in Example 1.

[0048] Comparative Example 8 (Sugar flow was immediately initiated after inoculation with helper bacteria to control viscosity reduction, without utilizing medium viscosity to promote synthesis) The difference from Example 1 is that in step (2), sucrose feeding is started immediately after the auxiliary bacteria are inoculated (i.e., sucrose feeding begins at the 12th hour, instead of waiting until the 36th hour), and the total sucrose feeding rate is 50 g / L (uniform feeding rate for 12~72 hours). A viscosity reduction strategy is implemented throughout the process, and the viscosity is not allowed to rise naturally. The other conditions are the same as in Example 1.

[0049] Test metrics: (a) Viscosity reduction and efficiency improvement indicators (1) Apparent viscosity of fermentation broth at the end point (mPa·s): rotational viscometer method, Brookfield DV2T type, 25℃, shear rate 100 s -1 ; (2) Liamocins yield (g / L): High performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD), C18 column (4.6×250 mm, 5 μm), acetonitrile-water gradient elution, flow rate 1.0 mL / min, ELSD drift tube temperature 40℃, carrier gas pressure 3.5 bar, the liamocins content in the fermentation broth was quantified by external standard method, and this content is the liamocins yield in the fermentation broth; (3) Surface tension reduction rate (%): The surface tension of the fermentation broth was measured by the Wilhelmy plate method and a surface tension meter. The reduction rate was calculated based on the surface tension of pure water of 72 mN / m. (4) Purity of Liamocins product (%): HPLC-ELSD method, same as (2) method, quantify the percentage of liamocins component in the mass of dried product by external standard method; (5) Emulsification index (%): Take 5 mL of 1% aqueous solution of liamocins product and 5 mL of vegetable oil, vortex for 2 min and let stand for 24 h, and measure the percentage of emulsion layer height to total height; The results are shown in Table 1 below: Table 1 Example 1 3202 6.8 42 65.2 48 Example 2 2815 8.5 48 67.1 52 Example 3 3680 5.2 35 60.5 40 Example 4 2596 9.0 52 68.3 55 Example 5 3115 7.2 44 63.8 46 Example 6 2949 7.8 45 65.8 49 Comparative Example 1 6803 0 0 — — Comparative Example 2 4101 0 25 — — Comparative Example 3 3911 4.8 30 57.8 38 Comparative Example 4 4498 5.5 28 55.6 36 Comparative Example 5 5195 2.1 15 52.0 30 Comparative Example 6 4804 4.0 22 53.8 32 Comparative Example 7 3817 5.5 36 62.1 42 Comparative Example 8 2526 7.4 50 65.9 50 As shown in Table 1, the final apparent viscosity of the fermentation broth in Examples 1-6 of this invention was 2596-3680 mPa·s, which was 45.9%-61.8% lower than that in Comparative Example 1; the liamocins yield was 5.2-9.0 g / L, the surface tension reduction rate was 35%-52%, the liamocins product purity was 60.5%-68.3%, and the emulsification index was 40%-55%. It can be seen that the liamocins synthesized endogenously by the auxiliary bacteria can effectively reduce the viscosity of the fermentation broth and endow the product with surface activity and emulsification function. Compared with Comparative Example 2, the final viscosity of Example 1 (3202 mPa·s) was lower than that of Comparative Example 2 (4101 mPa·s), and the surface tension reduction rate (42%) was higher than that of Comparative Example 2 (25%). It can be seen that the triple viscosity reduction mechanism of liamocins (reducing surface tension to improve gas-liquid mass transfer, competitively forming hydrogen bonds with polysaccharide chain hydroxyl groups to weaken the intermolecular hydrogen bond network, and micellar steric hindrance to prevent entanglement between polysaccharide chains) has a better viscosity reduction effect than the single surface tension reduction mechanism of Tween-80. Moreover, Example 1 additionally obtained liamocins product, realizing the transformation of viscosity reduction cost into product value-added. Compared with Comparative Examples 3 and 4, the liamocins yield of Example 1 (6.8 g / L) was higher than that of Comparative Examples 3 (4.8 g / L) and 4 (5.5 g / L), the endpoint viscosity (3202 mPa·s) was lower than that of Comparative Examples 3 (3911 mPa·s) and 4 (4498 mPa·s), and the surface tension reduction rate (42%) was higher than that of Comparative Examples 3 (30%) and 4 (28%). It can be seen that in staggered inoculation, the main producing bacteria are inoculated first and the auxiliary bacteria are inoculated later, which is conducive to the auxiliary bacteria synthesizing more liamocins and exerting a better viscosity reduction effect. Compared to Comparative Example 5, the liamocins yield of Example 1 (6.8 g / L) was 3.2 times that of Comparative Example 5 (2.1 g / L), the final viscosity (3202 mPa·s) was significantly lower than that of Comparative Example 5 (5195 mPa·s), and the surface tension reduction rate (42%) was significantly higher than that of Comparative Example 5 (15%). This indicates that the metabolic byproducts of the main producing bacteria were insufficient to support the synthesis of sufficient liamocins by the auxiliary bacteria, and the addition of sucrose as a dedicated carbon source for the auxiliary bacteria was key to achieving effective viscosity reduction. Compared to Comparative Example 6, the final viscosity of Example 1 was significantly lower than that of Comparative Example 6 (4804 mPa·s), the purity of the liamocins product (65.2%) was higher than that of Comparative Example 6 (53.8%), and the surface tension reduction rate (42%) was higher than that of Comparative Example 6 (22%). This indicates that controlling the pH within the range of 4.8-5.5 during the viscosity reduction period can prevent the auxiliary bacteria from transforming into mycelial form, thus maintaining the liamocins synthesis capacity and viscosity reduction effect.Compared to Comparative Example 7, the final viscosity of Example 1 (3202 mPa·s) was lower than that of Comparative Example 7 (3817 mPa·s), and the surface tension reduction rate (42%) was higher than that of Comparative Example 7 (36%). This shows that online viscosity monitoring combined with sucrose flow acceleration adjustment can ensure that liamocins synthesis accurately reaches the viscosity peak, which is superior to fixed-rate addition. The final viscosity (2526 mPa·s) and liamocins yield (7.4 g / L) of Comparative Example 8 were both better than those of Example 1, but according to the data in Table 2 below, its polysaccharide yield was significantly lower.

[0050] (II) Polysaccharide quality indicators (6) Total polysaccharide yield (g / L): Phenol-sulfuric acid method, with glucose as standard, the supernatant of the fermentation broth was centrifuged and appropriately diluted before determination; (7) Polysaccharide molecular weight distribution (kDa): High performance gel permeation chromatography (HPGPC), Shodex OHpak SB-804HQ column, 0.1 mol / L NaNO3 mobile phase, flow rate 0.5 mL / min, standard curve was prepared using Pullulan series standards; (8) Total polysaccharide content (%) of polysaccharide products: Phenol-sulfuric acid method, with glucose as standard, weigh the dried polysaccharide products for determination; The results are shown in Table 2 below: Table 2 Example 1 38.2 182 89.3 Example 2 35.5 158 87.1 Example 3 41.1 215 91.2 Example 4 34.9 147 85.4 Example 5 37.8 186 88.9 Example 6 39.3 191 90.1 Comparative Example 1 29.5 98 93.0 Comparative Example 2 33.0 132 72.2 Comparative Example 3 31.2 138 87.5 Comparative Example 4 26.5 118 85.8 Comparative Example 5 36.3 168 90.2 Comparative Example 6 35.2 162 84.6 Comparative Example 7 36.9 176 87.8 Comparative Example 8 30.5 143 83.5 As shown in Table 2, the total polysaccharide yield of Examples 1-6 of this invention was 34.9-41.1 g / L, which was 22.5%-44.2% higher than that of Comparative Example 1; the main peak of polysaccharide molecular weight was 147-215 kDa, which was 54.7%-126.3% higher than that of Comparative Example 1 (98 kDa). This shows that the synergistic fermentation of the two bacteria not only increased the polysaccharide yield, but also enabled the main producing bacteria to maintain aerobic metabolism due to the restoration of dissolved oxygen after viscosity reduction, resulting in more complete polysaccharide chain elongation and a significant increase in product molecular weight; the total polysaccharide content of the polysaccharide product was 85.4%-91.2%, which meets the purity requirements of food-grade and pharmaceutical-grade polysaccharides. Compared with Comparative Example 2, the total polysaccharide content of the polysaccharide product of Example 1 (89.3%) was significantly higher than that of Comparative Example 2 (72.2%). This shows that the residue of Tween-80 in the polysaccharide product led to a decrease in purity, while the method of this invention reduced viscosity through endogenous synthesis of liamocins, leaving no chemical reagent residues, resulting in a higher purity polysaccharide product. Compared with Comparative Examples 3 and 4, the total polysaccharide yield of Example 1 (38.2 g / L) was higher than that of Comparative Examples 3 (31.2 g / L) and 4 (26.5 g / L), and the main molecular weight peak of the polysaccharide (182 kDa) was higher than that of Comparative Examples 3 (138 kDa) and 4 (118 kDa). This indicates that staggered inoculation allows the dominant producing bacteria to preferentially establish population dominance and fully utilize carbon sources to synthesize high molecular weight polysaccharides. In contrast, simultaneous inoculation or reversal of the inoculation time both lead to increased competition for carbon sources between the two bacteria, inhibiting polysaccharide synthesis by the dominant producing bacteria. Compared with Comparative Example 5, the total polysaccharide yield of Example 1 (38.2 g / L) was higher than that of Comparative Example 5 (36.3 g / L), and the main molecular weight peak of the polysaccharide (182 kDa) was higher than that of Comparative Example 5 (168 kDa). This indicates that sucrose addition not only provides the auxiliary bacteria with a dedicated carbon source for liamocins synthesis but also indirectly promotes the synthesis of polysaccharides and the accumulation of high molecular weight products by enhancing the viscosity-reducing effect. Compared with Comparative Example 6, the total polysaccharide yield of Example 1 (38.2 g / L) was higher than that of Comparative Example 6 (35.2 g / L), and the total polysaccharide content of the polysaccharide product (89.3%) was higher than that of Comparative Example 6 (84.6%). This indicates that controlling the pH during the viscosity reduction period within the range of 4.8-5.5 is beneficial to the polysaccharide synthesis and product purity of the main producing bacteria. Compared with Comparative Example 8, the total polysaccharide yield of Example 1 (38.2 g / L) was significantly higher than that of Comparative Example 8 (30.5 g / L), and the main peak of polysaccharide molecular weight (182 kDa) was higher than that of Comparative Example 8 (143 kDa). This indicates that allowing the viscosity to rise naturally to a moderate level during the synthesis period is beneficial to the efficient synthesis of polysaccharides and the accumulation of high molecular weight products. Although the forced viscosity reduction throughout the process resulted in the lowest viscosity, it severely inhibited the polysaccharide synthesis of the main producing bacteria. Although the endpoint viscosity (2526 mPa·s) and liamocins yield (7.4 g / L) of Comparative Example 8 were better than those of Example 1, its polysaccharide yield was reduced by 20.2% compared to Example 1, and the main peak of polysaccharide molecular weight was reduced by 21.4% compared to Example 1. Therefore, the overall product value was lower than that of Example 1.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A method for reducing viscosity and enhancing efficiency in the fermentation of high-yield polysaccharide yeast, characterized in that, Includes the following steps: (1) Inoculate high-polysaccharide-producing yeast into a co-fermentation medium to start fermentation, wherein the inoculation amount of the main producing yeast is 3% to 8% of the fermentation liquid volume; (2) 8 to 18 hours after the start of fermentation, budding short-stem mold that produces liamocins is inoculated into the fermentation system as an auxiliary bacteria for co-fermentation. The amount of auxiliary bacteria inoculated is 1% to 5% of the fermentation liquid volume, and the ratio of viable bacteria between the main producing bacteria and the auxiliary bacteria at the time of inoculation is 2:1 to 6:

1. (3) Starting 28 to 44 hours after the start of fermentation, add sucrose solution to the fermentation system and adjust the pH to 4.8 to 5.

5. After fermentation for 60 to 80 hours, obtain the fermentation broth. The final apparent viscosity of the fermentation broth is less than 4000 mPa·s.

2. The method according to claim 1, characterized in that, The high-polysaccharide-producing yeast mentioned in step (1) is Kluyveromyces martensii H2, with accession number CGMCC NO.39233, deposited by the China General Microbiological Culture Collection Center, and deposited on June 15, 2026.

3. The method according to claim 1, characterized in that, The co-fermentation medium described in step (1) contains: 60-100 g / L glucose, 1.5-3.0 g / L ammonium sulfate, 1.0-2.0 g / L potassium dihydrogen phosphate, 0.3-1.0 g / L magnesium sulfate heptahydrate, 0.1-0.5 g / L calcium chloride, 5-15 mL / L vegetable oil, 2.0-4.0 g / L citric acid, 1.0-3.0 g / L sodium citrate, and an initial pH of 5.8-6.

5.

4. The method according to claim 1, characterized in that, The fermentation temperature in step (1) is 26~30℃, pH is 5.8~6.5, aeration rate is 0.8~1.2 vvm, and stirring speed is 150~250 rpm.

5. The method according to claim 1, characterized in that, The co-fermentation conditions described in step (2) are: temperature 24~28℃, pH 5.3~6.0, dissolved oxygen maintained at 15%~35% saturation, aeration rate 1.0~1.5 vvm, and stirring speed 180~280 rpm.

6. The method according to claim 1, characterized in that, In step (3), during the viscosity reduction period after the addition of sucrose, the temperature is 24~28℃, the dissolved oxygen is maintained at 35%~65% saturation, the aeration rate is 1.2~2.0 vvm, and the stirring speed is 150~220 rpm.

7. The method according to claim 1, characterized in that, The total amount of sucrose fed in step (3) is 25~50 g / L, and the flow rate is controlled to maintain the sucrose concentration in the fermentation broth at 5~20 g / L.

8. The method according to claim 1, characterized in that, It also includes product separation and purification steps: (a) Centrifuge the fermentation broth from step (3) of claim 1 at 6000~10000 rpm and 2~6℃ for 10~20 minutes to remove the cells and take the supernatant; let the supernatant stand for 4~12 hours, collect the liamocins sediment oil layer at the bottom, and remove residual cell debris by microfiltration of the middle supernatant at 0.22~0.45 μm; (b) The supernatant of microfiltration is concentrated under reduced pressure to 1 / 4 to 1 / 2 of the original volume, 2 to 4 times the volume of 90% to 96% ethanol is added and pre-cooled, and the mixture is allowed to stand at 0 to 6°C for 8 to 16 hours. The precipitate is collected by centrifugation to obtain crude polysaccharide and supernatant. (c) The supernatant from step (b) is combined with the liamocins sediment oil layer from step (a), and ethanol is added to a final concentration of 80% to 90%. After standing at low temperature, the precipitate is collected by centrifugation as a mixture of liamocins and low molecular weight oligosaccharides. (d) The crude polysaccharide was washed with anhydrous ethanol 1-3 times and then freeze-dried under vacuum to obtain the polysaccharide component; the mixture of liamocins and low molecular weight oligosaccharides was washed with n-hexane 1-2 times to remove residual vegetable oil, and then washed with anhydrous ethanol 1-2 times and then dried under vacuum to obtain the liamocins component.

9. A fermentation product prepared by the method according to any one of claims 1 to 8, characterized in that, The fermentation product contains a polysaccharide component mainly composed of β-1,3-glucan and a liamocins component, with a polysaccharide yield of not less than 30 g / L and a liamocins concentration of not less than 4 g / L.

10. The use of the fermentation product according to claim 9 in food preservation or functional dairy products.