Exopolysaccharide s. avenae, low viscosity triose gel prepared using the same, and beverage containing the same

Abstract

The present application belongs to the preparation of microbial polysaccharide, in particular to extracellular polysaccharide Sphingomonas, low viscosity triose gum prepared by the same and beverage containing the same. Extracellular polysaccharide Sphingomonas is inoculated into a sterilized fermentation medium containing carbon source, nitrogen source and necessary nutrients, and then aerated fermentation is carried out under the conditions of temperature 30-36 DEG C and pH 6.5-7.4. The fermentation broth after fermentation is subjected to biological deproteinization, and the fermentation broth after deproteinization is subjected to high-temperature and high-pressure homogenization and microwave treatment in sequence. The fermentation broth after microwave treatment is subjected to pH adjustment, stirring, centrifugation, alcohol extraction and drying to obtain low viscosity triose gum. The present application solves the technical problems of high viscosity of triose gum prepared by the prior art and unsuitability for making refreshing beverage, and has the advantages of low viscosity of the prepared triose gum product and good gel strength.

Description

Technical Field

[0001] This invention pertains to the preparation of microbial polysaccharides, specifically referring to an extracellular polymeric sphingosine monocytogenes, a low-viscosity triazine gum prepared using it, and beverages containing it. Background Technology

[0002] Sanzan gum is a microbial polysaccharide produced by fermenting *Sphingomonas sphingosine monocytogenes* as the inoculum and using starch or glucose as the main raw material, followed by extraction, drying, and pulverization. Due to its good gelling, emulsifying, thickening, acid-resistant, and high-temperature-resistant properties, Sanzan gum has broad application prospects in the food industry. The National Health Commission of China approved Sanzan gum as a new food additive in Announcement No. 4 of 2020, allowing its use as a thickener, stabilizer, and coagulant in meat sausages, fruit and vegetable juice (pulp) beverages, and plant protein beverages. The announcement also specified quality standards for Sanzan gum, such as a 1% potassium chloride aqueous solution viscosity ≥1600 mPa·s, a 1% aqueous solution viscosity ≥900 mPa·s, and a gel strength ≥25 g / cm³. 2 The current application of succinate in beverages mainly targets thickening concentrated milk beverages and plant protein drinks to achieve high viscosity, high strength, and strong suspension. For example, the patent document with application number CN202110530286.4 discloses that the viscosity of a 1% potassium chloride solution of succinate is above 1800 mPa·s, the viscosity of a 1% aqueous solution is 1050-1193 mPa·s, and the gel strength is 74.1-215 g / cm³. 2 between.

[0003] Refreshing beverages are a general term for various soft drinks that use drinking water as a base and control the balance of sweetness and acidity, turbidity, effervescence, cooling sensation, and aftertaste through formulation and processing. They leave no sticky or heavy feeling after drinking and provide a clean, refreshing, thirst-quenching, and invigorating multi-sensory experience. There is no explicit classification of refreshing beverages in current national or industry standards. Food flavor and beverage processing research reports generally categorize beverages into "Refreshing Type" and "Rich / Viscous Type" based on texture and body, a secondary sensory sub-classification widely used in formulation development, sensory evaluation, and market research. Refreshing beverages have the following characteristics: First, low viscosity, low solids, and no heavy, sticky feeling; second, mild acidity and light sweetness, without being cloying; third, a source of refreshing stimulation (CO2 bubbles, citric acid, mint, low temperature, and the slight bitterness of light tea); fourth, a clean aftertaste without any lingering heavy flavors, primarily for quenching thirst and cutting through greasiness; and fifth, low protein, low emulsifiers and stabilizers, and no high-viscosity matrices such as milk or grain concentrates. However, existing Sanzan gum, due to its viscous texture and heavy gelatinous feel, cannot meet the core requirements of refreshing beverages: "low additives, low viscosity, high transparency, and resistance to acid and ions." Existing Sanzan gum has the following problems when applied to refreshing beverages: First, it has a heavy, sticky feel that clings to the tongue and throat; its strong molecular chains and high aqueous solution viscosity mean that even a small amount adds to a sticky, gelatinous feel, ruining the light and refreshing taste of the beverage; second, its large molecular network encapsulates aroma substances, hindering the release of fruit and tea aromas and resulting in a dull flavor. It has poor shelf stability in acidic environments. When stored for a long time in low pH systems such as fruit juice, lemon tea, and plum vinegar, the main chain glycosidic bonds are hydrolyzed, the viscosity decreases rapidly, and fruit pulp settles, separates, and clumps at the bottom of the bottle. Thirdly, calcium and magnesium ions are prone to cross-link with side chain carboxyl groups to produce white flocculent precipitates.

[0004] While it is theoretically feasible to modify the synthetic pathway of Sphingomonas sphingosine monophosphate to directionally reduce the molecular weight and viscosity of tristan gum in refreshing beverages, its application in the food and beverage sector presents several challenges: First, according to the Food Safety Law and the Administrative Measures for the Safety Review of New Food Raw Materials, the fermentation of genetically modified microorganisms to produce food additives requires toxicological and genomic safety assessments and approval as new food raw materials, a process that is lengthy and costly. Second, the strains exhibit poor stability and low yields. Third, genetic modification simultaneously alters the branching of polysaccharides and the ratio of monosaccharides, potentially compromising tristan gum's core beverage functions, such as acid resistance, heat and cold resistance, and stable effervescence, in addition to reducing viscosity. It may also lead to sedimentation, decreased light transmittance, and other defects in product flavor and application performance. Fourth, the research and development and production processes are highly complex.

[0005] For the reasons mentioned above, the current method of preparing low-viscosity tristan gum using existing high-yield sphingosine monocytogenes fermentation and post-processing methods, although it cannot meet the requirements of low-viscosity tristan gum for refreshing beverages, remains the mainstream process for preparing low-viscosity tristan gum due to its compliance, low cost, and controllable product indicators. There is an urgent need in this field to develop a method that can produce high-yield, low-viscosity tristan gum with good gel strength from the fermentation source, so as to facilitate the preparation of refreshing beverages and effectively maintain product stability. Summary of the Invention

[0006] The purpose of this invention is to provide an extracellular polymeric sphingosine monocytogenes, a low-viscosity triazine gel prepared using it, and a beverage containing it. This strain can effectively utilize inorganic nitrogen sources. The triazine gel prepared using this strain has low viscosity and good gel strength. The refreshing beverage containing it has a refreshing taste, no sticky feeling, high aroma release, strong suspension ability, and good stability.

[0007] One objective of this invention is to provide an extracellular polymeric sphingosine monocytogenes bacterium, the Latin name of which is [missing information]. Sphingomonas Sanxanigenens The accession number is CGMCC No.38199.

[0008] The strain in this invention was deposited on March 30, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The abbreviation of the depository is CGMCC, and the accession number is CGMCC No. 38199.

[0009] The strain of this invention was obtained by the applicant through screening of the original strain using methods such as ultraviolet irradiation and inorganic nitrogen source tolerance culture. The original strain has the Latin name [missing information]. Sphingomonas Sanxanigenens The extracellular polymeric sphingosine monoclonal antibody, with accession number CGMCC No. 20172, is described. The strain in this invention is a Gram-negative bacterium, rod-shaped, irregularly arranged, with raised, round, white, moist, opaque colonies and neat edges. This strain can be used to synthesize low-viscosity triazine gel with good gel strength using an inorganic nitrogen source.

[0010] The second objective of this invention is to prepare a low-viscosity triazine gel using the aforementioned extracellular polymeric sphingosine monophosphate fermentation. The fermentation broth containing triazine gel prepared using the extracellular polymeric sphingosine monophosphate strain described in this invention has a viscosity of 2800–3600 mPa·s. The low-viscosity triazine gel prepared after purification of the fermentation broth was tested using the method in Appendix A of the National Health Commission's Announcement No. 4 of 2020. The results showed that the viscosity of the low-viscosity triazine gel in a 1% potassium chloride solution was 1000–1600 mPa·s, the viscosity in a 1% aqueous solution was 400–900 mPa·s, and the gel strength was 10–25 g / cm³. 2 .

[0011] A third objective of this invention is to provide a beverage containing low-viscosity triacinol, preferably a refreshing beverage. According to the applicant's experiments, adding 0.2‰ to 0.6‰ of the low-viscosity triacinol prepared according to this invention can achieve a stable suspension effect in the preparation of refreshing beverages.

[0012] The fourth objective of this invention is to provide the application of extracellular polymeric sphingosine monocytogenes in the fermentation preparation of low-viscosity triazine gel.

[0013] To ensure the normal growth of the microbial strain during fermentation and the successful synthesis of the final product, while also facilitating subsequent extraction, the preferred technical approach is to use inorganic nitrogen as the nitrogen source during fermentation. The advantages of using inorganic nitrogen as the nitrogen source are reflected in fermentation control, post-extraction purification, and product quality: First, the fermentation broth contains fewer impurities, significantly reducing the pressure on subsequent biological deproteinization; second, the composition is simple and stable, the fermentation process is controllable, and the viscosity and yield of the fermentation gel are stable across batches; third, it reduces the viscosity of the fermentation broth, improving mass transfer and oxygen supply in high-viscosity systems; fourth, it reduces foam formation, decreasing the use of defoamers and improving product quality; fifth, it reduces cell autolysis, lowering the release of intracellular impurities; sixth, it has low raw material and production costs and is resistant to storage; and seventh, it produces fewer byproducts, resulting in a lighter-colored product after alcohol extraction and drying, facilitating application in various scenarios.

[0014] To improve the purity of triazine gum by thoroughly removing impurities from the fermentation broth, the viscosity of the prepared triazine gum is controlled in a targeted manner to ensure product stability and optimize extraction efficiency. The preferred technical approach is to perform post-extraction on the fermentation broth after fermentation. This involves deproteinizing the fermentation broth using a biological method, followed by high-temperature and high-pressure homogenization and microwave treatment. The microwave-treated broth is then subjected to pH adjustment, stirring, centrifugation, alcohol extraction, and drying to obtain low-viscosity triazine gum. The process of preparing triazine gum from the microwave-treated fermentation broth is a conventional technique.

[0015] The main purpose of biological deproteinization is to lyse intact bacterial cells, releasing intracellular proteins, nucleic acids, and other impurities; degrade macromolecular proteins, breaking down polysaccharide-protein cross-linked flocs; and kill residual live bacteria to prevent subsequent microbial contamination of the product. The preferred technical approach is as follows: After fermentation, adjust the pH and temperature of the fermentation broth, add lysozyme, and react for 2-4 hours; after the lysozyme hydrolysis, adjust the pH and temperature of the fermentation broth, add alkaline protease, and react for 3-6 hours; after the alkaline protease hydrolysis, adjust the pH and temperature of the fermentation broth, add pepsin, and react for 2-3 hours. The main benefits of this treatment are: firstly, the bacterial cells are thoroughly broken down, and intracellular proteins are hydrolyzed into small polypeptides, which are easily separated and removed by centrifugation; secondly, the turbidity of the fermentation broth is significantly reduced, filtration resistance is greatly reduced, and the load on homogenization and alcohol extraction equipment is lessened; thirdly, the crude protein and ash content of the finished product, Sanzan gum, meets the standards, eliminating turbidity and odor caused by protein; and fourthly, the biological treatment conditions are mild, avoiding excessive disruption of the Sanzan gum polysaccharide backbone and ensuring controllable viscosity.

[0016] The core function of enzymatic hydrolysis is to gently remove proteins, break down bacterial flocs, and kill residual bacteria, thus eliminating key protein impurities that affect the purity and transparency of polysaccharides at the source, preventing precipitation and stratification during subsequent alcohol precipitation and concentration. The main function of lysozyme hydrolysis is to release proteins, nucleic acids, and peptidoglycan impurities encapsulated within the bacterial cells; and to kill residual live bacteria from fermentation, preventing bacterial proliferation during subsequent storage and processing that could cause polysaccharide turbidity and spoilage. A more preferred technical approach is to adjust the pH of the fermentation broth to 4.5–6.5 after fermentation, add lysozyme, and react at 40℃–60℃ for 2–4 hours. The lysozyme activity is approximately 200,000 u / m, and the amount added is 0.01%–0.05% of the fermentation broth volume.

[0017] The main function of alkaline protease hydrolysis is to hydrolyze the large intracellular and bacterial proteins released after lysing into small short peptides; to break down polysaccharide-protein cross-linked flocs, eliminate turbidity in the liquid, and improve polysaccharide clarity. A preferred method is to adjust the pH of the fermentation broth to 8.0–8.5 after the lysozyme hydrolysis, add alkaline protease, and react at 40°C–60°C for 3–6 hours. The alkaline protease activity is approximately 200,000 u / m, and the amount of lysozyme added is 0.01%–0.2% of the fermentation broth volume.

[0018] The main function of pepsin hydrolysis is to completely inactivate lysozyme and alkaline protease under acidic conditions. Pepsin then breaks down bacterial proteins and acidic protein complexes, dissociating polysaccharides from the acidic protein binding complex, thus achieving deproteinization of the acidic fermentation broth. A preferred method is to add pepsin to the fermentation broth after the alkaline protease hydrolysis reaction and react at 36℃~38℃ for 2~3 hours. The pepsin activity is approximately 1000 NFU / g, and the amount of pepsin added is 0.01%~0.2% of the fermentation broth volume.

[0019] The main functions of high-temperature and high-pressure homogenization are: first, to break up incompletely dispersed mycelial clumps and cell aggregates after enzymatic hydrolysis through mechanical shearing, cavitation, and impact effects, allowing residual intact cells to be fully exposed, and trace amounts of undegraded protein to be further decomposed in the subsequent microwave stage; second, to break up stubborn thick-walled mycelia and aggregated cells, disperse protein-polysaccharide colloidal flocs, reduce the overall viscosity of the fermentation broth, and reduce filtration resistance; and third, to ensure uniform particle size of the homogenized liquid, resulting in consistent microwave heating and preventing excessive degradation of high-molecular-weight polysaccharides by localized high temperatures. The preferred technical approach is to perform high-temperature and high-pressure homogenization under the following conditions: temperature = 70℃~90℃, pressure = 25Mpa~35Mpa, for a total of two cycles.

[0020] The main functions of microwave treatment are: first, to rapidly and thoroughly inactivate all enzyme preparations; the rapid overall heating of the microwave completely inactivates lysozyme and protease, preventing the continuous degradation of the polysaccharide backbone by enzymes during subsequent stirring, centrifugation, and alcohol extraction, thus avoiding a decrease in polysaccharide molecular weight and viscosity loss; second, to deeply sterilize and prevent contamination of the finished product; the instantaneous and uniform heating inside the microwave kills residual live bacteria and spores from fermentation, eliminating the need for long-term high-temperature cooking, reducing the risk of high-temperature degradation of polysaccharides, and ensuring the storage stability of the finished product; and third, to further dissociate colloidal impurities, as microwaves break down proteins and polysaccharides. The colloidal double layer facilitates the flocculation and precipitation of small molecule proteins and peptides, making subsequent filtration and alcohol precipitation easier to separate and remove them, further improving the purity and transmittance of polysaccharides; fourthly, the molecular weight of polysaccharides can be appropriately controlled. Controllable short-time microwaves can slightly break the side chains of polysaccharides, reduce the viscosity of the liquid, and improve the water solubility of the product, while not damaging the main chain structure and functional activity of the polysaccharides; the preferred technical means is that the microwave treatment conditions are: temperature = 40℃~60℃, pH = 4.5~5.5, microwave frequency = 2450MHz, and material transmission speed = 1~5 m / s.

[0021] The main benefits of microwave treatment in an acidic environment are: first, acidification pretreatment followed by microwave treatment achieves dual deproteinization through acid-induced flocculation and microwave thermal dissociation, significantly improving polysaccharide purity; second, precise control of polysaccharide degradation during microwave heating by acid type and pH ensures stable production of low-viscosity triacylglycerol; third, the synergistic acid-microwave action rapidly inactivates enzymes and deeply sterilizes, solving the problems of incomplete enzyme inactivation and insufficient sterilization caused by microwave alone; and fourth, chelation / precipitation of metal ions eliminates polysaccharide cross-linking and re-adhesion caused by microwave high temperature, resulting in better liquid flowability, improved efficiency of subsequent centrifugation and alcohol extraction, and reduced ethanol consumption. The preferred technical implementation method is to adjust the pH to 4.5–5.5 using 10%–20% citric acid, acetic acid, or phosphoric acid.

[0022] To meet the requirements of cell growth and end product synthesis during fermentation, the preferred technical approach is the application of extracellular polymeric sphingosine monocytogenes in the fermentation preparation of low-viscosity triazine gum, which includes the following steps: A. Inoculate the production strain into a sterilized fermentation medium containing carbon source, nitrogen source, and necessary nutrients; the production strain selected is named [Latin name missing]. Sphingomonas Sanxanigenens The extracellular polymeric sphingosine monocytogenes strain with accession number CGMCC No. 38199 was selected as the nitrogen source, and inorganic nitrogen was used. B. Under conditions of 30℃~36℃ and pH=6.5~7.4, aeration and stirring fermentation are carried out; C. Fermentation ends when the viscosity of the fermentation broth no longer increases or the fermentation cycle does not exceed 60 hours. D. The fermentation broth from step C is subjected to post-extraction to prepare low-viscosity triazine gum.

[0023] Furthermore, the fermentation medium in step A consists of the following raw materials in the following mass percentages: 3%–5% glucose or sucrose; 0.3%–0.5% potassium nitrate; 0.3%–0.5% ammonium sulfate; 0.1%–0.2% potassium dihydrogen phosphate; 0.1%–0.2% dipotassium hydrogen phosphate; 0.015%–0.025% magnesium sulfate; 1–5 ppm manganese sulfate; 5–10 ppm zinc chloride; 10–15 ppm ferrous sulfate; 0.03%–0.05% defoamer; the balance is sterile water.

[0024] To facilitate continuous industrial production, the preferred technical approach is that, in step A, the Latin name is... Sphingomonas Sanxanigenens After activation and expansion culture, the extracellular polymeric sphingosine monoclonal bacteria with accession number CGMCC No.38199 was inoculated into sterilized fermentation medium at an inoculation rate of 8% to 12%.

[0025] The main function of strain activation is to restore the physiological and metabolic activity of dormant strains, obtain sufficient high-activity seed cells, ensure the concentration of the starting strain for fermentation, screen out weak, mutated, and degenerated strains, ensure the genetic stability of the strain, adapt to the fermentation environment, reduce environmental stress, lower the risk of contamination by other microorganisms during fermentation, unify the viability of batches of strains, and stabilize the quality of polysaccharide products. The preferred technical method is that the activation conditions involve using a strain with the Latin name... Sphingomonas Sanxanigenens Extracellular polymeric sphingosine monocytogenes with accession number CGMCC No.38199 was inoculated into activation medium and activated for 16 to 24 hours at a temperature of 30℃~36℃ and a rotation speed of 170 rpm~220 rpm. The activation culture medium consists of the following raw materials in the following mass percentages: Sucrose 1.0%–1.5%; peptone 0.3%–0.5%; yeast extract 0.1%–0.15%; potassium dihydrogen phosphate 0.1%–0.2%; dipotassium hydrogen phosphate 0.1%–0.2%; magnesium sulfate 0.015%–0.025%; manganese sulfate 1–5 ppm; zinc chloride 5–10 ppm; ferrous sulfate 10–15 ppm; defoamer 0.03%–0.05%; balance: sterile water.

[0026] To ensure the smooth progress of the expansion culture and meet the economic needs of industrial production, the preferred technical implementation is that the expansion culture involves inoculating the activated seed liquid into the expansion culture medium at an inoculation rate of 1% to 12%, and culturing for 16 to 24 hours at a temperature of 30℃ to 36℃, an aeration rate of 40 cubic meters / hour to 100 cubic meters / hour, and a tank pressure of 0.008 MPa to 0.12 MPa. The expansion culture medium consists of the following raw materials in the following mass percentages: Sucrose 1.0%–1.5%; potassium nitrate 0.3%–0.5%; ammonium sulfate 0.3%–0.5%; potassium dihydrogen phosphate 0.1%–0.2%; dipotassium hydrogen phosphate 0.1%–0.2%; magnesium sulfate 0.015%–0.025%; manganese sulfate 1–5 ppm; zinc chloride 5–10 ppm; ferrous sulfate 10–15 ppm; defoamer 0.03%–0.05%; balance: sterile water.

[0027] The main functions of phased control and gradual increase of ventilation during fermentation are: 1) to match the changing oxygen consumption of cell growth, maintain safe dissolved oxygen, and prevent yield reduction due to hypoxia; 2) to enhance mass transfer in the high-viscosity polysaccharide fermentation broth, eliminate anaerobic dead zones, and ensure energy supply for polysaccharide synthesis; 3) to smoothly discharge CO2 metabolic waste gas, stabilize intracellular metabolic pathways, and improve polysaccharide conversion rate; 4) to control foaming in stages, prevent liquid escape contamination, reduce cell shear damage, and reduce impurities in later extraction; and 5) to stabilize the molecular weight and viscosity of polysaccharides. The preferred technical implementation is as follows: the aeration rate for aeration and stirring fermentation in step B is as follows: 0–10 hours: 600 cubic meters / hour to 1000 cubic meters / hour; 11–20 hours: 1000 cubic meters / hour to 1600 cubic meters / hour; 21–30 hours: 1600 cubic meters / hour to 2400 cubic meters / hour; 31–40 hours: 2400 cubic meters / hour to 3200 cubic meters / hour; 41–50 hours: 3200 cubic meters / hour to 4000 cubic meters / hour; 51 ~ Tank discharge: 4000 cubic meters / hour ~ 3600 cubic meters / hour.

[0028] A more preferred technical implementation is that the stirring speed in step B is as follows: 0-10 hours: 30-60 rpm; 11–20 hours: 60–90 revolutions per minute; 21–30 hours: 90–120 rpm; 31–40 hours: 120–150 rpm; 41–50 hours: 150–180 rpm; 51~Can placement: 135~165 rpm.

[0029] The applicant used the method described in Appendix A of Announcement No. 4 of 2020 issued by the National Health Commission to test the viscosity of the 1% potassium chloride solution, the viscosity of the 1% aqueous solution, and the gel strength of the low-viscosity triazine gel in this invention. The specific process is as follows: 1. Determination of viscosity (1% KCl solution) (1) Instruments and equipment ① Analytical balance: accurate to 0.001g.

[0030] ② High-speed mixer.

[0031] ③ Bruker viscometer, with a measurement error of ±5%, or other viscometers with equivalent performance.

[0032] (2) Measurement conditions ① Rotor model: No. 3 rotor.

[0033] ② Rotor speed: 60 r / min.

[0034] ③Measurement temperature: 25±1℃.

[0035] (3) Analysis steps ① Preparation of a solution containing 1% sample and 1% potassium chloride Accurately weigh 3g of the sample and 3g of potassium chloride (accurate to 0.001g) using clean, dry weighing paper, and mix them thoroughly. Pour 300mL of distilled water into a container. Place the container under a stirrer, turn on the stirrer at 8000 rpm, slowly add the mixed sample into the container, and start timing. Stir continuously for 15 minutes. Then stop stirring, remove the container, and agitate the solution a few times with a stirring rod or similar object.

[0036] ② Measurement Place a solution containing 1% sample and 1% potassium chloride in a tall beaker, and measure and read the viscosity value under the above-described test conditions.

[0037] 2. Determination of viscosity (1% aqueous solution) (1) Instruments and equipment ① Analytical balance: accurate to 0.001g.

[0038] ② High-speed mixer.

[0039] ③ Bruker viscometer, with a measurement error of ±5%, or other viscometers with equivalent performance.

[0040] (2) Measurement conditions ① Rotor model: No. 3 rotor.

[0041] ② Rotor speed: 60 r / min.

[0042] ③Measurement temperature: 25±1℃.

[0043] (3) Analysis steps ① Solution preparation Accurately weigh 3g of the sample (accurate to 0.001g) using clean, dry weighing paper. Pour 300mL of distilled water into a container. Place the container under a stirrer, turn on the stirrer at 8000 rpm, slowly add the sample into the container, and start timing. Stir continuously for 15 minutes. Then stop stirring, remove the container, and agitate the solution a few times with a stirring rod or similar object.

[0044] ② Measurement Place the 1% sample solution in a tall beaker, and measure and read the viscosity value under the above-mentioned test conditions.

[0045] 3. Determination of gel strength (1) Instruments and equipment ① Analytical balance: accurate to 0.001g.

[0046] ② Incubator (temperature range: 5℃~50℃).

[0047] ③ Gel strength meter.

[0048] ④ Water bath (temperature control range: room temperature to 100℃).

[0049] (2) Test conditions ① Probe shape and size: 1.0cm 2 Stainless steel piston cylinder.

[0050] ② Probe movement speed: 10mm / s.

[0051] (3) Analysis steps ① Sample preparation Weigh 3g of sample (accurate to 0.001g) and slowly add it to a container containing 300mL of distilled water while stirring at 8000r / min. Stir for 15min. Pour the sample solution into a tall beaker and heat it in a 95℃ water bath. Stir intermittently with a glass rod 3 times, 5-10 times each time. After heating for 30min, remove the beaker, remove the foam on the top, and pour the gel solution into a flat-bottomed container while it is still hot, to a height of 4cm. Let it stand and cool naturally until it gels. Then place it in a constant temperature incubator at 20℃ for 20h before testing.

[0052] ② Measurement Three parallel samples were measured using a gel strength meter, and the arithmetic mean was taken.

[0053] The essential features and significant technological advancements of this invention are as follows: 1. This invention discloses a substance with the Latin name... Sphingomonas Sanxanigenens The extracellular polymeric sphingosine monocytogenes strain with accession number CGMCC No. 38199, when used to prepare a tristan gum, has a low viscosity value and good gel strength. It not only meets the technical requirements for beverage additives, but also produces beverages with good stability. According to the applicant's experiments, adding 0.2‰ to 0.6‰ of the low-viscosity tristan gum prepared by this invention can play a stabilizing and suspending role in the preparation of refreshing beverages.

[0054] 2. During fermentation, based on the characteristics of the microbial strain, staged ventilation control is adopted. First, it matches the changes in oxygen consumption during cell growth, maintains safe dissolved oxygen, and prevents yield reduction due to hypoxia. Second, it enhances mass transfer in the high-viscosity polysaccharide fermentation broth, eliminates anaerobic dead zones, and ensures energy supply for polysaccharide synthesis. Third, it steadily discharges CO2 metabolic waste gas, stabilizes intracellular metabolic pathways, and improves polysaccharide conversion rate. Fourth, it controls foaming in stages to prevent liquid escape contamination, reduce cell shear damage, and reduce impurities in later extraction. Fifth, it stabilizes the molecular weight and viscosity of polysaccharides.

[0055] 3. Inorganic nitrogen is used as the nitrogen source during fermentation, which reduces protein and colloidal impurities from the source. This not only stabilizes fermentation and precisely controls the viscosity of the tannin (the viscosity of the fermentation liquid after fermentation is 2800-3600 mPa.s), but also simplifies the extraction process after protein removal, centrifugation, and alcohol extraction. The finished product has good light transmittance, low protein residue, and light color, which effectively matches the requirements of refreshing beverages for colloidal clarification and low heaviness. At the same time, it reduces the overall production cost of raw materials, enzyme preparations, and defoamers.

[0056] 4. The fermentation broth is enzymatically hydrolyzed by lysozyme, alkaline protease, and pepsin. This process serves three purposes: first, it gently removes proteins, breaks down bacterial flocs, and kills residual bacteria; second, it removes core protein impurities that affect the purity and transparency of polysaccharides from the source; and third, it avoids precipitation and stratification during subsequent alcohol precipitation and concentration.

[0057] 5. The main functions of microwave treatment are: first, to rapidly and thoroughly inactivate all enzyme preparations, preventing continuous degradation of the polysaccharide backbone by enzymes during subsequent concentration and alcohol precipitation, thus avoiding a decrease in polysaccharide molecular weight and viscosity loss; second, to provide instantaneous and uniform heating inside the microwave, killing residual live bacteria and spores from fermentation, eliminating the need for long-term high-temperature cooking, reducing the risk of polysaccharide degradation at high temperatures, and ensuring the stability of the finished product during storage; third, to further dissociate colloidal impurities, as microwaves disrupt the colloidal double layer formed by proteins and polysaccharides, causing small molecule proteins and peptides to flocculate and precipitate, making them easier to separate and remove during subsequent filtration and alcohol precipitation, further improving the purity and transmittance of polysaccharides; and fourth, to appropriately control the molecular weight of polysaccharides, as controllable short-duration microwaves can slightly break the polysaccharide side chains, reducing the viscosity of the liquid and improving the water solubility of the product, while not damaging the polysaccharide backbone structure and functional activity.

[0058] 6. According to the applicant's tests, the viscosity of a 1% potassium chloride solution of the low-viscosity triazine prepared using this invention is 1000–1600 mPa·s, the viscosity of a 1% aqueous solution is 400–900 mPa·s, and the gel strength is 10–25 g / cm³. 2 The low-viscosity tri-adhesive of this invention is applied to the preparation of refreshing beverages. The resulting refreshing beverages have the advantages of a refreshing taste, no sticky feeling, high aroma release, strong suspension ability and good stability.

[0059] The strains in this invention were deposited on March 30, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The depository is referred to as CGMCC. Detailed Implementation

[0060] The present invention will be further described below with reference to embodiments, but this is not intended to limit the present invention. The scope of protection of the present invention shall be determined by the contents of the claims. Any equivalent technical means substitution made in accordance with the specification shall not depart from the scope of protection of the present invention. Example 1

[0061] An extracellular polymeric sphingosine monocytogenes, the Latin name of this species is... Sphingomonas Sanxanigenens The accession number is CGMCC No.38199.

[0062] The low-viscosity triazine gel prepared by fermentation with the aforementioned extracellular polymeric sphingosine monoclonal antibody (SPM) had a viscosity of 3400 mPa·s in the fermentation broth containing triazine gel prepared using SPM in this embodiment. The low-viscosity triazine gel prepared after purification of the fermentation broth was tested using the method in Appendix A of the National Health Commission Announcement No. 4 of 2020. The results showed that the viscosity of the low-viscosity triazine gel in a 1% potassium chloride solution was 1510 mPa·s, the viscosity in a 1% aqueous solution was 740 mPa·s, and the gel strength was 16.3 g / cm³. 2 .

[0063] A refreshing beverage containing low-viscosity triglycerides.

[0064] The application of extracellular polymeric sphingosine monocytogenes in the fermentation preparation of low-viscosity triazine gum includes the following steps: A. Inoculate the production strain into a sterilized fermentation medium containing carbon source, nitrogen source, and necessary nutrients; the production strain selected is named [Latin name missing]. Sphingomonas Sanxanigenens The extracellular polymeric sphingosine monocytogenes strain with accession number CGMCC No. 38199 was used, with inorganic nitrogen as the nitrogen source; the fermentation medium in step A consisted of the following raw materials in the indicated mass percentages: 4% glucose or sucrose; 0.4% potassium nitrate; 0.4% ammonium sulfate; 0.15% potassium dihydrogen phosphate; 0.15% dipotassium hydrogen phosphate; 0.02% magnesium sulfate; 3 ppm manganese sulfate; 7.5 ppm zinc chloride; 12.5 ppm ferrous sulfate; 0.04% defoamer; balance: sterile water; In step A, the Latin name is Sphingomonas Sanxanigenens After activation and expansion culture, the extracellular polymeric sphingosine monocytogenes of bacteria with accession number CGMCC No. 38199 was inoculated into sterilized fermentation medium at an inoculation rate of 10%.

[0065] The activation condition is to use the Latin name as Sphingomonas Sanxanigenens Extracellular polymeric sphingosine monocytogenes with accession number CGMCCNo.38199 was inoculated into activation medium and activated for 20 hours at 33°C and 195 rpm. The activation culture medium consists of the following raw materials in the following mass percentages: Sucrose 1.35%; peptone 0.4%; yeast extract 0.13%; potassium dihydrogen phosphate 0.15%; dipotassium hydrogen phosphate 0.15%; magnesium sulfate 0.02%; manganese sulfate 3 ppm; zinc chloride 7.5 ppm; ferrous sulfate 13 ppm; defoamer 0.04%; balance: sterile water.

[0066] The expansion culture involves inoculating the activated seed culture into the expansion culture medium at an inoculum rate of 5.5%, and culturing for 20 hours at a temperature of 33℃, an aeration rate of 700 cubic meters per hour, and a tank pressure of 0.06 MPa. The culture medium for expanded culture consists of the following raw materials in the following mass percentages: Sucrose 1.3%; potassium nitrate 0.4%; ammonium sulfate 0.4%; potassium dihydrogen phosphate 0.15%; dipotassium hydrogen phosphate 0.15%; magnesium sulfate 0.02%; manganese sulfate 3 ppm; zinc chloride 7.5 ppm; ferrous sulfate 13 ppm; defoamer 0.04%; balance: sterile water.

[0067] B. Under conditions of 33℃ and pH=7.0, aeration and stirring fermentation were carried out. The aeration rate for aeration and stirring fermentation in step B is as follows: 0-10 hours: 800 cubic meters / hour; 11–20 hours: 1300 cubic meters / hour; 21–30 hours: 2000 cubic meters / hour; 31–40 hours: 2800 cubic meters / hour; 41–50 hours: 3600 cubic meters / hour; 51 ~ Tank discharge: 3800 cubic meters / hour.

[0068] The stirring speed in step B is as follows: 0-10 hours: 45 revolutions per minute; 11–20 hours: 75 rpm; 21–30 hours: 105 revolutions per minute; 31–40 hours: 135 rpm; 41–50 hours: 165 revolutions per minute; 51 ~ Can placement: 150 rpm.

[0069] C. Fermentation ends when the viscosity of the fermentation broth no longer increases or the fermentation cycle does not exceed 60 hours. D. The fermentation broth from step C is subjected to post-extraction to prepare low-viscosity triac gum. This involves biologically deproteinizing the fermentation broth, followed by high-temperature, high-pressure homogenization and microwave treatment. The microwave-treated broth is then adjusted to pH 2.0 with a 15% hydrochloric acid, sulfuric acid, or nitric acid solution and stirred until fiber precipitates. The resulting triac gum fibers are obtained by centrifugation. These fibers are then added to an 82.5% ethanol solution and adjusted to pH 7.0 with a 25% potassium hydroxide solution. After centrifugation, wet triac gum is obtained. This wet triac gum is dried at 57°C for 6 hours and then pulverized to obtain low-viscosity triac gum specifically for refreshing beverages.

[0070] The biological deproteinization process involves adjusting the pH of the fermentation broth to 5.5 after fermentation, adding lysozyme, and reacting at 50°C for 3 hours. The lysozyme activity is approximately 200,000 u / m, and the amount added is 0.03% of the fermentation broth volume. After the lysozyme enzymatic hydrolysis reaction, the pH of the fermentation broth is adjusted to 8.3, and alkaline protease is added and reacted at 50°C for 4.5 hours. The alkaline protease activity is approximately 200,000 u / m, and the amount added is 0.12% of the fermentation broth volume. After the alkaline protease enzymatic hydrolysis reaction, pepsin is added to the fermentation broth and reacted at 37°C for 2.5 hours. The pepsin activity is approximately 1000 NFU / g, and the amount added is 0.1% of the fermentation broth volume.

[0071] The conditions for high-temperature and high-pressure homogenization were: temperature = 80℃, pressure = 30 MPa, and two cycles.

[0072] The microwave treatment conditions are: temperature = 50℃, pH = 5.0, pH = 5.0 adjusted with 15% citric acid, acetic acid or phosphoric acid; microwave frequency = 2450MHz, material transport speed = 3m / s. Example 2

[0073] The difference between this embodiment and Embodiment 1 is that: An extracellular polymeric sphingosine monocytogenes, the Latin name of this species is... Sphingomonas Sanxanigenens The accession number is CGMCC No.38199.

[0074] The low-viscosity triazine gel prepared by fermentation with the above-mentioned extracellular polymeric sphingosine monophosphate (SPMP) showed a viscosity of 3200 mPa·s in the fermentation broth containing triazine gel prepared using the SPMP in this example. The low-viscosity triazine gel prepared after purification of the fermentation broth was tested using the method in Appendix A of the National Health Commission Announcement No. 4 of 2020. The results showed that the viscosity of the low-viscosity triazine gel in a 1% potassium chloride solution was 1400 mPa·s, the viscosity in a 1% aqueous solution was 610 mPa·s, and the gel strength was 18 g / cm³. 2 .

[0075] The application of extracellular polymeric sphingosine monocytogenes in the fermentation preparation of low-viscosity triazine gum includes the following steps: A. The fermentation medium is composed of the following raw materials in the following mass percentages: 3% glucose or sucrose; 0.3% potassium nitrate; 0.3% ammonium sulfate; 0.1% potassium dihydrogen phosphate; 0.1% dipotassium hydrogen phosphate; 0.015% magnesium sulfate; 1 ppm manganese sulfate; 5 ppm zinc chloride; 10 ppm ferrous sulfate; 0.03% defoamer; the balance is sterile water. In step A, the Latin name is Sphingomonas Sanxanigenens After activation and expansion culture, the extracellular polymeric sphingosine monocytogenes of CGMCC No. 38199 was inoculated into sterilized fermentation medium at an inoculation rate of 8%.

[0076] The activation condition is to use the Latin name as Sphingomonas Sanxanigenens Extracellular polymeric sphingosine monocytogenes with accession number CGMCC No. 38199 was inoculated into activation medium and activated for 16 hours at 30°C and 170 rpm. The activation culture medium consists of the following raw materials in the following mass percentages: Sucrose 1.0%; peptone 0.3%; yeast extract 0.1%; potassium dihydrogen phosphate 0.1%; dipotassium hydrogen phosphate 0.1%; magnesium sulfate 0.015%; manganese sulfate 1 ppm; zinc chloride 5 ppm; ferrous sulfate 10 ppm; defoamer 0.03%; balance: sterile water.

[0077] The expansion culture involves inoculating the activated seed culture at a rate of 1% into the expansion culture medium, and culturing for 16 hours at a temperature of 30℃, a ventilation rate of 40 cubic meters per hour, and a tank pressure of 0.008 MPa. The culture medium for expanded culture consists of the following raw materials in the following mass percentages: Sucrose 1.0%; potassium nitrate 0.3%; ammonium sulfate 0.3%; potassium dihydrogen phosphate 0.1%; dipotassium hydrogen phosphate 0.1%; magnesium sulfate 0.015%; manganese sulfate 1 ppm; zinc chloride 5 ppm; ferrous sulfate 10 ppm; defoamer 0.03%; balance: sterile water.

[0078] B. Under conditions of 30℃ and pH=6.5, aeration and stirring fermentation were carried out. The aeration rate for aeration and stirring fermentation in step B is as follows: 0-10 hours: 600 cubic meters / hour; 11–20 hours: 1000 cubic meters / hour; 21–30 hours: 1600 cubic meters / hour; 31–40 hours: 2400 cubic meters / hour; 41–50 hours: 3200 cubic meters / hour; 51 ~ Tank discharge: 4000 cubic meters / hour.

[0079] The stirring speed in step B is as follows: 0-10 hours: 30 revolutions per minute; 11–20 hours: 60 revolutions per minute; 21–30 hours: 90 rpm; 31–40 hours: 120 rpm; 41–50 hours: 150 rpm; 51 ~ Can placement: 135 rpm.

[0080] C. Fermentation ends when the viscosity of the fermentation broth no longer increases or the fermentation cycle does not exceed 60 hours. D. The fermentation broth from step C is subjected to post-extraction to prepare low-viscosity triac gum. This involves biologically deproteinizing the fermentation broth, followed by high-temperature, high-pressure homogenization and microwave treatment. The microwave-treated broth is then adjusted to pH 1.0 with a 10% hydrochloric acid, sulfuric acid, or nitric acid solution and stirred until fiber precipitates. The resulting triac gum fibers are obtained by centrifugation. These fibers are then added to a 70% ethanol solution and adjusted to pH 6.0 with a 10% potassium hydroxide solution. After centrifugation, wet triac gum is obtained. This wet triac gum is dried at 55°C for 4 hours and then pulverized to obtain low-viscosity triac gum specifically for refreshing beverages.

[0081] The biological deproteinization method involves adjusting the pH of the fermentation broth to 4.5 after fermentation, adding lysozyme, and reacting at 40℃ for 2 hours. The lysozyme activity is approximately 200,000 u / m, and the amount added is 0.01% of the fermentation broth volume. After the lysozyme enzymatic hydrolysis reaction, the pH of the fermentation broth is adjusted to 8.0, and alkaline protease is added and reacted at 40℃ for 3 hours. The alkaline protease activity is approximately 200,000 u / m, and the amount added is 0.01% of the fermentation broth volume. After the alkaline protease enzymatic hydrolysis reaction, pepsin is added to the fermentation broth and reacted at 36℃ for 2 hours. The pepsin activity is approximately 1000 NFU / g, and the amount added is 0.01% of the fermentation broth volume.

[0082] The conditions for high-temperature and high-pressure homogenization were: temperature = 70℃, pressure = 25 MPa, and two cycles.

[0083] The microwave treatment conditions are: temperature = 40℃, pH = 4.5, pH = 4.5 adjusted with 10% citric acid, acetic acid or phosphoric acid; microwave frequency = 2450MHz, material transport speed = 1 m / s.

[0084] The rest of the content is the same as in Example 1. Example 3

[0085] The difference between this embodiment and Embodiment 1 is that: An extracellular polymeric sphingosine monocytogenes, the Latin name of this species is... Sphingomonas Sanxanigenens The accession number is CGMCC No.38199.

[0086] The low-viscosity triazine gel prepared by fermentation with the aforementioned extracellular polymeric sphingosine monophosphate (SPMP) was tested using the method described in Appendix A of the National Health Commission's Announcement No. 4 of 2020. The results showed that the viscosity of the low-viscosity triazine gel prepared by fermentation with SPMP was 3000 mPa·s. After purification, the low-viscosity triazine gel was tested using the method described in Appendix A of the National Health Commission's Announcement No. 4 of 2020. The results showed that the viscosity of the low-viscosity triazine gel in a 1% potassium chloride solution was 1280 mPa·s, the viscosity of a 1% aqueous solution was 670 mPa·s, and the gel strength was 13.7 g / cm³. 2 .

[0087] The application of extracellular polymeric sphingosine monocytogenes in the fermentation preparation of low-viscosity triazine gum includes the following steps: A. The fermentation medium is composed of the following raw materials in the following mass percentages: 5% glucose or sucrose; 0.5% potassium nitrate; 0.5% ammonium sulfate; 0.2% potassium dihydrogen phosphate; 0.2% dipotassium hydrogen phosphate; 0.025% magnesium sulfate; 5 ppm manganese sulfate; 10 ppm zinc chloride; 15 ppm ferrous sulfate; 0.05% defoamer; the balance is sterile water. In step A, the Latin name is Sphingomonas Sanxanigenens After activation and expansion culture, the extracellular polymeric sphingosine monocytogenes of bacteria with accession number CGMCC No. 38199 was inoculated into sterilized fermentation medium at an inoculation rate of 12%.

[0088] The activation condition is to use the Latin name as Sphingomonas Sanxanigenens The extracellular polymeric sphingosine monocytogenes with accession number CGMCCNo.38199 was inoculated into activation medium and activated for 24 hours at a temperature of 36℃ and a rotation speed of 220 rpm. The activation culture medium consists of the following raw materials in the following mass percentages: Sucrose 1.5%; peptone 0.5%; yeast extract 0.15%; potassium dihydrogen phosphate 0.2%; dipotassium hydrogen phosphate 0.2%; magnesium sulfate 0.025%; manganese sulfate 5 ppm; zinc chloride 10 ppm; ferrous sulfate 15 ppm; defoamer 0.05%; balance: sterile water.

[0089] The expansion culture involves inoculating the activated seed culture into the expansion culture medium at an inoculum of 12%, and culturing for 24 hours at a temperature of 36℃, an aeration rate of 100 cubic meters per hour, and a tank pressure of 0.12 MPa. The culture medium for expanded culture consists of the following raw materials in the following mass percentages: Sucrose 1.5%; potassium nitrate 0.5%; ammonium sulfate 0.5%; potassium dihydrogen phosphate 0.2%; dipotassium hydrogen phosphate 0.2%; magnesium sulfate 0.025%; manganese sulfate 5 ppm; zinc chloride 10 ppm; ferrous sulfate 15 ppm; defoamer 0.05%; balance: sterile water.

[0090] B. Under conditions of 36℃ and pH=7.4, aeration and stirring fermentation were carried out. The aeration rate for aeration and stirring fermentation in step B is as follows: 0–10 hours: 1000 cubic meters / hour; 11–20 hours: 1600 cubic meters / hour; 21–30 hours: 2400 cubic meters / hour; 31–40 hours: 3200 cubic meters / hour; 41–50 hours: 4000 cubic meters / hour; 51 ~ Tank discharge: 3600 cubic meters / hour.

[0091] The stirring speed in step B is as follows: 0-10 hours: 60 revolutions per minute; 11–20 hours: 90 rpm; 21–30 hours: 120 rpm; 31–40 hours: 150 rpm; 41–50 hours: 180 rpm; 51 ~ Can placement: 165 rpm.

[0092] C. Fermentation ends when the viscosity of the fermentation broth no longer increases or the fermentation cycle does not exceed 60 hours. D. The fermentation broth from step C is subjected to post-extraction to prepare low-viscosity triac gum. This involves biologically deproteinizing the fermentation broth, followed by high-temperature, high-pressure homogenization and microwave treatment. The microwave-treated broth is then adjusted to pH 3.0 with a 20% hydrochloric acid, sulfuric acid, or nitric acid solution and stirred until fiber precipitates. The resulting triac gum fibers are obtained by centrifugation. These fibers are then added to a 95% ethanol solution and adjusted to pH 8.0 with a 40% potassium hydroxide solution. After centrifugation, wet triac gum is obtained. This wet triac gum is dried at 60°C for 8 hours and then pulverized to obtain low-viscosity triac gum specifically for refreshing beverages.

[0093] The biological deproteinization method involves adjusting the pH of the fermentation broth to 6.5 after fermentation, adding lysozyme, and reacting at 60℃ for 4 hours. The lysozyme activity is approximately 200,000 u / m, and the amount of lysozyme added is 0.05% of the fermentation broth volume. After the lysozyme enzymatic hydrolysis reaction, the pH of the fermentation broth is adjusted to 8.5, and alkaline protease is added and reacted at 60℃ for 6 hours. The alkaline protease activity is approximately 200,000 u / m, and the amount of lysozyme added is 0.2% of the fermentation broth volume. After the alkaline protease enzymatic hydrolysis reaction, pepsin is added to the fermentation broth and reacted at 38℃ for 3 hours. The pepsin activity is approximately 1000 NFU / g, and the amount of pepsin added is 0.2% of the fermentation broth volume.

[0094] The conditions for high-temperature and high-pressure homogenization were: temperature = 90℃, pressure = 35 MPa, and two cycles.

[0095] The microwave treatment conditions are: temperature = 60℃, pH = 5.5, pH = 5.5 adjusted with 20% citric acid, acetic acid or phosphoric acid; microwave frequency = 2450MHz, material transport speed = 5m / s.

[0096] The rest of the content is the same as in Example 1. Example 4

[0097] The difference between this embodiment and Embodiment 1 is that: An extracellular polymeric sphingosine monocytogenes, the Latin name of this species is... Sphingomonas Sanxanigenens The accession number is CGMCC No.38199.

[0098] The low-viscosity triazine gel prepared by fermentation with the aforementioned extracellular polymeric sphingosine monophosphate (SPMP) showed a viscosity of 3500 mPa·s in the fermentation broth containing triazine gel prepared using the SPMP in this embodiment. The low-viscosity triazine gel prepared after purification of the fermentation broth was tested using the method in Appendix A of the National Health Commission's Announcement No. 4 of 2020. The results showed that the viscosity of the low-viscosity triazine gel in a 1% potassium chloride solution was 1360 mPa·s, the viscosity in a 1% aqueous solution was 700 mPa·s, and the gel strength was 20.4 g / cm³. 2 .

[0099] The application of extracellular polymeric sphingosine monocytogenes in the fermentation preparation of low-viscosity triazine gum includes the following steps: A. The fermentation medium is composed of the following raw materials in the following mass percentages: 3.5% glucose or sucrose; 0.35% potassium nitrate; 0.35% ammonium sulfate; 0.12% potassium dihydrogen phosphate; 0.12% dipotassium hydrogen phosphate; 0.018% magnesium sulfate; 2 ppm manganese sulfate; 6 ppm zinc chloride; 11 ppm ferrous sulfate; 0.035% defoamer; balance: sterile water; In step A, the Latin name is Sphingomonas Sanxanigenens After activation and expansion culture, the extracellular polymeric sphingosine monocytogenes of bacteria with accession number CGMCC No. 38199 was inoculated into sterilized fermentation medium at an inoculation rate of 9%.

[0100] The activation condition is to use the Latin name as Sphingomonas Sanxanigenens Extracellular polymeric sphingosine monocytogenes with accession number CGMCC No. 38199 was inoculated into activation medium and activated for 18 hours at 32°C and 180 rpm. The activation culture medium consists of the following raw materials in the following mass percentages: Sucrose 1.1%; peptone 0.35%; yeast extract 0.11%; potassium dihydrogen phosphate 0.12%; dipotassium hydrogen phosphate 0.12%; magnesium sulfate 0.018%; manganese sulfate 2 ppm; zinc chloride 6 ppm; ferrous sulfate 11 ppm; defoamer 0.035%; balance: sterile water.

[0101] The expansion culture involves inoculating the activated seed culture into the expansion culture medium at an inoculum rate of 3%, and culturing for 18 hours at a temperature of 31℃, an aeration rate of 50 cubic meters per hour, and a tank pressure of 0.009 MPa. The culture medium for expanded culture consists of the following raw materials in the following mass percentages: Sucrose 1.1%; potassium nitrate 0.35%; ammonium sulfate 0.35%; potassium dihydrogen phosphate 0.12%; dipotassium hydrogen phosphate 0.12%; magnesium sulfate 0.018%; manganese sulfate 2 ppm; zinc chloride 6 ppm; ferrous sulfate 10-15 ppm; defoamer 0.03%-0.05%; balance is sterile water.

[0102] B. Under conditions of 31℃ and pH=6.8, aeration and stirring fermentation were carried out. The aeration rate for aeration and stirring fermentation in step B is as follows: 0-10 hours: 700 cubic meters / hour; 11–20 hours: 1200 cubic meters / hour; 21–30 hours: 1800 cubic meters / hour; 31–40 hours: 2600 cubic meters / hour; 41–50 hours: 3300 cubic meters / hour; 51 ~ Tank discharge: 3700 cubic meters / hour.

[0103] The stirring speed in step B is as follows: 0-10 hours: 40 revolutions per minute; 11–20 hours: 70 rpm; 21–30 hours: 100 revolutions per minute; 31–40 hours: 130 rpm; 41–50 hours: 160 rpm; 51 ~ Can placement: 145 rpm.

[0104] C. Fermentation ends when the viscosity of the fermentation broth no longer increases or the fermentation cycle does not exceed 60 hours. D. The fermentation broth from step C is subjected to post-extraction to prepare low-viscosity triac gum. This involves biologically deproteinizing the fermentation broth, followed by high-temperature, high-pressure homogenization and microwave treatment. The microwave-treated broth is then adjusted to pH 1.5 with a 12% hydrochloric acid, sulfuric acid, or nitric acid solution and stirred until fiber precipitates. The resulting triac gum fibers are obtained by centrifugation. These fibers are then added to a 75% ethanol solution and adjusted to pH 6.5 with an 18% potassium hydroxide solution. After centrifugation, wet triac gum is obtained. This wet triac gum is dried at 55–60°C for 4–8 hours and then pulverized to obtain low-viscosity triac gum specifically for refreshing beverages.

[0105] The biological deproteinization process involves adjusting the pH of the fermentation broth to 5.0 after fermentation, adding lysozyme, and reacting at 45℃ for 2.5 hours. The lysozyme activity is approximately 200,000 u / m, and the amount added is 0.02% of the fermentation broth volume. After the lysozyme enzymatic hydrolysis reaction, the pH of the fermentation broth is adjusted to 8.1, and alkaline protease is added and reacted at 45℃ for 3.5 hours. The alkaline protease activity is approximately 200,000 u / m, and the amount added is 0.05% of the fermentation broth volume. After the alkaline protease enzymatic hydrolysis reaction, pepsin is added to the fermentation broth and reacted at 36.5℃ for 2.3 hours. The pepsin activity is approximately 1000 NFU / g, and the amount added is 0.015% of the fermentation broth volume.

[0106] The conditions for high-temperature and high-pressure homogenization were: temperature = 75℃, pressure = 28 MPa, and two cycles were performed.

[0107] The microwave treatment conditions are: temperature = 45℃, pH = 4.8, pH = 4.8 adjusted with 13% citric acid, acetic acid or phosphoric acid; microwave frequency = 2450MHz, material transport speed = 2m / s.

[0108] The rest of the content is the same as in Example 1. Example 5

[0109] The difference between this embodiment and Embodiment 1 is that: An extracellular polymeric sphingosine monocytogenes, the Latin name of this species is... Sphingomonas Sanxanigenens The accession number is CGMCC No.38199.

[0110] The low-viscosity triazine gel prepared by fermentation with the above-mentioned extracellular polymeric sphingosine monophosphate (SPMP) was obtained. The viscosity of the fermentation broth containing triazine gel prepared using SPMP in this example was 2900 mPa·s. The low-viscosity triazine gel prepared after purification of the fermentation broth was tested using the method in Appendix A of the National Health Commission Announcement No. 4 of 2020. The results showed that the viscosity of the low-viscosity triazine gel in a 1% potassium chloride solution was 1410 mPa·s, the viscosity in a 1% aqueous solution was 520 mPa·s, and the gel strength was 15.5 g / cm³. 2 .

[0111] The application of extracellular polymeric sphingosine monocytogenes in the fermentation preparation of low-viscosity triazine gum includes the following steps: A. The fermentation medium is composed of the following raw materials in the following mass percentages: 4.5% glucose or sucrose; 0.45% potassium nitrate; 0.45% ammonium sulfate; 0.18% potassium dihydrogen phosphate; 0.18% dipotassium hydrogen phosphate; 0.023% magnesium sulfate; 4 ppm manganese sulfate; 9 ppm zinc chloride; 14 ppm ferrous sulfate; 0.045% defoamer; balance: sterile water; In step A, the Latin name is Sphingomonas Sanxanigenens After activation and expansion culture, the extracellular polymeric sphingosine monoclonal bacteria with accession number CGMCC No. 38199 was inoculated into sterilized fermentation medium at an inoculation rate of 11%.

[0112] The activation condition is to use the Latin name as Sphingomonas Sanxanigenens Extracellular polymeric sphingosine monocytogenes with accession number CGMCCNo.38199 was inoculated into activation medium and activated for 22 hours at 35°C and 210 rpm. The activation culture medium consists of the following raw materials in the following mass percentages: Sucrose 1.4%; peptone 0.45%; yeast extract 0.14%; potassium dihydrogen phosphate 0.18%; dipotassium hydrogen phosphate 0.18%; magnesium sulfate 0.022%; manganese sulfate 4 ppm; zinc chloride 9 ppm; ferrous sulfate 14 ppm; defoamer 0.0455%; balance: sterile water.

[0113] The expansion culture involves inoculating the activated seed culture at a rate of 10% into the expansion culture medium, and culturing for 22 hours at a temperature of 35℃, an aeration rate of 90 cubic meters per hour, and a tank pressure of 0.11 MPa. The culture medium for expanded culture consists of the following raw materials in the following mass percentages: Sucrose 1.4%; potassium nitrate 0.45%; ammonium sulfate 0.45%; potassium dihydrogen phosphate 0.18%; dipotassium hydrogen phosphate 0.18%; magnesium sulfate 0.022%; manganese sulfate 4 ppm; zinc chloride 9 ppm; ferrous sulfate 14 ppm; defoamer 0.045%; balance: sterile water.

[0114] B. Under conditions of 35℃ and pH=7.2, aeration and stirring fermentation were carried out. The aeration rate for aeration and stirring fermentation in step B is as follows: 0-10 hours: 900 cubic meters / hour; 11–20 hours: 1500 cubic meters / hour; 21–30 hours: 2200 cubic meters / hour; 31–40 hours: 3000 cubic meters / hour; 41–50 hours: 3800 cubic meters / hour; 51 ~ Tank discharge: 3900 cubic meters / hour.

[0115] The stirring speed in step B is as follows: 0-10 hours: 55 revolutions per minute; 11–20 hours: 85 revolutions per minute; 21–30 hours: 115 revolutions per minute; 31–40 hours: 145 rpm; 41–50 hours: 174 revolutions per minute; 51 ~ Can placement: 160 rpm.

[0116] C. Fermentation ends when the viscosity of the fermentation broth no longer increases or the fermentation cycle does not exceed 60 hours. D. The fermentation broth from step C is subjected to post-extraction to prepare low-viscosity tri-tan gum. This involves biologically deproteinizing the fermentation broth, followed by high-temperature, high-pressure homogenization and microwave treatment. The microwave-treated broth is then adjusted to pH 1.0–3.0 with 18% hydrochloric acid, sulfuric acid, or nitric acid solution and stirred until fiber precipitates. The resulting tri-tan gum fibers are obtained by centrifugation. These fibers are then added to a 90% ethanol solution and adjusted to pH 7.5 with 35% potassium hydroxide solution. After centrifugation, wet tri-tan gum is obtained. This wet tri-tan gum is dried at 58°C for 7 hours and then pulverized to obtain low-viscosity tri-tan gum specifically for refreshing beverages.

[0117] The biological deproteinization method involves adjusting the pH of the fermentation broth to 5.3 after fermentation, adding lysozyme, and reacting at 53℃ for 3.5 hours. The lysozyme activity is approximately 200,000 u / m, and the amount of lysozyme added is 0.045% of the fermentation broth volume. After the lysozyme enzymatic hydrolysis reaction, the pH of the fermentation broth is adjusted to 8.4, and alkaline protease is added and reacted at 55℃ for 5 hours. The alkaline protease activity is approximately 200,000 u / m, and the amount of lysozyme added is 0.018% of the fermentation broth volume. After the alkaline protease enzymatic hydrolysis reaction, pepsin is added to the fermentation broth and reacted at 37.5℃ for 2.8 hours. The pepsin activity is approximately 1000 NFU / g, and the amount of pepsin added is 0.18% of the fermentation broth volume.

[0118] The conditions for high-temperature and high-pressure homogenization were: temperature = 88℃, pressure = 32 MPa, and two cycles.

[0119] The microwave treatment conditions are: temperature = 55℃, pH = 5.3, pH = 5.3 adjusted with 18% citric acid, acetic acid or phosphoric acid; microwave frequency = 2450MHz, material transport speed = 4m / s.

[0120] The rest of the content is the same as in Example 1.

[0121] The applicant measured the viscosity of the tristan gum fermentation broth prepared in Examples 1-5 and the performance indicators of the purified low-viscosity tristan gum. The specific results are as follows:

Claims

1. Extracellular polymeric sphingosine monocytogenes, characterized in that... Its Latin name is Sphingomonas Sanxanigenens The accession number is CGMCC No.38199.

2. Low-viscosity triazine gel prepared by fermentation of the extracellular polymeric sphingosine monocytogenes as described in claim 1.

3. The low-viscosity tri-adhesive according to claim 2, characterized in that... The viscosity of a 1% potassium chloride solution of low-viscosity triazine is 1000–1600 mPa·s, and the viscosity of a 1% aqueous solution is 400–900 mPa·s.

4. The low-viscosity tri-adhesive according to claim 2, characterized in that... The gel strength of low-viscosity triazine is 10–25 g / cm³. 2 .

5. A beverage containing low-viscosity triac gum as described in any one of claims 2 to 4.

6. The beverage according to claim 5, characterized in that... The beverage is a refreshing drink.

7. The application of the extracellular polymeric sphingosine monocytogenes according to claim 1 in the fermentation preparation of low-viscosity triazine gel.

8. The application according to claim 7, characterized in that... Inorganic nitrogen is used as the nitrogen source in fermentation.

9. The application according to claim 7, characterized in that... The post-fermentation broth is extracted by biological deproteinization. The deproteinized broth is then subjected to high-temperature and high-pressure homogenization and microwave treatment. The microwave-treated broth is then subjected to pH adjustment, stirring, centrifugation, alcohol extraction, and drying to obtain low-viscosity triazine gum.

10. The application according to claim 9, characterized in that... The biological deproteinization method involves adjusting the pH and temperature of the fermentation broth after fermentation, adding lysozyme and reacting for 2-4 hours; adjusting the pH and temperature of the fermentation broth after the lysozyme hydrolysis reaction, adding alkaline protease and reacting for 3-6 hours; and adjusting the pH and temperature of the fermentation broth after the alkaline protease hydrolysis reaction, adding pepsin and reacting for 2-3 hours.

11. The application according to claim 10, characterized in that... The lysozyme enzymatic hydrolysis reaction involves adjusting the pH of the fermentation broth after fermentation to 4.5–6.5, adding lysozyme, and reacting at 40℃–60℃ for 2–4 hours. The lysozyme activity is approximately 200,000 u / m, and the amount of lysozyme added is 0.01%–0.05% of the fermentation broth volume.

12. The application according to claim 10, characterized in that... The alkaline protease hydrolysis reaction involves adjusting the pH of the fermentation broth to 8.0–8.5 after the lysozyme hydrolysis reaction, adding alkaline protease, and reacting at a temperature of 40℃–60℃ for 3–6 hours. The alkaline protease activity is approximately 200,000 u / m, and the amount of lysozyme added is 0.01%–0.2% of the fermentation broth volume.

13. The application according to claim 10, characterized in that... The pepsin hydrolysis reaction involves adding pepsin to the fermentation broth after the alkaline protease hydrolysis reaction is completed, and reacting at a temperature of 36℃~38℃ for 2~3 hours. The pepsin activity is approximately 1000 NFU / g, and the amount of pepsin added is 0.01%~0.2% of the fermentation broth volume.

14. The application according to claim 9, characterized in that... The conditions for high-temperature and high-pressure homogenization are: temperature = 70℃~90℃, pressure = 25Mpa~35Mpa, for a total of two times.

15. The application according to claim 9, characterized in that... The microwave treatment conditions are: temperature = 40℃~60℃, pH = 4.5~5.5, microwave frequency = 2450MHz, and material transport speed = 1~5 m / s.

16. The application according to claim 15, characterized in that... Adjust the pH to 4.5–5.5 using 10%–20% citric acid, acetic acid, or phosphoric acid.

17. The application according to any one of claims 7 to 16, characterized in that... Includes the following steps: A. Inoculate the production strain into a sterilized fermentation medium containing carbon source, nitrogen source, and necessary nutrients; the production strain selected is named [Latin name missing]. Sphingomonas Sanxanigenens The extracellular polymeric sphingosine monocytogenes strain with accession number CGMCC No. 38199 was selected as the nitrogen source, and inorganic nitrogen was used. B. Under conditions of 30℃~36℃ and pH=6.5~7.4, aeration and stirring fermentation are carried out; C. Fermentation ends when the viscosity of the fermentation broth no longer increases or the fermentation cycle does not exceed 60 hours. D. The fermentation broth from step C is subjected to post-extraction to prepare low-viscosity triazine gum.

18. The application according to claim 17, characterized in that... The fermentation medium in step A consists of the following raw materials in the following mass percentages: 3%–5% glucose or sucrose; 0.3%–0.5% potassium nitrate; 0.3%–0.5% ammonium sulfate; 0.1%–0.2% potassium dihydrogen phosphate; 0.1%–0.2% dipotassium hydrogen phosphate; 0.015%–0.025% magnesium sulfate; 1–5 ppm manganese sulfate; 5–10 ppm zinc chloride; 10–15 ppm ferrous sulfate; 0.03%–0.05% defoamer; the balance is sterile water.

19. The application according to claim 17, characterized in that... In step A, the Latin name is... Sphingomonas Sanxanigenens After activation and expansion culture, the extracellular polymeric sphingosine monoclonal bacteria with accession number CGMCC No.38199 was inoculated into sterilized fermentation medium at an inoculation rate of 8% to 12%.

20. The application according to claim 19, characterized in that... The activation condition is to use the Latin name as Sphingomonas Sanxanigenens Extracellular polymeric sphingosine monocytogenes with accession number CGMCC No.38199 was inoculated into activation medium and activated for 16 to 24 hours at a temperature of 30℃~36℃ and a rotation speed of 170 rpm~220 rpm. The activation culture medium consists of the following raw materials in the following mass percentages: Sucrose 1.0%–1.5%; peptone 0.3%–0.5%; yeast extract 0.1%–0.15%; potassium dihydrogen phosphate 0.1%–0.2%; dipotassium hydrogen phosphate 0.1%–0.2%; magnesium sulfate 0.015%–0.025%; manganese sulfate 1–5 ppm; zinc chloride 5–10 ppm; ferrous sulfate 10–15 ppm; defoamer 0.03%–0.05%; balance: sterile water.

21. The application according to claim 19, characterized in that... The aforementioned expansion culture involves inoculating the activated seed liquid into the expansion culture medium at an inoculation rate of 1% to 12%, and culturing for 16 to 24 hours at a temperature of 30℃ to 36℃, an aeration rate of 40 cubic meters / hour to 100 cubic meters / hour, and a tank pressure of 0.008 MPa to 0.12 MPa. The expansion culture medium consists of the following raw materials in the following mass percentages: Sucrose 1.0%–1.5%; potassium nitrate 0.3%–0.5%; ammonium sulfate 0.3%–0.5%; potassium dihydrogen phosphate 0.1%–0.2%; dipotassium hydrogen phosphate 0.1%–0.2%; magnesium sulfate 0.015%–0.025%; manganese sulfate 1–5 ppm; zinc chloride 5–10 ppm; ferrous sulfate 10–15 ppm; defoamer 0.03%–0.05%; balance: sterile water.

22. The application according to claim 17, characterized in that... The aeration rate for aeration and stirring fermentation in step B is as follows: 0–10 hours: 600 cubic meters / hour to 1000 cubic meters / hour; 11–20 hours: 1000 cubic meters / hour to 1600 cubic meters / hour; 21–30 hours: 1600 cubic meters / hour to 2400 cubic meters / hour; 31–40 hours: 2400 cubic meters / hour to 3200 cubic meters / hour; 41–50 hours: 3200 cubic meters / hour to 4000 cubic meters / hour; 51 ~ Tank discharge: 4000 cubic meters / hour ~ 3600 cubic meters / hour.

23. The application according to claim 17, characterized in that... The stirring speed in step B is as follows: 0-10 hours: 30-60 rpm; 11–20 hours: 60–90 revolutions per minute; 21–30 hours: 90–120 rpm; 31–40 hours: 120–150 rpm; 41–50 hours: 150–180 rpm; 51~Can placement: 135~165 rpm.

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