Method for detecting camellia oil fermentation of bumblebee candida humilis

Abstract

The present application relates to the technical field of fermentation detection, and particularly relates to a camellia oil fermentation detection method of bear-born candida, which comprises the following steps: preparing camellia oil emulsion; mixing the camellia oil emulsion with bear-born candida seed liquid, and feeding gas to obtain fermentation liquid; detecting the stirring shaft torque of the fermentation tank; adjusting the feeding rate of the camellia oil emulsion; detecting the attenuation rate of the volume oxygen transfer coefficient of the fermentation liquid after the feeding rate is adjusted; adjusting the aeration mode of the fermentation liquid, including adjusting the gas-liquid contact time or carrying out pulse aeration; continuously monitoring the oil-water liquid level potential of the fermentation liquid after the aeration mode is adjusted; determining whether the fermentation liquid reaches the fermentation end point according to the change curve of the oil-water liquid level potential; if the fermentation liquid reaches the fermentation end point, determining the stirring speed of the stirring shaft; and obtaining the bear-born candida after fermentation. The present application realizes the improvement of the accuracy of fermentation process control.

Description

Technical Field

[0001] This invention relates to the field of fermentation detection technology, and in particular to a method for detecting camellia oil fermentation of *Candida bacillus*. Background Technology

[0002] In existing technologies, conventional physicochemical indicators mainly include acid value, peroxide value, iodine value, and saponification value. Acid value reflects the content of free fatty acids in oils, peroxide value reflects the degree of oxidation of oils, iodine value characterizes the degree of unsaturation of oils, and saponification value is related to the average molecular weight of oils. Conventional physicochemical indicator detection is mostly offline, which is cumbersome and time-consuming, and cannot reflect the dynamic changes in the fermentation process in real time. The accuracy and stability of rapid detection methods still need to be improved, making it difficult to meet the real-time monitoring needs of industrial production.

[0003] Chinese Patent Publication No. CN118547029A discloses a method for producing sophorolipids by fermentation of Candida albicans and its application, including the following steps: (1) Candida albicans is inoculated into a seed culture medium and fermented to obtain a primary seed liquid. The primary seed liquid is then inoculated into the seed culture medium again and fermented to form a bacterial suspension, i.e., a secondary seed liquid; (2) Fermentation culture medium is added to the fermenter, and the secondary seed liquid obtained in step (1) is inoculated into the fermentation culture medium; (3) During the fermentation process, the dissolved oxygen value of the fermenter is maintained at 30-35% for 0-48h, 35-40% for 48-96h, and 40-45% for 96-138h. Among them, glucose and supplemental culture medium are added every 6h after 24h of fermentation. After 96h of fermentation, the lower layer of sophorolipids is separated while fermenting until 138h of fermentation is completed, and sophorolipid fermentation liquid is obtained. Therefore, it can be seen that the method and application of producing sophorolipids by fermentation of Candida albicans has the problem that when Candida albicans ferments camellia oil substrate with high oil content, the rheological properties of the fermentation broth change due to the combination of oil and cell, which in turn causes a nonlinear decrease in dissolved oxygen transfer efficiency, making it impossible to accurately obtain the true fermentation state. Summary of the Invention

[0004] Therefore, the present invention provides a method for detecting camellia oil fermentation by *Candida bacillus*, in order to overcome the problem in the prior art that when *Candida bacillus* ferments camellia oil substrates with high oil content, the rheological properties of the fermentation broth change due to the combination of oil and cell, which in turn causes a nonlinear decrease in dissolved oxygen transfer efficiency, making it impossible to accurately obtain the true fermentation state.

[0005] To achieve the above objectives, the present invention provides a method for detecting camellia oil fermentation of *Candida bacillus*, comprising: Camellia oil was pre-emulsified to prepare camellia oil emulsion; The camellia oil emulsion was mixed by introducing bumblebee Candida seed liquid, and gas was introduced to carry out fermentation to obtain fermentation liquid; The torque of the stirring shaft of the fermentation tank during the introduction of the camellia oil emulsion was detected; The feeding rate of the camellia oil emulsion is adjusted according to the torque of the stirring shaft of the fermentation tank; The rate of decrease in the volumetric dissolved oxygen coefficient of the fermentation broth after adjusting the feeding rate was detected. The aeration mode of the fermentation broth is adjusted according to the decay rate of the dissolved oxygen coefficient, including adjusting the gas-liquid contact time or performing pulse aeration. After adjusting the ventilation mode, the oil-water surface potential of the fermentation broth is continuously monitored; The fermentation broth is determined to have reached the fermentation endpoint based on the change curve of the oil-water liquid surface potential. If the fermentation broth reaches the fermentation endpoint, the stirring speed of the stirring shaft is determined based on the oil-water surface potential. The fermentation broth is stirred according to the stirring speed of the stirring shaft to obtain fermented bumblebee Candida albicans.

[0006] Furthermore, adjusting the feeding rate of the camellia oil emulsion according to the torque of the stirring shaft of the fermentation tank includes: Obtain the torque of the stirring shaft of the fermenter; The torque of the stirring shaft is compared with the preset torque; If the torque of the stirring shaft is greater than or equal to the preset torque, the feeding rate of the camellia oil emulsion is reduced.

[0007] Further, adjusting the aeration mode of the fermentation broth according to the decay rate of the dissolved oxygen coefficient includes: The rate of decrease in the volumetric dissolved oxygen coefficient of the fermentation broth after adjusting the feeding rate is obtained. The decay rate is compared with a preset first rate and a preset second rate, respectively; If the attenuation rate is greater than the preset second rate, then pulse ventilation is performed; If the attenuation rate is greater than or equal to the preset first rate and less than or equal to the preset second rate, then the gas-liquid contact time is increased. If the attenuation rate is less than the preset first rate, then normal ventilation is performed.

[0008] Furthermore, the decay rate is the ratio of the difference between the volumetric dissolved oxygen coefficient at the start of a unit detection time and the volumetric dissolved oxygen coefficient at the end of a unit detection time to the unit detection time.

[0009] Furthermore, the pulse ventilation is a cyclical operation that alternates between ventilation and cessation of ventilation within a preset time period.

[0010] Furthermore, the increase in gas-liquid contact time is determined based on the difference between the decay rate and the preset first rate.

[0011] Further, determining whether the fermentation broth has reached the fermentation endpoint based on the change curve of the oil-water surface potential includes: Continuously monitor the oil-water level potential; Plot the curves showing the change in oil-water surface potential over time. If the curve of the oil-water liquid surface potential change over time shows an inflection point where the second derivative is zero, and the absolute value of the potential at this inflection point is lower than the preset potential, and the second derivative changes from a negative value to a positive value at the inflection point, then the fermentation broth is determined to have reached the fermentation endpoint.

[0012] Further, determining the stirring speed of the stirring shaft based on the oil-water surface potential includes: After determining that fermentation has reached its end point, the potential of the oil-water liquid surface is obtained; The potential of the oil-water liquid level is compared with the preset potential; If the potential of the oil-water liquid surface is less than the preset potential, then the stirring speed of the stirring shaft is increased.

[0013] Furthermore, the stirring speed of the stirring shaft is negatively correlated with the absolute value of the oil-water surface potential.

[0014] Furthermore, the oleic acid content in the camellia oil is 74%–87% by mass.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: the method of the present invention forms a stable emulsion by pre-treating camellia oil through emulsification, which weakens the non-specific hydrophobic adsorption between high-viscosity oil and *Candida bumblebee* cells, reduces the oil film coating rate on the cell surface, and thus alleviates the problems of apparent viscosity surge and shear thinning behavior instability in the fermentation broth; on this basis, the feeding rate of the camellia oil emulsion is controlled by the torque of the stirring shaft of the fermenter as a dynamic feedback signal. When the torque increases, indicating that oil phase enrichment leads to local micro-region retention and the initial appearance of mass transfer barriers, the feeding rate is reduced to avoid an exponential increase in dissolved oxygen transfer resistance, thus ensuring the synchronization of cell metabolic activity and substrate utilization efficiency; By detecting the decay rate of the dissolved oxygen coefficient, the oxygen transfer efficiency of the fermentation broth can be detected. The lipases and surfactant peptides secreted by *Candida bumblebee* continuously modify the oil-water interface, leading to a systematic decrease in the interfacial charge density. The inflection point of the second derivative of its potential change curve precisely corresponds to multiple physiological critical points such as intracellular lipid accumulation saturation, secondary metabolic shift, and cell self-flocculation initiation. The stirring speed of the stirring shaft is set in reverse based on the absolute value of the endpoint potential. After the endpoint is determined, a low-disturbance, stable interface stirring mode is implemented. The lower the absolute value of the potential, the smaller the interfacial tension and the easier it is for oil droplets to coalesce. By increasing the stirring speed, the upward floating and stratification of the oil phase can be delayed, thereby improving the accuracy of fermentation control.

[0016] Furthermore, the method of the present invention reduces the feeding rate of camellia oil emulsion according to the torque of the stirring shaft of the fermenter. Since camellia oil is a hydrophobic carbon source, if the feeding rate is too fast, the high concentration of oil droplets will coat the cells, hindering oxygen transfer, or causing excessive fatty acid accumulation in the cells. The greater the resistance to oxygen transfer from the bubbles to the liquid, the better. By reducing the feeding rate of camellia oil emulsion, the substrate is prevented from causing toxicity or metabolic inhibition to Candida bumblebee, thus preventing energy metabolism disorders and improving the accuracy of fermentation control.

[0017] Furthermore, the method of the present invention adjusts the aeration mode of the fermentation broth according to the decay rate of the volumetric dissolved oxygen coefficient. If the decay rate is greater than the preset second rate, the fermentation broth is metabolically vigorous but mass transfer is limited. By performing pulse ventilation, the oil film surrounding the bubbles is broken by utilizing the bubble breakage and redistribution at the moment of gas cessation. If the decay rate is greater than or equal to the preset first rate and less than or equal to the preset second rate, the bubbles are severely merged. By generating microbubbles and increasing the gas-liquid contact time, the oxygen transfer efficiency is precisely restored and maintained. If the decay rate is less than the preset first rate, it indicates that the mass transfer state of the fermentation broth is good, and the conventional continuous ventilation mode is maintained to meet the metabolic needs.

[0018] Furthermore, the method of the present invention determines whether the fermentation broth has reached the fermentation endpoint by analyzing the change curve of the oil-water liquid surface potential. When the second derivative of the potential change curve shows a significant inflection point and the absolute value of the potential continues to decrease below the preset potential, it indicates that the intracellular lipid accumulation has reached saturation, and the cell metabolism shifts from primary lipid synthesis to the generation of secondary metabolites. At the same time, the increased hydrophobicity of the cell surface triggers self-flocculation, at which point the fermentation is determined to have entered the endpoint stage. Based on the quantitative result of the absolute value of the endpoint potential, the stirring speed in the later stage of production is set in reverse. The lower the absolute value of the potential, the smaller the interfacial tension and the stronger the tendency of oil droplet aggregation. By increasing the stirring speed, a suitable turbulence intensity is formed, which effectively slows down the rate of oil phase floating and stratification, maintains the multiphase dispersion stability of the fermentation system, and improves the accuracy of endpoint determination and the reliability of subsequent process connection. Attached Figure Description

[0019] Figure 1 This is an overall flowchart of the camellia oil fermentation detection method using *Candida bacillus* from bumblebees, as described in an embodiment of the present invention. Figure 2 The flowchart illustrates the process of adjusting the feeding rate of camellia oil emulsion in the detection method of camellia oil fermentation using bumblebee Candida albicans according to an embodiment of the present invention. Figure 3 This is a flowchart illustrating the process of adjusting the aeration mode of the fermentation broth in the detection method of camellia oil fermentation using *Candida bacillus* from bumblebees, according to an embodiment of the present invention. Figure 4 This is a flowchart illustrating the method for detecting the fermentation of camellia oil by bumblebee Candida albicans in an embodiment of the present invention, specifically for determining whether the fermentation broth has reached the fermentation endpoint. Detailed Implementation

[0020] To make the objectives and advantages of the present invention clearer, the present invention will be further described below with reference to embodiments; it should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0021] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. Those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0022] It should be noted that in the description of this invention, the terms "upper", "lower", "left", "right", "inner", "outer", etc., which indicate directions or positional relationships, are based on the directions or positional relationships shown in the accompanying drawings. This is only for the convenience of description and is not intended to indicate or imply that the device or element must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, it should not be construed as a limitation of this invention.

[0023] Furthermore, it should be noted that, in the description of this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0024] Please see Figure 1 , Figure 2 , Figure 3 as well as Figure 4 The diagrams shown are, respectively, the overall flowchart of the camellia oil fermentation detection method using *Candida bacillus* from bumblebees according to an embodiment of the present invention, the flowchart for adjusting the feeding rate of the camellia oil emulsion, the flowchart for adjusting the aeration mode of the fermentation broth, and the flowchart for determining whether the fermentation broth has reached the fermentation endpoint. The camellia oil fermentation detection method using *Candida bacillus* from bumblebees according to an embodiment of the present invention includes: Camellia oil was pre-emulsified to prepare camellia oil emulsion; The camellia oil emulsion was mixed by introducing bumblebee Candida seed liquid, and gas was introduced to carry out fermentation to obtain fermentation liquid; The torque of the stirring shaft of the fermentation tank during the introduction of the camellia oil emulsion was detected; The feeding rate of the camellia oil emulsion is adjusted according to the torque of the stirring shaft of the fermentation tank; The rate of decrease in the volumetric dissolved oxygen coefficient of the fermentation broth after adjusting the feeding rate was detected. The aeration mode of the fermentation broth is adjusted according to the decay rate of the dissolved oxygen coefficient, including adjusting the gas-liquid contact time or performing pulse aeration. After adjusting the ventilation mode, the oil-water surface potential of the fermentation broth is continuously monitored; The fermentation broth is determined to have reached the fermentation endpoint based on the change curve of the oil-water liquid surface potential. If the fermentation broth reaches the fermentation endpoint, the stirring speed of the stirring shaft is determined based on the oil-water surface potential. The fermentation broth is stirred according to the stirring speed of the stirring shaft to obtain fermented bumblebee Candida albicans.

[0025] Specifically, the process of emulsifying camellia oil to prepare a camellia oil emulsion includes: Tween 80 polyoxyethylene dehydrated sorbitan monooleate was selected as the emulsifier; Emulsifier is added at a rate of 0.2% to 5% of the mass of camellia oil to form a crude emulsion; Camellia oil emulsion with an average particle size of 200-300 nm was prepared by using a high-speed shear emulsifier with a rotation speed of 12000-15000 rpm and an emulsification time of 15-20 min.

[0026] Specifically, the bumblebee Candida seed culture is prepared by inoculating the activated strain into YPD medium and culturing it at 30°C and 200 rpm in a shaker for 18-24 hours until the OD600 reaches 1.2-1.5.

[0027] Specifically, the preparation method of bumblebee *Candida albicans* seed culture includes: The bumblebee Candida prolifera strain stored in glycerol tubes at -80℃ was streaked onto YPD solid medium and incubated at 28℃ for 48–72 hours. Single colonies were picked and inoculated into 50ml LYPD liquid medium and cultured at 28℃ with shaking at 180rpm for 16-20 hours as primary seed culture. Transfer the primary seed at a 5% inoculation rate to 200 mL of seed culture medium and culture at 28°C and 200 rpm for 12–16 hours to obtain the secondary seed. When the cell density of the secondary seed reaches 1×10⁸–2×10⁸ CFU / mL, it can be used for fermenter inoculation.

[0028] Specifically, the seed culture medium consists of: 30 g / L glucose, 15 g / L peptone, 8 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, and pH 6.0.

[0029] In practice, the method of this invention pre-treats camellia oil to form a stable emulsion, weakening the non-specific hydrophobic adsorption between high-viscosity oil and *Candida bumblebee* cells, reducing the oil film coating rate on the cell surface, thereby alleviating the problems of sudden increase in apparent viscosity and instability of shear thinning behavior in the fermentation broth. Based on this, the feed rate of the camellia oil emulsion is controlled by the torque of the fermenter's stirring shaft as a dynamic feedback signal. When the torque increases, indicating oil phase enrichment leading to local micro-region retention and the initial appearance of mass transfer barriers, the feed rate is reduced to avoid an exponential increase in dissolved oxygen transfer resistance, ensuring the synchronicity of cell metabolic activity and substrate utilization efficiency. The volumetric dissolved oxygen is monitored. The coefficient decay rate is used to detect the oxygen transfer efficiency of the fermentation broth. The lipases and surfactant peptides secreted by *Candida bumblebee* continuously modify the oil-water interface, leading to a systematic decrease in the interfacial charge density. The inflection point of the second derivative of its potential change curve precisely corresponds to multiple physiological critical points such as intracellular lipid accumulation saturation, secondary metabolic shift, and cell self-flocculation initiation. The stirring speed of the stirring shaft is set in reverse based on the absolute value of the endpoint potential. After the endpoint is determined, a low-disturbance, stable interface stirring mode is implemented. The lower the absolute value of the potential, the smaller the interfacial tension and the easier it is for oil droplets to coalesce. By increasing the stirring speed, the upward floating and stratification of the oil phase can be delayed, thereby improving the accuracy of fermentation control.

[0030] Specifically, adjusting the feeding rate of the camellia oil emulsion according to the torque of the stirring shaft of the fermentation tank includes: Obtain the torque of the stirring shaft of the fermenter; The torque of the stirring shaft is compared with the preset torque; If the torque of the stirring shaft is greater than or equal to the preset torque, the feeding rate of the camellia oil emulsion is reduced.

[0031] Specifically, a silicone tube is used to connect the peristaltic pump, and the feed tube is a stainless steel tube with an inner diameter of 2mm. The insertion depth of the stainless steel tube is 5cm below the liquid surface, and the outlet direction is parallel to the stirring shaft and downward.

[0032] Specifically, a torque sensor is installed on the stirring shaft of the fermenter to detect the total driving torque during the stirring process in real time. The baseline torque is pre-calibrated when there is no feeding (stirring only), and then the torque during feeding is detected in real time. The difference between the two is calculated as the pure feeding disturbance torque.

[0033] Specifically, 3L of fermentation medium is placed in a 5L fermenter, and activated Candida bumblebee seed liquid is inoculated at an inoculation amount of 10%. Under the condition that the diameter-to-height ratio of the 5L fermenter is 1:2, the general range of the preset torque is [0.01N·m, 0.5N·m], and the preferred embodiment of the preset torque is 0.2N·m.

[0034] Those skilled in the art will understand that the range of preset torque and the preferred embodiment provided in this embodiment are the values ​​that best address the technical problem solved by the present invention, under the conditions that 3L of fermentation medium is placed in a 5L fermenter, activated Candida bumblebee seed liquid is inoculated, the inoculation amount is 10%, and the diameter-to-height ratio of the 5L fermenter is 1:2. In actual applications or experiments, those skilled in the art can make adaptive adjustments to the preset torque according to the actual application environment and application scenario.

[0035] In practice, if the difference between the torque and the preset torque exceeds 0.01 N·m, the feeding rate of the camellia oil emulsion will be reduced by 5%. The base value of the feeding rate is 1% to 3% of the initial volume of the fermentation medium added per hour. For example, when the detected torque is 0.25 N·m and the preset torque is 0.2 N·m, the initial feeding rate is 10 ml / h, then the feeding rate will be reduced by 25%, from 10 ml / h to 7.5 ml / h.

[0036] In practice, the method of the present invention reduces the feeding rate of camellia oil emulsion according to the torque of the stirring shaft of the fermenter. Since camellia oil is a hydrophobic carbon source, if the feeding rate is too fast, the high concentration of oil droplets will coat the cells, hindering oxygen transfer, or causing excessive fatty acid accumulation in the cells. The greater the resistance to oxygen transfer from the bubbles to the liquid, the better. By reducing the feeding rate of camellia oil emulsion, the substrate is avoided from producing toxicity or metabolic inhibition on bumblebee Candida albicans, preventing energy metabolism disorders and improving the accuracy of fermentation control.

[0037] Specifically, adjusting the aeration mode of the fermentation broth according to the decay rate of the dissolved oxygen coefficient includes: The rate of decrease in the volumetric dissolved oxygen coefficient of the fermentation broth after adjusting the feeding rate is obtained. The decay rate is compared with a preset first rate and a preset second rate, respectively; If the attenuation rate is greater than the preset second rate, then pulse ventilation is performed; If the attenuation rate is greater than or equal to the preset first rate and less than or equal to the preset second rate, then the gas-liquid contact time is increased. If the attenuation rate is less than the preset first rate, then normal ventilation is performed.

[0038] Specifically, the decay rate is the ratio of the difference between the volumetric dissolved oxygen coefficient at the start of a unit detection time and the volumetric dissolved oxygen coefficient at the end of a unit detection time to the unit detection time.

[0039] Specifically, the unit detection time is 1 second.

[0040] Specifically, the pulse ventilation is a cyclical operation that alternates between ventilation and cessation of ventilation within a preset time period.

[0041] Specifically, the increase in gas-liquid contact time is determined based on the difference between the decay rate and the preset first rate.

[0042] Specifically, the volumetric dissolved oxygen coefficient (VOC) of the fermentation broth represents the ability of oxygen to be transferred from the gas phase to a unit volume of liquid phase per unit time. It is a core indicator for measuring the oxygen supply capacity and equipment efficiency of a fermentation system. The VOC is a well-known existing technology, so its specific calculation formula will not be elaborated here. The VOC can be understood as how many times the liquid in the tank can be "completely replaced" with oxygen per hour under standard driving force. A higher value indicates a greater amount of oxygen that can be transferred per unit time.

[0043] Specifically, the volumetric dissolved oxygen coefficient is detected using a polarographic dissolved oxygen electrode installed inside the fermenter. The electrode response time is less than 10 seconds, and data is collected every 30 seconds. The unit detection duration is set to 5 minutes, and data points at 10 consecutive collection times are collected to calculate the decay rate. The preset time period for pulse ventilation is 60 seconds, with a 40-second ventilation period and a 20-second aeration stop period. The ventilation flow rate is maintained at 1 vvm. During the aeration stop period, the bubbles in the fermenter break and redistribute due to buoyancy differences, breaking the stable encapsulation of the bubbles by the oil film and restoring the gas-liquid mass transfer interface.

[0044] Specifically, conventional ventilation is a continuous and stable ventilation mode, with the ventilation flow rate maintained at 0.5-1.0 vvm. The ventilation gas is a mixture of air and pure oxygen, with the proportion of pure oxygen adjusted according to the fermentation stage: 10%-20% pure oxygen in the early stage of fermentation, 20%-30% pure oxygen in the middle stage of fermentation, and 30%-40% pure oxygen in the later stage of fermentation, in order to match the oxygen uptake requirements of bumblebee Candida albicans at different growth stages.

[0045] In practice, a 5L fermenter is filled with 3L of fermentation medium, and activated *Candida bumblebee* seed culture is inoculated at a rate of 10%. Under the condition that the diameter-to-height ratio of the 5L fermenter is 1:2, the preset first rate is generally taken within the range of [3%h]. -1 6%h -1 The preferred embodiment of the preset first rate is 5%h. -1 The preset second rate generally takes values ​​within the range of [12%h]. -1 18%h -1 The preferred embodiment of the preset second rate is 15%h. -1 .

[0046] Those skilled in the art will understand that the range of preset first rate and preset second rate provided in this embodiment, as well as the preferred embodiment, are the values ​​that best address the technical problem solved by the present invention under the conditions that 3L of fermentation medium is placed in a 5L fermenter, activated Candida albicans seed liquid is inoculated, the inoculation amount is 10%, and the diameter-to-height ratio of the 5L fermenter is 1:2. In actual applications or experiments, those skilled in the art can make adaptive adjustments to the preset first rate and preset second rate according to the actual application environment and application scenario.

[0047] In practice, when the decay rate is between a preset first rate and a preset second rate, if the difference between the decay rate and the preset first rate increases by 1%h... -1This increases the gas-liquid contact time by 1 second. In this embodiment, the increase in gas-liquid contact time is achieved by extending the residence path of the gas in the fermentation broth. An annular gas distributor is added to the bottom of the fermenter, with 12-16 micropores evenly arranged on the distributor. The pore diameter is 0.5-1.0 mm. After the gas passes through the micropores and forms tiny bubbles, it moves along a spiral upward path, thereby extending the gas-liquid contact time. For example, when the decay rate is 8%h... -1 The gas-liquid contact time is increased by 3 seconds. The annular gas distributor extends the rising path of the bubbles by adjusting the micropore diameter and the tilt angle of the spiral guide plate. The spiral guide plate of the annular gas distributor is made of polytetrafluoroethylene and the surface is treated with superhydrophobicity, with a contact angle greater than 150°.

[0048] In practice, the method of the present invention adjusts the aeration mode of the fermentation broth according to the decay rate of the volumetric dissolved oxygen coefficient. If the decay rate is greater than the preset second rate, the fermentation broth is metabolically vigorous but mass transfer is limited. Pulsed aeration is performed to break the lipid film surrounding the bubbles by utilizing the bubble breakage and redistribution at the moment of gas cessation. If the decay rate is greater than or equal to the preset first rate and less than or equal to the preset second rate, the bubbles are severely merged. Microbubbles are generated and the gas-liquid contact time is increased to achieve precise recovery and maintenance of oxygen transfer efficiency. If the decay rate is less than the preset first rate, it indicates that the mass transfer state of the fermentation broth is good, and the conventional continuous aeration mode is maintained to meet the metabolic needs.

[0049] Specifically, determining whether the fermentation broth has reached the fermentation endpoint based on the change curve of the oil-water surface potential includes: Continuously monitor the oil-water level potential; Plot the curves showing the change in oil-water surface potential over time. If the curve of the oil-water liquid surface potential change over time shows an inflection point where the second derivative is zero, and the absolute value of the potential at this inflection point is lower than the preset potential, and the second derivative changes from a negative value to a positive value at the inflection point, then the fermentation broth is determined to have reached the fermentation endpoint.

[0050] Specifically, determining the stirring speed of the stirring shaft based on the oil-water surface potential includes: After determining that fermentation has reached its end point, the potential of the oil-water liquid surface is obtained; The potential of the oil-water liquid level is compared with the preset potential; If the potential of the oil-water liquid surface is less than the preset potential, then the stirring speed of the stirring shaft is increased.

[0051] Specifically, the stirring speed of the stirring shaft is negatively correlated with the absolute value of the oil-water surface potential.

[0052] Specifically, the oleic acid content in the camellia oil is 74% to 87% by mass.

[0053] Specifically, the oil-water surface potential is detected using an insertion-type micropotential sensor. The probe is inserted 3-5 cm below the liquid surface in the fermenter, with a detection accuracy of ±0.1 mV and a data acquisition frequency of once per minute. The first and second derivatives are calculated. When the second derivative changes from negative to positive after crossing zero, and the absolute value of the potential at this inflection point is less than -15 mV, the fermentation is considered to have reached its end.

[0054] Specifically, the selectable range of the preset potential is [-20mV, -10mV], and the preferred embodiment of the preset potential is -15mV.

[0055] Specifically, the basic value for the stirring speed of the stirring shaft is 100-150 rpm; In implementation, when the oil-water surface potential is less than the preset potential, for every 1mV that the preset potential exceeds the oil-water surface potential, the current stirring speed of the stirring shaft is increased by 5rpm. In one possible embodiment, the oil-water surface potential is -17mV and the current stirring speed of the stirring shaft is 120rpm. Then the increased stirring speed of the stirring shaft is 120rpm + (-15mV - (-17mV)) / 1mV × 5rpm = 130rpm. The maximum stirring speed of the stirring shaft does not exceed 250rpm.

[0056] In practice, the method of this invention determines whether the fermentation broth has reached the fermentation endpoint by analyzing the change curve of the oil-water surface potential. When the second derivative of the potential change curve shows a significant inflection point and the absolute value of the potential continues to decrease to below a preset threshold, it indicates that the intracellular lipid accumulation has reached saturation, and the cell metabolism shifts from primary lipid synthesis to the generation of secondary metabolites. At the same time, the increased hydrophobicity of the cell surface triggers self-flocculation, at which point the fermentation is determined to have entered the endpoint stage. Based on the quantitative result of the absolute value of the endpoint potential, the stirring speed in the later stage of production is set in reverse. The lower the absolute value of the potential, the smaller the interfacial tension and the stronger the tendency of oil droplet aggregation. By increasing the stirring speed, a suitable turbulence intensity is formed, which effectively slows down the rate of oil phase floating and stratification, maintains the multiphase dispersion stability of the fermentation system, and improves the accuracy of endpoint determination and the reliability of subsequent process connection.

[0057] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A method for detecting the fermentation of camellia oil by *Candida baccata*, characterized in that, include: Camellia oil was pre-emulsified to prepare camellia oil emulsion; The camellia oil emulsion was mixed by introducing bumblebee Candida seed liquid, and gas was introduced to carry out fermentation to obtain fermentation liquid; The torque of the stirring shaft of the fermentation tank during the introduction of the camellia oil emulsion was detected; The feeding rate of the camellia oil emulsion is adjusted according to the torque of the stirring shaft of the fermentation tank; The rate of decrease in the volumetric dissolved oxygen coefficient of the fermentation broth after adjusting the feeding rate was detected. The aeration mode of the fermentation broth is adjusted according to the decay rate of the dissolved oxygen coefficient, including adjusting the gas-liquid contact time or performing pulse aeration. After adjusting the ventilation mode, the oil-water surface potential of the fermentation broth is continuously monitored; The fermentation broth is determined to have reached the fermentation endpoint based on the change curve of the oil-water liquid surface potential. If the fermentation broth reaches the fermentation endpoint, the stirring speed of the stirring shaft is determined based on the oil-water surface potential. The fermentation broth is stirred according to the stirring speed of the stirring shaft to obtain fermented bumblebee Candida albicans.

2. The method for detecting camellia oil fermentation of *Candida bacillus* according to claim 1, characterized in that, Adjusting the feeding rate of the camellia oil emulsion according to the torque of the stirring shaft of the fermentation tank includes: Obtain the torque of the stirring shaft of the fermenter; The torque of the stirring shaft is compared with the preset torque; If the torque of the stirring shaft is greater than or equal to the preset torque, the feeding rate of the camellia oil emulsion is reduced.

3. The method for detecting camellia oil fermentation of *Candida baccata* from bumblebees according to claim 2, characterized in that, Adjusting the aeration mode of the fermentation broth according to the decay rate of the dissolved oxygen coefficient includes: The rate of decrease in the volumetric dissolved oxygen coefficient of the fermentation broth after adjusting the feeding rate is obtained. The decay rate is compared with a preset first rate and a preset second rate, respectively; If the attenuation rate is greater than the preset second rate, then pulse ventilation is performed; If the attenuation rate is greater than or equal to the preset first rate and less than or equal to the preset second rate, then the gas-liquid contact time is increased. If the attenuation rate is less than the preset first rate, then normal ventilation is performed.

4. The method for detecting camellia oil fermentation of *Candida bacillus* according to claim 3, characterized in that, The decay rate is the ratio of the difference between the volumetric dissolved oxygen coefficient at the start of a unit detection time and the volumetric dissolved oxygen coefficient at the end of a unit detection time to the unit detection time.

5. The method for detecting camellia oil fermentation of *Candida bacillus* according to claim 4, characterized in that, The pulse ventilation is a cyclical operation that alternates between ventilation and cessation of ventilation within a preset time period.

6. The method for detecting camellia oil fermentation of *Candida bacillus* according to claim 5, characterized in that, The increase in gas-liquid contact time is determined based on the difference between the decay rate and the preset first rate.

7. The method for detecting camellia oil fermentation of *Candida bacillus* according to claim 6, characterized in that, Determining whether the fermentation broth has reached the fermentation endpoint based on the oil-water surface potential change curve includes: Continuously monitor the oil-water level potential; Plot the curves showing the change in oil-water surface potential over time. If the curve of the oil-water liquid surface potential change over time shows an inflection point where the second derivative is zero, and the absolute value of the potential at this inflection point is lower than the preset potential, and the second derivative changes from a negative value to a positive value at the inflection point, then the fermentation broth is determined to have reached the fermentation endpoint.

8. The method for detecting camellia oil fermentation of *Candida bacillus* according to claim 7, characterized in that, Determining the stirring speed of the stirring shaft based on the oil-water surface potential includes: After determining that fermentation has reached its end point, the potential of the oil-water liquid surface is obtained; The potential of the oil-water liquid level is compared with the preset potential; If the potential of the oil-water liquid surface is less than the preset potential, then the stirring speed of the stirring shaft is increased.

9. The method for detecting camellia oil fermentation of *Candida bacillus* according to claim 8, characterized in that, The stirring speed of the stirring shaft is negatively correlated with the absolute value of the oil-water surface potential.

10. The method for detecting camellia oil fermentation of *Candida bacillus* according to claim 9, characterized in that, The oleic acid content in the camellia oil is 74%–87% by mass.

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