Method for producing fermented vegetable oil by semi-continuous fed-batch fermentation process

Abstract

The invention relates to a method for producing fermented vegetable oil by a semi-continuous fed-batch fermentation process, which comprises the following steps: firstly, inoculating a strain into a fermentation culture medium for thallus propagation under proper conditions, then mixing with crude vegetable oil according to a certain proportion, fermenting at proper temperature, and discharging part of fermentation liquor after the fermentation index reaches a set value, thereby obtaining the fermented vegetable oil. And returning a water phase containing active thalli to the fermentation tank, and meanwhile, supplementing fresh vegetable oil crude oil with the same mass as the water phase to continue fermentation. And repeating the steps until the time of the acid value of the oil phase of the fermentation liquid reaching the set value exceeds 24 hours or is more than two times of the time of the fermentation reaching the set value after the previous oil supplement, ending the fermentation, and centrifugally separating and purifying the separated oil phase to obtain the fermented vegetable oil. The production efficiency of the fermented vegetable oil can be greatly improved, the single-batch production yield is improved, the stability of the quality of the fermented oil is ensured, the production cost is saved, and the method can be widely applied to the cosmetic and skin care field, the food field or the pharmaceutical industry.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a method for producing fermented vegetable oil by a semi-continuous fed-batch fermentation process. Background Art

[0002] Microbial fermentation mainly includes four modes: batch fermentation, fed-batch fermentation, semi-continuous fed-batch fermentation and continuous fed-batch fermentation. Batch fermentation: All substrates and strains are put into the fermentation at one time, and no feeding or discharge is done during the whole process. The operation is simple but the efficiency is low. The substrate concentration gradually decreases with metabolism, and the yield is eventually limited by insufficient nutrition. Fed-batch fermentation: Batch or continuous addition of substrates but no discharge to maintain substrate concentration and improve yield and efficiency. However, there are problems such as long fermentation cycle, accumulation of harmful metabolites, and inhibition of product synthesis, and the amount of feed is mostly dependent on experience. Continuous / semi-continuous fed-batch fermentation: continuous or intermittent feeding and release of fermentation liquid to keep the volume, substrate and product concentration in the tank constant. It can extend the cycle, avoid metabolite accumulation and product inhibition, and is widely used in the production of ethanol, amino acids, etc., but requires high equipment sealing and strains are prone to variation. The existing technology mostly adopts batch or fed-batch mode, which requires operations such as cleaning and sterilization between batches, consumes a lot of resources and has low efficiency. Existing vegetable oil fermentation models all use the efficacy of fermented vegetable oil as the evaluation standard, lacking consideration and attention to fermentation yield and fermentation efficiency. So far, there have been no reports on the use of semi-continuous fermentation processes to produce plant fermented oils.

[0003] Chinese invention patent application CN117180143A uses two-stage yeast to ferment shea butter step by step, with a long total cycle and low production efficiency. The fermented oil output per unit volume of fermentation liquid is only 200-300g / kg, and the fermentation efficiency is only 1.39-2.08g / kg / h; Chinese invention patent application CN117737137A improves the emulsification capacity of vegetable oil through batch fermentation, but long-term fermentation leads to decomposition of triglycerides, excessive acidity and quality decline, and the fermented sacha inchi oil output per unit mass of fermentation liquid is only 333g / kg, and the fermentation efficiency is 2.31g / kg / h.

[0004] Chinese Patent Application CN118286117A discloses a fed-batch fermentation mode. In this invention, glucose, bacteria, rapeseed oil, and macadamia nut oil are first added to a fermentation tank and fermented with low-speed stirring at 28-32 degrees for 70-74 hours. Then, rapeseed oil and macadamia nut oil are added again, and after low-speed stirring and fermenting for 158-162 hours, steps such as heating, sterilization, cooling, and centrifugation are carried out to obtain fermented oil. Under the fed-batch fermentation mode, the output and production efficiency of the mixed oil of fermented rapeseed oil and fermented macadamia nut oil are both doubled compared to the batch fermentation mode. Ignoring the seed preparation time (not given in the patent), based on the data given in Examples 1-3, the output of the fermented mixed oil reaches 684-686 g / kg, and the fermentation efficiency is 2.908-3 g / kg / h. Summary of the Invention

[0005] The first aspect of the present invention provides a method for producing fermented vegetable oil by a semi-continuous fed-batch fermentation process, comprising the following steps: Step 1: Preparation of seed liquid: Inoculate the strain into a seed medium for bacterial culture to obtain seed liquid; Step 2: Preparation of primary fermentation liquid: Inoculate the seed liquid into a fermentation medium for strain expansion culture to obtain primary fermentation liquid; Step 3: Vegetable oil fermentation: Mix the crude vegetable oil with the primary fermentation liquid in proportion and carry out vegetable oil fermentation to obtain fermentation liquid; Step 4: Regularly take samples during the vegetable oil fermentation process to detect the acid value of the oil phase in the fermentation liquid. After the acid value reaches the set value, take out 4-60% of the total fermentation liquid, centrifuge to separate the oil phase and the cell-containing aqueous phase. The oil phase is reserved, the cell-containing aqueous phase is mixed with the fermentation liquid, and then crude vegetable oil with the same mass as the separated oil phase is added and fermentation continues; Step 5: Repeat Step 4 until the time when the acid value of the oil phase in the fermentation liquid reaches the set value exceeds 24 hours or is more than twice the time when the acid value of the fermentation liquid reaches the set value after the previous round of oil supplementation, then end the fermentation and separate the oil phase for reserve; Step 6: Mix the oil phases separated in Step 4 and Step 5, and after centrifugation, heating, and filtration for purification, obtain fermented vegetable oil.

[0006] After the acid value of the oil phase in the fermentation broth reaches the set value, the present invention repeatedly discharges a part of the fermentation broth, centrifuges to remove the oil phase, returns the water phase containing active bacteria to the fermentation tank, and simultaneously supplements fresh crude vegetable oil of equal mass. On the one hand, it can extend the fermentation cycle and increase the yield; on the other hand, it can better control the accumulation of metabolic by-products and is more conducive to the separation and purification of products; on the third hand, it can better utilize the nutrients in the culture medium, reduce waste, and at the same time reduce the time and energy consumption of material preparation and equipment preparation, improve production efficiency, and reduce production costs. This method is applicable to various microorganisms including but not limited to yeast, mold, bacteria, etc. The application fields of the fermented vegetable oil produced by this method include but not limited to the fields of cosmetic skin care, food, and the pharmaceutical industry.

[0007] The strains include but are not limited to any one of yeast, bacteria or mold, or any combination thereof.

[0008] The test of the acid value refers to the QS-TM-16 standard.

[0009] The set value is 3 - 30 mgKOH / g.

[0010] More preferably, the set value is 8 - 25 mgKOH / g.

[0011] Most preferably, the set value is 10 - 20 mgKOH / g.

[0012] The mass ratio of the crude vegetable oil to the primary fermentation broth is (1~4):(1~4).

[0013] Preferably, the mass ratio of the crude vegetable oil to the primary fermentation broth is (1~2):(1~2).

[0014] Most preferably, the mass ratio of the crude vegetable oil to the primary fermentation broth is (1~1.5):(1~1.5).

[0015] In step 4, 4 - 60% of the total fermentation broth is taken out.

[0016] More preferably, in step 4, 10 - 50% of the total fermentation broth is taken out.

[0017] Most preferably, in step 4, 20 - 30% of the total fermentation broth is taken out.

[0018] The crude vegetable oil includes at least one of sunflower oil, shea butter, perilla oil, babassu oil, jojoba oil, camellia seed oil, and peony seed oil.

[0019] The cell concentration OD600 (absorbance value at 600 nm wavelength) in the seed liquid is 3 - 60.

[0020] Preferably, the cell concentration OD600 in the seed liquid is 20 - 40.

[0021] The cell concentration OD600 in the primary fermentation broth is 20 - 100.

[0022] More preferably, the cell concentration OD600 in the primary fermentation broth is 40 - 80.

[0023] Most preferably, the cell concentration OD600 in the primary fermentation broth is 50 - 60.

[0024] The second aspect of the present invention provides an application of a method for producing fermented vegetable oil by a semi - continuous fed - batch fermentation process, which is applied to the fields of cosmetic skin care, food, or the pharmaceutical industry.

[0025] Beneficial effects: 1. By monitoring that the acid value of the fermented vegetable oil reaches the set standard, repeatedly discharging part of the fermentation broth, centrifuging to extract the oil phase, retaining the water phase with active cells and returning it to the fermentation tank, and at the same time supplementing the same mass of crude vegetable oil, the fermentation cycle can be extended and the yield can be increased.

[0026] 2. The process method of the present invention can better control the accumulation of metabolic by - products and is more conducive to the separation and purification of products.

[0027] 3. The process method of the present invention can make better use of the nutrient components of the culture medium, reduce waste, and at the same time reduce the time and energy consumption of material preparation and equipment preparation, improve production efficiency, and reduce production costs.

[0028] 4. The present invention is suitable for the fermentation of various crude vegetable oils.

[0029] 5. The process method of the present invention can increase the yield by 10% - 92.8%, and by repeatedly discharging and feeding, the quality of the fermented vegetable oil is ensured and the stability of the product is improved. Description of the drawings

[0030] Figure 1 is the flow chart of the semi - continuous fermentation for producing fermented vegetable oil of the present invention.

[0031] Figure 2 is the curve graph of the acid value change during the fermentation process of Examples 1 - 3 of the present invention.

[0032] Figure 3 is the curve graph of the acid value change during the fermentation process of Examples 4 - 6 of the present invention.

[0033] Figure 4 is the curve graph of the acid value change during the fermentation process of Examples 7 - 9 of the present invention. Detailed implementation manners

[0034] Examples 1 - 9, Comparative Example 1 - 1, Comparative Example 1 - 2, Comparative Examples 2 - 9 A method for producing fermented vegetable oil by a semi - continuous fed - batch fermentation process, the flow chart is as Figure 1 shown, and the process parameters are shown in Table 1. The steps are as follows: Step 1: Preparation of seed liquid. Take a 500 - ml glass Erlenmeyer flask, fill it with 150 g of seed - liquid medium, and sterilize it at 121 °C for 30 minutes.

[0035] Inoculate 1.8 ml of glycerol - stored Candida apicola strain into the sterilized seed - medium flask, culture it at 25 °C and 200 rpm for 36 h to obtain a seed liquid with an OD600 of 31; Step 2: Preparation of the first - stage fermentation broth. Prepare a liquid fermentation medium in a 5 - liter glass stirred - tank fermenter, sterilize it at 121 °C for 30 minutes, and then inoculate the seed liquid cultured in Step 1 into the fermenter at an inoculation amount of 5% for cell mass expansion (25 °C, 600 rpm, aeration rate 0.6 vvm, 25 h) to obtain a first - stage fermentation broth with an OD600 of 45 (Table 1, quality of the first - stage fermentation broth); Step 3: Mix the crude vegetable oil with the first - stage fermentation broth for vegetable - oil fermentation to obtain a fermentation broth; Step 4: During the vegetable - oil fermentation process, sample every 1 - 4 hours according to the increasing rate of acid value, monitor the acid value of the oil phase in the fermentation broth. After the acid value reaches the set value (Table 1, set acid value), take out a part of the fermentation broth (Table 1, single - discharge amount), perform centrifugal separation to obtain an oil phase and an aqueous phase, reserve the oil phase, mix the aqueous phase with the fermentation broth, and then add crude vegetable oil with the same mass as the separated oil phase to continue fermentation; Step 5: Repeat Step 4 until the duration when the acid value of the oil phase in the fermentation broth reaches the set value exceeds 24 h or is twice that of the previous time, then the fermentation ends, and the oil phase is separated and reserved; Step 6: Mix the oil phases separated in Step 4 and Step 5, and obtain the fermented vegetable oil after centrifugation, heating, and filtration.

[0036] Table 1

[0037] Performance test method The wetting angle refers to the angle at the three - phase junction of solid, liquid, and gas. The angle from the solid - liquid interface through the liquid interior to the gas - liquid interface is called the contact angle, also known as the wetting angle. The wetting angle of fermented oil (Oil Spreading Angle or Oil Displacement Angle) is one of the important indicators for evaluating the performance of biosurfactants. This angle directly reflects the ability of the surfactant to reduce the surface tension of the liquid and promote the spreading at the oil - water interface.

[0038] The surface tension and wetting angle of the fermentation oils in Examples 1-9, Comparative Example 1-1, Comparative Example 1-2, and Comparative Examples 2-9 were tested using a surface tensiometer. The test results are shown in Table 2.

[0039] Table 2

[0040] Table 3

[0041] Comparing the productivity of different experimental schemes and the percentage increase in the yield of the examples compared to the comparative examples, the data are shown in Table 3. The process method of the present invention can increase the yield by 10%-92.8%. Moreover, by repeatedly discharging and replenishing materials, the quality of the fermented vegetable oil is ensured, and the stability of the product is improved.

[0042] The data in Table 2 show that the performance of the fermentation oils in the examples is not significantly different from that of the comparative examples, indicating that the performance of the fermented vegetable oils obtained by continuous fermentation is relatively stable.

Claims

1. A method for producing fermented vegetable oil by a semi-continuous fed-batch fermentation process, characterized in that: The following steps are involved: Step 1: inoculating the bacterial strain into a seed culture medium for bacterial culture to obtain a seed solution; Step 2: inoculating the seed liquid into the fermentation medium for bacterial expansion to obtain a primary fermentation liquid; Step 3: mixing the crude vegetable oil with the primary fermentation liquid in proportion, fermenting the vegetable oil to obtain a fermentation liquid; Step 4: during the vegetable oil fermentation process, samples are taken regularly to detect the acid value of the oil phase in the fermentation liquid. When the acid value reaches the set value, the fermentation liquid accounting for 4-60% of the total fermentation liquid is taken out, and centrifuged to obtain the oil phase and the bacterial cell-containing aqueous phase, the oil phase is reserved, the bacterial cell-containing aqueous phase is mixed with the fermentation liquid, and then the vegetable oil crude oil of the same mass as the separated oil phase is added to continue the fermentation; Step 5: Repeat step 4 until the time for the acid value of the oil phase in the fermentation liquid to reach the set value exceeds 24 hours or the time for the acid value of the fermented oil after the previous round of oil supplementation to reach the set value is more than twice, then the fermentation is terminated and the oil phase is separated for later use; Step 6: mixing the oil phases separated in step 4 and step 5, and obtaining fermented vegetable oil after purification; The set value is 3-30 mgKOH / g.

2. The method for producing fermented vegetable oil by semi-continuous fed-batch fermentation process according to claim 1, characterized in that: The mass ratio of the crude vegetable oil to the primary fermentation liquid is (1-4): (1-4).

3. The method for producing fermented vegetable oil by semi-continuous fed-batch fermentation process according to claim 1, characterized in that: The set value is 8-25 mgKOH / g.

4. The method for producing fermented vegetable oil by semi-continuous fed-batch fermentation process according to claim 1, characterized in that: In step 4, 4-60% of the fermentation liquid is taken out.

5. The method for producing fermented vegetable oil by semi-continuous fed-batch fermentation process according to claim 1, characterized in that: The crude vegetable oil comprises at least one of sunflower seed oil, shea butter, perilla seed oil, babassu seed oil, jojoba oil, camellia seed oil and peony seed oil.

6. The method for producing fermented vegetable oil by semi-continuous fed-batch fermentation process according to claim 1, characterized in that: The bacterial cell concentration OD600 in the seed solution is 3-60.

7. The method for producing fermented vegetable oil by semi-continuous fed-batch fermentation process according to claim 1, characterized in that: The bacterial cell concentration OD600 in the primary fermentation liquid is 20-100.

8. An application of the method for producing fermented vegetable oil by semi-continuous fed-batch fermentation process according to any one of claims 1 to 7, characterized in that: Used in cosmetics and skin care, food or pharmaceutical industries.

Images