A method for producing a fermented vegetable oil by a semi-continuous fed-batch fermentation process

Abstract

The present application relates to a kind of semi-continuous fed-batch fermentation process production fermented vegetable oil method, first inoculate strain to fermentation medium under appropriate conditions and carry out cell propagation, then mixed with vegetable oil crude oil according to certain proportion, fermentation is carried out at appropriate temperature, after fermentation index reaches set value, part of fermentation liquor is discharged, oil phase is taken out by centrifugation, water phase containing active bacteria is returned to fermenter, while the same mass of fresh vegetable oil crude oil is supplemented to continue fermentation.So repeat, until the time length when the oil phase acid value of fermentation liquor reaches set value is more than 24h or the fermentation after oil supplement in previous round reaches set value more than 2 times, fermentation is ended, oil phase is separated, and centrifugal separation purification is carried out to obtain fermented vegetable oil.The productivity of fermented vegetable oil can be greatly improved, the production yield of single batch is increased, while the stability of fermented oil quality is ensured, production cost is saved, and it can be widely applied in the field of cosmetics, food field or pharmaceutical industry.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial fermentation, and particularly relates to a method for producing fermented vegetable oil by semi-continuous fed-batch fermentation process. BACKGROUND

[0002] Microbial fermentation mainly includes four modes of batch fermentation, fed-batch fermentation, semi-continuous fed-batch fermentation and continuous fed-batch fermentation. Batch fermentation: all substrates and strains are put in at one time, and no feeding or discharging is performed throughout the process. It is simple to operate but low in efficiency, and the substrate concentration gradually decreases with metabolism, finally limiting the yield due to insufficient nutrition. Fed-batch fermentation: the substrate is added in batches or continuously but not discharged, which maintains the substrate concentration, improves the yield and efficiency. However, there are problems such as long fermentation period, accumulation of harmful metabolites, and inhibition of product synthesis, and the feeding amount depends on experience. Continuous / semi-continuous fed-batch fermentation: continuous or intermittent feeding and discharging of fermentation broth, maintaining constant volume, substrate and product concentration in the tank. 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 the strain is easy to vary. The existing technology mostly adopts batch or fed-batch mode, which requires cleaning, sterilization and other operations between batches, resulting in large resource consumption and low efficiency. The existing fermentation mode of vegetable oil takes the efficacy of fermented vegetable oil as the evaluation standard, lacks consideration and concern for fermentation yield and fermentation efficiency, and there is no report on semi-continuous fermentation process for producing fermented vegetable oil.

[0003] Chinese invention patent application CN117180143A adopts two-stage yeast step-by-step fermentation of shea nut oil, and the total period is long, the production efficiency is low, the fermented oil yield of unit volume of fermentation broth 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 by batch fermentation, but long-time fermentation leads to decomposition of triglyceride, high acid value and decreased quality, and the fermented mufu oil yield of unit mass of fermentation broth is only 333g / kg, and the fermentation efficiency is 2.31g / kg / h.

[0004] Chinese invention patent application CN118286117A discloses a fed-batch fermentation mode. The invention first adds glucose, bacterial cells, rapeseed oil, and macadamia nut oil into a fermentation tank for low-speed stirring fermentation at 28-32 degrees for 70-74 hours, then adds rapeseed oil and macadamia nut oil again, and after low-speed stirring fermentation for 158-162 hours, performs heating, sterilization, cooling, and centrifugation to obtain fermented oil. In the fed-batch fermentation mode, the yield and production efficiency of the mixed oil of fermented rapeseed oil and fermented macadamia nut oil are doubled compared to the batch fermentation mode. Ignoring the seed preparation time (not given in the patent), according to the data given in Examples 1-3, the yield of fermented mixed oil reaches 684-686 g / kg, and the fermentation efficiency is 2.908-3 g / kg / h. SUMMARY

[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:

[0006] Step 1: Preparation of seed solution: inoculate the bacterial strain into the seed culture medium for bacterial culture to obtain the seed solution;

[0007] Step 2: Preparation of primary fermentation solution: inoculate the seed solution into the fermentation culture medium for bacterial strain expansion to obtain the primary fermentation solution;

[0008] Step 3: Vegetable oil fermentation: mix the vegetable oil crude oil with the primary fermentation solution in a certain proportion, and perform vegetable oil fermentation to obtain the fermentation solution;

[0009] Step 4: During the vegetable oil fermentation process, periodically take samples to detect the acid value of the oil phase in the fermentation solution. After the acid value reaches the set value, take out 4-60% of the fermentation solution, centrifuge to separate the oil phase and the bacterial cell-containing water phase, reserve the oil phase, mix the bacterial cell-containing water phase with the fermentation solution, and add vegetable oil crude oil with the same mass as the separated oil phase to continue fermentation;

[0010] Step 5: Repeat Step 4 until the time when the acid value of the oil phase in the fermentation solution reaches the set value exceeds 24 hours or is more than twice the time when the acid value of the fermented oil reaches the set value after the previous round of oil addition, end the fermentation, and separate the oil phase for reserve;

[0011] Step 6: Mix the oil phases separated in Step 4 and Step 5, centrifuge, heat, filter, and purify to obtain the fermented vegetable oil.

[0012] The present application can prolong the fermentation period, improve the yield, better control the accumulation of metabolic by-products, and be more conducive to the separation and purification of the product, better utilize the nutritional components of the culture medium, reduce waste, reduce the time and energy consumption of material preparation and equipment preparation, improve production efficiency, and reduce production cost. The method is suitable for various microorganisms including but not limited to yeast, mold, bacteria, etc. The application field of the fermented vegetable oil produced by the method includes but is not limited to the fields of cosmetics and skin care, food, and medicine.

[0013] The bacteria include but are not limited to any one of yeast, bacteria, or mold, or any combination thereof.

[0014] The acid value is tested according to the QS-TM-16 standard.

[0015] The set value is 3-30 mgKOH / g.

[0016] More preferably, the set value is 8-25 mgKOH / g.

[0017] Most preferably, the set value is 10-20 mgKOH / g.

[0018] The mass ratio of the vegetable oil crude oil to the primary fermentation liquid is (1-4):(1-4).

[0019] Preferably, the mass ratio of the vegetable oil crude oil to the primary fermentation liquid is (1-2):(1-2).

[0020] Most preferably, the mass ratio of the vegetable oil crude oil to the primary fermentation liquid is (1-1.5):(1-1.5).

[0021] In step 4, 4-60% of the total fermentation liquid is taken out.

[0022] More preferably, in step 4, 10-50% of the total fermentation liquid is taken out.

[0023] Most preferably, in step 4, 20-30% of the total fermentation liquid is taken out.

[0024] The vegetable oil crude oil includes at least one of sunflower seed oil, shea butter, perilla seed oil, babassu seed oil, jojoba oil, camellia seed oil, and peony seed oil.

[0025] The concentration of the bacteria in the seed liquid is OD600 (absorbance at 600 nm wavelength) of 3-60.

[0026] Preferably, the concentration of the bacteria in the seed liquid is OD600 of 20-40.

[0027] The concentration of the bacteria in the primary fermentation liquid is OD600 of 20-100.

[0028] More preferably, the concentration of the bacteria in the primary fermentation liquid is OD600 of 40-80.

[0029] Most preferably, the concentration of the bacteria in the primary fermentation liquid is OD600 of 50-60.

[0030] The second aspect of the present application provides an application of a method for producing a fermented vegetable oil by a semi-continuous fed-batch fermentation process, which is applied in the field of cosmetic skin care, food or pharmaceutical industry.

[0031] Advantages:

[0032] 1. By monitoring the acid value of the fermented vegetable oil to reach the set standard, repeatedly discharging part of the fermentation liquid, centrifuging to obtain the oil phase, and returning the water phase with active bacteria to the fermentation tank while supplementing the same mass of crude vegetable oil, the fermentation period can be prolonged and the yield can be improved.

[0033] 2. The process method of the present application can better control the accumulation of metabolic by-products, which is more conducive to the separation and purification of the product.

[0034] 3. The process method of the present application can better utilize the nutritional 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 cost.

[0035] 4. The present application is suitable for the fermentation of various vegetable oils.

[0036] 5. The process method of the present application can increase the yield by 10%-92.8%, and through repeated discharging and feeding, the quality of the fermented vegetable oil is ensured and the stability of the product is improved. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is a flow chart of the semi-continuous fermentation for producing a fermented vegetable oil of the present application.

[0038] Figure 2 is a curve graph of the acid value change in the fermentation process of Example 1-Example 3 of the present application.

[0039] Figure 3 is a curve graph of the acid value change in the fermentation process of Example 4-Example 6 of the present application.

[0040] Figure 4 is a curve graph of the acid value change in the fermentation process of Example 7-Example 9 of the present application. DETAILED DESCRIPTION

[0041] Examples 1-9, Comparative Examples 1-1, Comparative Example 1-2, Comparative Examples 2-9

[0042] A method for producing a fermented vegetable oil by a semi-continuous fed-batch fermentation process, the flow chart is shown as Figure 1 The process parameters are shown in Table 1, and the steps are as follows:

[0043] Step 1: Seed liquid preparation, take 500ml glass triangular flask, pour 150g seed liquid medium, sterilize at 121℃ for 30 minutes.

[0044] 1.8ml of glycerol tube frozen Candida bombicola strain is inoculated into the sterilized seed culture medium flask, and cultured at 25℃, 200rpm for 36h to obtain seed liquid with OD600 of 31;

[0045] Step 2: Preparation of primary fermentation liquid, prepare liquid fermentation medium in a 5-liter glass stirring fermentation tank, sterilize at 121℃ for 30 minutes, then inoculate the seed liquid prepared in step 1 into the fermentation tank at an inoculation amount of 5% for cell expansion (25℃, 600rpm, aeration amount 0.6vvm, 25h), to obtain primary fermentation liquid with OD600 of 45 (Table 1, primary fermentation liquid mass);

[0046] Step 3: Mix the vegetable oil crude oil with the primary fermentation liquid to ferment the vegetable oil, and obtain the fermentation liquid;

[0047] Step 4: During the fermentation of vegetable oil, sample every 1-4 hours according to the increase rate of acid value, monitor the acid value of the oil phase in the fermentation liquid, and after the acid value reaches the set value (Table 1, set acid value), take out part of the fermentation liquid (Table 1, single discharge amount), centrifugal separation to obtain oil phase and water phase, the oil phase is reserved, and the water phase is mixed with the fermentation liquid, and the same mass of vegetable oil crude oil as the separated oil phase is added, and the fermentation is continued;

[0048] Step 5: Repeat step 4 until the time length of the acid value of the oil phase in the fermentation liquid reaching the set value is more than 24h or is twice the previous time, and the fermentation is ended, and the oil phase is separated and reserved;

[0049] Step 6: Mix the oil phases separated from steps 4 and 5, centrifugal heating and filtration to obtain the fermented vegetable oil.

[0050] Table 1

[0051]

[0052] Performance test method

[0053] The wetting angle refers to the angle between the solid-liquid interface and the gas-liquid interface in the three-phase junction of solid, liquid and gas, which is also called the contact angle. The oil spreading angle or oil displacement angle of the fermented oil is one of the important indicators for evaluating the performance of the biosurfactant, which directly reflects the ability of the surfactant to reduce the surface tension of the liquid and promote the spreading of the oil-water interface.

[0054] The surface tension and wetting angle of the fermented oil in Examples 1-9, Comparative Examples 1-1, 1-2 and 2-9 were tested by using a surface tension meter, and the test results are shown in Table 2.

[0055] Table 2

[0056]

[0057] Table 3

[0058]

[0059] The production rates of different experimental schemes and the proportion of the yield of the examples compared with the comparative examples are shown in Table 3. The process method of the present application can increase the yield by 10%-92.8%, and through repeated feeding, the quality of the fermented vegetable oil is ensured, and the stability of the product is improved.

[0060] The data in Table 2 shows that the performance of the fermented oil in the examples is not much different from that in the comparative examples, indicating that the performance of the fermented vegetable oil obtained by continuous fermentation is relatively stable.

Claims

1. A method of producing a fermented vegetable oil by a semi-continuous fed-batch fermentation process, characterized in that, The method comprises the following steps: Step 1: inoculating the strain into a seed culture medium to culture the strain, and obtaining a seed liquid; The strain is Candida bombicola; Step 2: inoculating the seed liquid into a fermentation culture medium to expand the strain, and obtaining a primary fermentation liquid; the concentration of the strain in the primary fermentation liquid is OD600 of 40-60; Step 3: mixing the plant oil crude oil and the primary fermentation liquid at a mass ratio of (1-1.5):(1-1.5) to ferment the plant oil, and obtaining a fermentation liquid; Step 4: periodically sampling and detecting the acid value of the oil phase in the fermentation liquid during the plant oil fermentation; after the acid value reaches a set value of 15-20 mgKOH / g, 20-30% of the fermentation liquid is taken out, centrifuged to obtain an oil phase and a water phase containing the strain, the oil phase is reserved, the water phase containing the strain is mixed with the fermentation liquid, and the same mass of plant oil crude oil as the separated oil phase is added to continue the fermentation; Step 5: repeating step 4 until the time when the acid value of the oil phase in the fermentation liquid reaches the set value of 15-20 mgKOH / g is more than 24 h or the time when the acid value of the fermented oil after the oil supplement in the previous round reaches the set value of 15-20 mgKOH / g is more than twice the time of the previous round, the fermentation is ended, and the oil phase is separated and reserved; Step 6: mixing the oil phases separated in steps 4 and 5, and obtaining a fermented plant oil after purification.

2. The process for producing a fermented vegetable oil according to claim 1, wherein, The plant oil crude oil comprises at least one of sunflower seed oil, shea nut oil, perilla seed oil, babassu seed oil, jojoba oil, camellia seed oil, and peony seed oil.

3. The process for producing a fermented vegetable oil according to claim 1, wherein, The concentration of the strain in the seed liquid is OD600 of 3-60.

4. The process as claimed in claim 1, wherein the semi-continuous fed-batch fermentation process for production of the fermented plant oil is carried out at a temperature in the range of 25-35°C. The concentration of the strain in the primary fermentation liquid is OD600 of 40-50.

5. Use of a process for the production of a fermented vegetable oil according to any one of claims 1 to 4, characterized in that, The fermented plant oil is applied in the fields of cosmetic skin care, food, and medicine.

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