Double-compartment mixed bacteria fermentation device capable of collecting pure bacteria on line

Abstract

The utility model provides a double-compartment mixed bacteria fermentation device capable of collecting pure bacteria on line. The double-compartment mixed bacteria fermentation device comprises a centrifugal tube, a dialysis device is arranged in the centrifugal tube, a centrifugal tube cover is arranged at the top of the centrifugal tube, a fixing hole is formed in the centrifugal tube cover, and an S-shaped one-way valve is fixed by the centrifugal tube cover through the fixing hole. The double-compartment mixed bacteria fermentation device capable of collecting pure bacteria on line solves the problems that the volume ratio of two chambers of an existing mixed bacteria fermentation device is too large in difference, the effect of simulating mixed bacteria fermentation is poor, and cost is high, the device is easy to build, a pump does not need to be installed, the design is ingenious, wine alcoholic fermentation can be guaranteed to be completed smoothly, and the cost is low. And two types of florae can be effectively separated while metabolite exchange exists, so that online collection of pure bacteria is realized.

Description

Technical Field

[0001] The utility model relates to the technical field of brewing bacteria fermentation devices, in particular to a double-compartment mixed bacteria fermentation device capable of collecting pure bacteria online. Background Art

[0002] The conventional wine fermentation process is to add a single commercial brewing yeast to the grape juice to complete the alcohol fermentation. Although it can effectively reduce the risk of the wine fermentation process and ensure the quality of the wine, it will also lead to a single aroma of the wine, aggravate the homogeneity of the flavor, and lose the characteristics of the wine. Therefore, in recent years, researchers have introduced non-brewing yeasts with outstanding aroma production ability into grape juice and mixed fermentation with brewing yeast. This can not only successfully complete the alcohol fermentation, but also give the wine more complex flavor characteristics through the secondary metabolism of non-brewing yeasts, shape the flavor characteristics of the wine, and effectively improve the situation of flavor homogeneity of domestic wines.

[0003] The mixed fermentation culture of brewing yeast / non-brewing yeast has become a hot topic in current research and is widely used in production practice. However, in this mixed fermentation system, non-brewing yeast and brewing yeast will compete for nutrients, occupy ecological niches, and have cell-to-cell contact, which makes the wine produced unstable in aroma and uncontrollable in quality.

[0004] In order to solve the above problems, it is necessary to understand the ecological interactions of different species on the aroma of wine and clarify the interaction mechanism between the two strains in the mixed fermentation system. Based on this, researchers have designed some double-compartment fermentation devices for mixed fermentation, which are mainly divided into left and right double compartments and inner and outer double compartments. Both can separate the two different bacterial communities through biofilms, which can not only simulate the environment of mixed fermentation, but also help to accurately control the quality of wine. However, there are some problems with the existing double-compartment fermentation devices. For example, although the inner and outer double compartment fermentation device is easy to build, the volume ratio of the two chambers is too different, and the effect of simulating mixed fermentation is poor; although the left and right double compartment fermentation device has a good simulation effect, it needs to install a pump separately to promote the exchange of substances between the two chambers, and the cost of experimental materials is relatively high. Utility Model Content

[0005] The utility model aims to provide a double-compartment mixed bacteria fermentation device capable of collecting pure bacteria online, which solves the problems of large difference in volume ratio between the two chambers of the existing mixed bacteria fermentation device, poor effect of simulated mixed bacteria fermentation and high cost.

[0006] The technical solution adopted by the utility model is: a double-compartment mixed bacteria fermentation device capable of collecting pure bacteria online, comprising a centrifuge tube, a dialysis device is arranged inside the centrifuge tube, a centrifuge tube cover is arranged on the top of the centrifuge tube, the centrifuge tube cover is provided with a fixing hole, and the centrifuge tube cover fixes an S-type one-way valve through the fixing hole.

[0007] The technical solution adopted by the utility model is also characterized by:

[0008] Furthermore, the interior of the dialysis device is an inner chamber, and the exterior of the dialysis device is an outer chamber.

[0009] Furthermore, a plurality of holes are opened on the top of the dialysis device, and the hole diameters are 0.8 mm to 1.2 mm.

[0010] Furthermore, a fixing hole is provided between the center of the centrifuge tube cover and the edge of the cover.

[0011] Furthermore, the diameter of the fixed hole is adapted to the outer diameter of the S-type one-way valve.

[0012] Furthermore, the end of the S-shaped one-way valve passes through the fixed hole and extends into the centrifuge tube.

[0013] The beneficial effects of the utility model are:

[0014] 1. The utility model provides a double-compartment mixed bacteria fermentation device that can collect pure bacteria online. The device is easy to build, does not require pumps, and has a smart design. It can not only ensure the smooth completion of wine alcohol fermentation, but also effectively separate two types of bacteria while exchanging metabolites, and realize the online collection of pure bacteria. The device can further clarify the individual effects of the ecological interaction between strains in the mixed bacteria fermentation system and clarify the interaction mechanism of the mixed bacteria fermentation system, thereby realizing the accurate regulation of wine aroma and producing more excellent quality specialty wines.

[0015] 2. Compared with the previous devices, the fermentation device of the utility model has a small fermentation volume and is simple to build. All experimental materials except the dialysis membrane in the inner chamber can be reused. There is no need to prepare materials again for secondary use, which saves manpower, material and financial resources for the experiment and improves the efficiency of scientific research work.

[0016] 3. Compared with the left and right double compartment fermentation device, the utility model has a smart design, does not require an additional pump, and is simple to build and easy to operate.

[0017] 4. Compared with the double compartments of inner and outer chambers, the fermentation volume ratio of the two chambers of the fermentation device of the utility model is smaller, the volume is closer, and the effect of simulating mixed bacteria fermentation is better.

[0018] 5. The fermentation device of the utility model can effectively separate two different bacterial communities and realize the online collection of pure bacteria without affecting the exchange of metabolites and nutrients in the two chambers. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a structural schematic diagram of the fermentation device of the utility model;

[0020] Figure 2aIt is a schematic diagram of the centrifuge tube cover structure of the fermentation device of the utility model;

[0021] Figure 2b It is a schematic diagram of the centrifuge tube structure of the fermentation device of the utility model;

[0022] Figure 2c It is a schematic diagram of the structure of the dialysis device of the fermentation device of the utility model;

[0023] Figure 2d It is a schematic diagram of the structure of an S-type one-way valve of a fermentation device of the utility model;

[0024] Figure 3 It is an application schematic diagram of the fermentation device of the utility model;

[0025] Figure 4a The NX09 in the inner chamber of the fermentation device of the utility model is diluted by 10 5 The colony picture after culturing on WLN plate for 5 days;

[0026] Figure 4b The F5 in the outer chamber of the fermentation device of the utility model is diluted by 10 5 The colony picture after culturing on WLN plate for 5 days;

[0027] Figure 5a It is a line graph of sugar consumption during the fermentation process of the fermentation device of the utility model;

[0028] Figure 5b It is a line graph of ethanol production during the fermentation process of the fermentation device of the utility model.

[0029] In the figure: 1. centrifuge tube cover, 2. centrifuge tube, 3. dialysis device, 4. S-type one-way valve. DETAILED DESCRIPTION

[0030] The utility model is described in detail below with reference to the accompanying drawings and specific implementation modes.

[0031] The utility model provides a double-compartment mixed bacteria fermentation device capable of collecting pure bacteria online: Figure 1 As shown, comprising a centrifuge tube 2, such as Figure 2b As shown, a dialysis device 3 is arranged inside the centrifuge tube 2, a centrifuge tube cover 1 is arranged on the top of the centrifuge tube 2, and the centrifuge tube cover 1 has a fixing hole, such as Figure 2c As shown, the centrifuge tube cover 1 fixes the S-type one-way valve 4 through the fixing hole.

[0032] The interior of the dialysis device 3 is the inner chamber, and the exterior of the dialysis device 3 is the outer chamber. Figure 2aAs shown, a fixing hole is opened between the center of the centrifuge tube cover 1 and the edge of the cover, the diameter of the fixing hole is adapted to the outer diameter of the S-type one-way valve 4, the end of the S-type one-way valve 4 passes through the fixing hole and extends into the centrifuge tube 2, and a plurality of holes are opened on the top of the dialysis device 3, and the hole diameter is 0.8mm-1.2mm.

[0033] The utility model is described in detail below with reference to the accompanying drawings and embodiments.

[0034] Example 1

[0035] A double-compartment mixed bacteria fermentation device capable of collecting pure bacteria online comprises a centrifuge tube 2, a dialysis device 3 is arranged inside the centrifuge tube 2, a centrifuge tube cover 1 is arranged on the top of the centrifuge tube 2, the centrifuge tube cover 1 has a fixing hole, the centrifuge tube cover 1 fixes an S-type one-way valve 4 through the fixing hole, the inside of the dialysis device 3 is an inner chamber, the outside of the dialysis device 3 is an outer chamber, a plurality of holes are arranged on the top of the dialysis device 3, and the hole diameter is 0.8 mm.

[0036] Example 2

[0037] A double-compartment mixed bacteria fermentation device capable of collecting pure bacteria online comprises a centrifuge tube 2, a dialysis device 3 is arranged inside the centrifuge tube 2, a centrifuge tube cover 1 is arranged on the top of the centrifuge tube 2, the centrifuge tube cover 1 is provided with a fixing hole, the centrifuge tube cover 1 fixes an S-type one-way valve 4 through the fixing hole, the inside of the dialysis device 3 is an inner chamber, the outside of the dialysis device 3 is an outer chamber, a plurality of holes are arranged on the top of the dialysis device 3, the hole diameter is 1 mm, and a fixing hole is arranged between the center of the centrifuge tube cover 1 and the edge of the cover.

[0038] Example 3

[0039] A double-compartment mixed bacteria fermentation device capable of collecting pure bacteria online comprises a centrifuge tube 2, a dialysis device 3 is arranged inside the centrifuge tube 2, a centrifuge tube cover 1 is arranged on the top of the centrifuge tube 2, the centrifuge tube cover 1 is provided with a fixing hole, the centrifuge tube cover 1 fixes an S-type one-way valve 4 through the fixing hole, the inside of the dialysis device 3 is an inner chamber, the outside of the dialysis device 3 is an outer chamber, a plurality of holes are provided on the top of the dialysis device 3, the hole diameter is 1.2 mm, a fixing hole is provided between the center of the centrifuge tube cover 1 and the edge of the cover, the aperture of the fixing hole is adapted to the outer diameter of the S-type one-way valve 4, and the end of the S-type one-way valve 4 extends into the centrifuge tube 2 through the fixing hole.

[0040] like Figure 3 As shown, the application method of the fermentation device of the utility model is as follows:

[0041] 1. Construction of the device:

[0042] 1) Take a 50mL screw-cap centrifuge tube and make a hole between the center of the centrifuge tube cap and the edge of the cap. The diameter of the hole is 8mm. Figure 2a , Figure 2bAs shown; Take a biotechnology-grade dialysis device with a volume of 5 mL and a pore size of 1000 kDa, open 5 holes at the top of the device, the diameter of the holes is 1 mm, and the number is 1, as shown in FIG. Figure 2c shown.

[0043] 2) Take an S-type check valve with an outer diameter of 8 mm, such as Figure 2d As shown, it is assembled with the punched centrifuge tube and the biotechnology-grade dialysis device. Figure 3 shown.

[0044] 2. Fermentation application of the device:

[0045] 1) Yeast activation, purification and expansion

[0046] Take 100 μL of -80℃ glycerol stock of commercial Saccharomyces cerevisiae F5 and Hanseniaspora osmophila NX09 (CCTCC NO: M 20232410) respectively in a test tube containing 5mL YPD medium and culture at 28℃ for 48h. Use an inoculation loop to dip the cultured bacterial liquid and streak it on WLN medium. After culture at 28℃ for 72h, obtain a purified single colony. Pick the purified single colony, inoculate it in 100mL YPD medium, place it in a shaker, and culture it at 28℃ and 160rpm / min for 48h to obtain a yeast starter.

[0047] 2) Preparation of simulated grape juice

[0048] 1L of the simulated grape juice is composed of the following ingredients: 100g glucose, 100g fructose, 3g malic acid, 0.2g citric acid, 1.14g potassium dihydrogen phosphate, 1.23g magnesium sulfate heptahydrate, 3.12g potassium sodium tartrate, 0.44g calcium chloride dihydrate, 198.2μg manganese chloride tetrahydrate, 135.5μg zinc chloride, 32μg ferrous chloride, 13.6μg cupric chloride, 5.7μg boric acid, 29.1μg cobalt nitrate, 24.2μg sodium molybdate, 10.8μg potassium iodide, 100mg inositol, 5mg pyridoxine hydrochloride, 2mg nicotinic acid, 1mg calcium pantothenate, 0.5mg nicotinic acid thiamine, 0.2mg p-aminophenyl Formic acid, 0.125 mg vitamin H, 0.2 mg folic acid, 0.2 mg riboflavin and 200 mg assimilable nitrogen amino acids (0.1 g alanine, 0.75 arginine, 0.15 g asparagine, 0.35 g aspartic acid, 0.5 g glutamic acid, 0.2 g glutamine, 0.05 g glycine, 0.15 g histidine, 0.2 g isoleucine, 0.3 g leucine, 0.25 g lysine, 0.15 g methionine, 0.15 g phenylalanine, 0.5 g proline, 0.4 g serine, 0.35 g threonine, 0.1 g tryptophan, 0.02 g tyrosine, 0.2 g valine, 0.1 g ammonium chloride). The simulated juice was adjusted to pH 3.5 and sterilized by filtration through a 0.22 μm membrane.

[0049] 3) Yeast inoculation and fermentation

[0050] The simulated grape juice was divided into the inner and outer chambers of the device, with the inner chamber containing 4 mL of liquid and the outer chamber containing 26 mL, ensuring that the inner and outer liquid levels were flush. F5 was inoculated into the outer chamber and NX09 was inoculated into the inner chamber, with the inoculation volume of 2×10 6 cells / mL. The fermentation diagram of the device is shown in Figure 3 As shown, the fermentation liquid was placed in a shaker for shaking fermentation (25°C, 150 rpm / min), and samples were taken from the inner and outer chambers during the fermentation process. The fermentation endpoint was considered when the sugar content in the fermentation liquid was ≤2 g / L.

[0051] 3. Device feasibility assessment

[0052] 1) Dilution and coating of yeast in inner and outer chambers

[0053] Take appropriate amount of bacterial solution from inner and outer chambers respectively into sterile centrifuge tubes and dilute them in sequence to 10 5 Afterwards, 50 μL of the diluted bacterial solution was spread on the WLN plate, and the plate was placed in a 28°C constant temperature incubator for 5 days before a single colony was obtained. Figure 4a This is the NX09 colony picture in the inner room (the colony is green and flat). Figure 4bThis is the F5 colony picture of the outer chamber (the colonies are white and convex). The results show that there is no cross-contamination between the two yeasts in the inner and outer chambers during the fermentation process, indicating that the device is effective in blocking the two bacterial communities.

[0054] 2) Determination of reducing sugar and ethanol content during fermentation

[0055] 1 mL of fermentation sample was taken and filtered using a 0.22 μm filter membrane and a syringe. The reducing sugar content and ethanol content in the fermentation sample were determined by high performance liquid chromatography. The high performance liquid chromatograph was Shimadzu LC-2050C 3D. HPX-87H chromatographic column was connected to RID-20A detector, and the detection conditions were: column temperature 60℃, flow rate 0.6mL / min, injection volume 20μL, mobile phase 10mmol / L sulfuric acid solution. According to the change curve of sugar content and ethanol content in the inner and outer chambers during the fermentation process, Figure 5a and Figure 5b As shown, alcohol fermentation was successfully completed within 10 days, and there was no significant difference in reducing sugar content and ethanol content between the inner and outer chambers on the same day. The results show that when the device is used for mixed bacterial fermentation, the inner and outer chambers can exchange nutrients with good exchange effect.

[0056] In summary, the application of this device for mixed bacteria fermentation can not only complete alcohol fermentation, but also effectively separate two types of bacteria and exchange nutrients at the same time. In addition, the device is simple to build and easy to sample, so this method is feasible for mixed bacteria fermentation.

Claims

1. A double-compartment mixed bacteria fermentation device capable of collecting pure bacteria online, characterized in that: The centrifuge tube (2) comprises a dialysis device (3) arranged inside the centrifuge tube (2), a centrifuge tube cover (1) arranged on the top of the centrifuge tube (2), the centrifuge tube cover (1) having a fixing hole, and the centrifuge tube cover (1) fixing an S-shaped one-way valve (4) through the fixing hole.

2. The dual-compartment mixed bacteria fermentation device capable of collecting pure bacteria online according to claim 1, characterized in that: The interior of the dialysis device (3) is an inner chamber, and the exterior of the dialysis device (3) is an outer chamber.

3. The dual-compartment mixed bacteria fermentation device capable of collecting pure bacteria online according to claim 1, characterized in that: The top of the dialysis device (3) is provided with a plurality of holes, and the hole diameters are 0.8 mm to 1.2 mm.

4. The dual-compartment mixed bacteria fermentation device capable of collecting pure bacteria online according to claim 1, characterized in that: A fixing hole is provided between the center of the centrifuge tube cover (1) and the edge of the cover.

5. The double-compartment mixed bacteria fermentation device capable of collecting pure bacteria online according to claim 1, characterized in that: The diameter of the fixed hole is adapted to the outer diameter of the S-type one-way valve (4).

6. The dual-compartment mixed bacteria fermentation device capable of collecting pure bacteria online according to claim 1, characterized in that: The end of the S-shaped one-way valve (4) passes through the fixing hole and extends into the centrifuge tube (2).

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