Chemostat culture device and method for controlling steady state of growth and metabolism of food microorganisms

Abstract

The invention discloses a chemostat culture device and method for controlling the steady state of growth and metabolism of food microorganisms, the device comprises a reactor and storage containers, the storage containers are divided into a first storage container, a second storage container and a third storage container, the first storage container is used for storing a fresh culture medium, and the second storage container and the third storage container are used for storing an old culture medium; the first storage container is connected with the reactor through a pipeline, and a first peristaltic pump is arranged on the pipeline so as to pump a fresh culture medium stored in the first storage container into the reactor; the reactor is connected with the second storage container and the third storage container through a pipeline and a three-way valve; a second peristaltic pump is arranged on the pipeline, so that the old culture medium fermented in the reactor is pumped into the second storage container and / or the third storage container. The device is provided with a plurality of storages and is matched with the peristaltic pump, so that the constant requirements of various growth rates of different food microorganisms in the logarithmic growth phase are met, and the growth and metabolism steady state of the food microorganisms is controlled.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering and microbiology, and particularly relates to a constant temperature culture device and method for controlling the growth and metabolic steady state of food microorganisms. Background Art

[0002] Food microorganisms play a vital role in food processing. They give food a diverse taste, rich nutritional value and long shelf life through fermentation, maturation and improvement processes. Therefore, it is particularly important to deeply study their metabolic conditions and related characteristics at different growth rates during their growth process.

[0003] The chemostat is a device for experimentally controlling cell growth rate, which provides an effective means to systematically study how growth rate affects cellular processes, including gene expression and metabolism, and the regulatory networks that control cell growth rate. Figure 1 As shown in the figure, the traditional chemostat includes a medium reservoir, a pump, a reactor, an outflow tube and a collection container. The principle is that when cells grow in a fixed volume of culture medium, the culture medium is continuously diluted by adding new culture medium from the medium reservoir to the reactor and removing old culture medium and cells at the same time. According to the cell growth coupled differential equation:

[0004] This differential equation describes the time-dependent evolution of the cell density (x) in the chemostat as a result of cell growth and cell removal by dilution (assuming that cell death is negligible). The cell growth rate (μ) depends on the external nutrient concentration (s), including the maximum growth rate (μ max ) and the half-maximal growth rate constant (K s ) variables. The dilution rate (D) is determined by the rate of medium addition and the rate of culture removal.

[0005] In steady state, the specific growth rate of the cells (i.e., the exponential growth rate constant) is equal to the rate at which the culture is diluted. By varying the dilution rate, steady-state cell populations can be established at different growth rates and under different conditions of nutrient limitation. Experimental control of growth rate using a chemostat is essential for understanding how cell physiology changes with growth rate.

[0006] However, the traditional chemostat culture device only uses the feeding and discharging functions of the chemostat and uses it as a continuous culture device. The main uses include strain domestication and screening of specific functional strains, but it has not yet been applied to control the growth and metabolic homeostasis of food microorganisms, and there is a lack of research on the metabolic conditions of food microorganisms at different growth rates. In addition, the traditional chemostat culture device cannot meet the chemostat culture needs of different food microorganisms, and no corresponding culture methods have been formed for different food microorganisms. Summary of the invention

[0007] In order to overcome the problems of the prior art, the present invention proposes a chemostat culture device and method for controlling the steady state of growth and metabolism of food microorganisms. The device is provided with multiple storage devices and cooperates with a peristaltic pump to complete feeding and discharging, and the stirring device allows the culture system to reach equilibrium faster. By adjusting the pump speed of the peristaltic pump, changing the gas introduced and adjusting the culture volume, the chemostat requirements of various growth rates of different food microorganisms in the logarithmic growth phase are met, so as to achieve the control of the steady state of growth and metabolism of food microorganisms and study them.

[0008] The object of the present invention is achieved in that:

[0009] A first aspect of the present invention provides a chemostat culture device for controlling the steady state of growth and metabolism of food microorganisms, comprising a reactor and storage containers, wherein the storage containers are divided into first, second and third storage containers, wherein the first storage container is used to store fresh culture medium, and the second storage container and the third storage container are used to store old culture medium;

[0010] The first storage container is connected to the reactor via a pipeline, and a first peristaltic pump is arranged on the pipeline to pump the fresh culture medium stored in the first storage container into the reactor;

[0011] The reactor is connected to the second and third storage containers through pipelines and three-way valves. A second peristaltic pump is arranged on the pipeline to pump the old culture medium after fermentation in the reactor into the second storage container and / or the third storage container.

[0012] Furthermore, the reactor is provided with a stirring device, wherein a stirring paddle is arranged inside the reactor tank and is completely immersed in the culture solution, and the stirring paddle is coaxially connected to a motor, which is arranged outside the reactor tank.

[0013] Furthermore, the reactor is also provided with a ventilation device and an online OD detection device, which are respectively used to transport gases including nitrogen, carbon dioxide and oxygen required for microbial growth into the reactor, and to monitor the OD value of the culture solution in real time.

[0014] The second aspect of the present invention provides a chemostat culture method for controlling the growth and metabolic steady state of food microorganisms using the device of the first aspect, comprising the following steps:

[0015] First, the strain is inoculated into the culture medium in the reactor, and the ventilation device and the stirring device are turned on at the same time. After the strain grows to the late logarithmic growth phase or the plateau phase, the first peristaltic pump is turned on to allow the fresh culture medium in the first storage container to flow into the reactor, the second peristaltic pump is turned on, and the three-way valve is adjusted to allow the old culture medium to flow from the reactor into the second storage container. The flow rate of the peristaltic pump is adjusted according to the required growth rate, and the flow rate of the first peristaltic pump is the same as that of the second peristaltic pump; the online OD detection device is turned on to detect the absorbance of the constant temperature system in the reactor at 600nm. When the absorbance value fluctuates within a very small range, it can be said that the constant temperature system has reached a steady state. At this time, the three-way valve is adjusted to allow the old culture medium to flow into the third storage container for subsequent experimental analysis.

[0016] Furthermore, the method is applicable to a variety of food microorganisms including Lactobacillus delbrueckii subsp. bulgaricus, Lactococcus lactis subsp. lactis and Kluyveromyces marxianus.

[0017] The advantages and beneficial effects of the present invention are:

[0018] 1. The constant temperature culture device of the present invention can limit the reactor volume, the first storage container volume, the second storage container volume, the first peristaltic pump flow rate range, and the second peristaltic pump flow rate range based on various growth rates of food microorganisms in the logarithmic growth phase, thereby meeting the constant temperature requirements of various growth rates of different food microorganisms in the logarithmic growth phase.

[0019] 2.OD 600 (optical density at 600nm) is a fast, simple and relatively accurate measurement method for evaluating cell concentration. The absorbance value is proportional to the cell concentration. Therefore, an online OD detection device is set in the reactor of the device of the present invention to monitor the change of cell concentration in real time, so as to judge whether the chemostat system has reached a state of equilibrium.

[0020] 3. In the constant culture method for controlling the steady state of growth and metabolism of food microorganisms using the device of the present invention, after the system reaches a state of equilibrium, the old culture medium is collected and the metabolic products of the bacteria therein are detected and analyzed. The substrate consumption rate and product generation rate of the strain at the growth rate can be calculated, and the growth rate of food microorganisms is matched with the metabolic rate. This plays an important role in studying the production process of fermented food and can also effectively guide the optimization of the fermentation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0022] Figure 1 It is a schematic diagram of the structure of the existing chemostat;

[0023] Figure 2is a schematic diagram of the structure of the chemostat culture device described in Example 1 of the present invention;

[0024] Figure 3-Figure 5 The chemostat culture of LBA3 at different growth rates described in the test example is shown;

[0025] Figure 6-Figure 8 The chemostat culture of BL19 at different growth rates described in the test example is shown;

[0026] Fig. 9 The phenylethanol production of Kluyveromyces marxii at different phenylalanine concentrations described in Example 3 is shown;

[0027] Fig.10 This is a graph showing the chemostat results corresponding to the maximum production rate of phenylethanol in Example 3. DETAILED DESCRIPTION

[0028] The implementation of the present invention is not limited to the following embodiments, and any form of modification and / or change made to the present invention will fall within the scope of protection of the present invention. In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. Unless otherwise specified, the methods used in the embodiments are common techniques in the art.

[0029] For numerical ranges, the endpoints of each range, the endpoints of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be deemed to be specifically disclosed herein.

[0030] The strain Lactobacillus delbrueckii subspecies bulgaricusA3 (Lactobacillus delbrueckii subspecies bulgaricusA3) involved in the embodiment of the present invention comes from traditional fermented milk and is isolated by the Food Flavor and Health Innovation Team of Beijing Technology and Business University; the strain Lactococcus lactis subsp.lactis IMAU11919 (Lactococcus lactis subspecies BL19) comes from traditional fermented milk and is purchased from the China General Microbiological Culture Collection Center (CGMCC), with a preservation number of CGMCC No.16242 and a preservation date of 2018.08.09; the strain Kluyveromyces marxianus N2-2 (Kluyveromyces marxianus N2-2) is purchased from the China Industrial Microbiological Culture Collection Center (CICC), with a preservation number of CICC 1953 and a preservation date of 2007.5.23.

[0031] The culture medium formula involved in the embodiment of the present invention includes: strain LBA3 uses MRS culture medium, the formula is: 10g / L protein Chen, 5g / L beef powder, 4g / L yeast powder, 20g glucose, 1ml Tween 80, 2g / L dipotassium hydrogen phosphate, 5g / L sodium acetate, 2g / L triammonium citrate, 0.2g / L magnesium sulfate, 0.05g / L manganese sulfate, dissolved in 1000ml deionized water, and sterilized at 121°C for 15min.

[0032] The strain BL19 uses M17 medium with the following formula: 5 g soy peptone, 5 g tryptone, 5 g beef extract powder, 19 g sodium β-glycerophosphate, 0.25 g magnesium sulfate, 0.5 g ascorbic acid, 2.5 g yeast extract powder, 5 g lactose, and 5 g glucose, dissolved in 1000 ml deionized water and sterilized at 121°C for 15 min.

[0033] Example 1

[0034] like Figure 2 As shown, this embodiment provides a chemostat culture device for controlling the growth and metabolic steady state of food microorganisms, including a reactor 1 and various storage containers, the storage containers are divided into first, second and third storage containers, wherein the first storage container 8 is used to store fresh culture medium, and the second storage container 9 and the third storage container 10 are used to store old culture medium.

[0035] The reactor 1 is provided with an aeration device 2, a stirring device 3 and an online OD detection device 4, wherein a stirring paddle is arranged inside the reactor tank and completely immersed in the culture solution, and the stirring paddle is coaxially connected with a motor, and the motor is arranged outside the reactor tank; the aeration device 2 and the online OD detection device 4 are respectively used to transport gases including nitrogen, carbon dioxide and oxygen required for microbial growth into the reactor, and to monitor the OD value of the culture solution in real time.

[0036] The first storage container 8 is connected to the reactor 1 through a pipeline, and a first peristaltic pump 5 is arranged on the pipeline to pump the fresh culture medium stored in the first storage container into the reactor; the reactor 1 is connected to the second and third storage containers through a pipeline and a three-way valve 7, and a second peristaltic pump 6 is arranged on the pipeline to pump the old culture medium after fermentation in the reactor into the second storage container 9 and / or the third storage container 10.

[0037] Example 2

[0038] This embodiment uses the device described in Example 1 to provide a chemostat culture method for controlling the growth and metabolic steady state of food microorganisms, including the following steps:

[0039] First, the strain is inoculated into the reactor 1, and the ventilation device 2 and the stirring device 3 are turned on at the same time. After the strain grows to the late logarithmic growth phase or the plateau phase, the first peristaltic pump 5 is turned on to allow the fresh culture medium in the first storage container 8 to flow into the reactor 1. The second peristaltic pump 6 is turned on, and the three-way valve 7 is adjusted to allow the old culture medium to flow from the reactor 1 into the second storage container 9. The flow rate of the peristaltic pump is adjusted according to the required growth rate. The flow rate of the first peristaltic pump 5 is the same as that of the second peristaltic pump 6. The online OD detection device 4 is turned on to detect the absorbance of the constant temperature system in the reactor 1 at 600 nm. When the absorbance value fluctuates within a very small range, it can be said that the constant temperature system has reached a steady state. At this time, the three-way valve is adjusted to allow the old culture medium to flow into the third storage container 10 for subsequent experimental analysis.

[0040] Test Case

[0041] In this test example, strain Lactobacillus delbrueckii subspecies bulgaricusA3 (LBA3) and strain Lactococcus lactis subsp. lactis IMAU11919 (BL19) were used as examples, and the method described in Example 2 was used to perform steady-state control on the growth and metabolism of food microorganisms.

[0042] like Figure 3 , Figure 4 , Figure 5 As shown, based on the growth rate of Lactobacillus delbrueckii subspecies bulgaricus LBA3 in the logarithmic growth phase in MRS medium, the growth rate is between 0.4 and 0.7 h. -1 By adjusting the culture constant volume and pump speed, the low (0.45h -1 ), medium (0.52h -1 ), high (0.65h -1 ) growth rate, indicating that the chemostat culture device can meet the chemostat culture requirements of Lactobacillus delbrueckii subspecies bulgaricus at different growth rates.

[0043] like Figure 6 , Figure 7 , Figure 8 As shown, based on the growth rate of Lactococcus lactis subsp. lactis BL19 in M17 medium during the logarithmic growth phase, the growth rate was between 0.6 and 1.5 h. -1 By adjusting the culture constant volume and pump speed, the low (0.65h -1 ), medium (1.22h -1 ), high (1.46h -1 ) growth rate, indicating that the constant temperature culture device can meet the constant temperature culture requirements of Lactococcus lactis at different growth rates.

[0044] Example 3

[0045] As a food microorganism, Kluyveromyces marxii is widely used in the brewing and food industries, and is well-known for synthesizing a large number of aromatic compounds, including fruit esters, carboxylic acids, ketones, furanones, alcohols and aromatic hydrocarbons. Among the above aromatic compounds, phenylethanol is often considered to be one of the most commercially valuable flavor molecules because of its rose-like aroma. It is obtained by Kluyveromyces marxii through the conversion of phenylalanine. The present application experimentally found that adding 2g / L of phenylalanine to YPD medium (10g yeast extract, 20g glucose, 20g peptone, dissolved in 1000ml deionized water, autoclaved at 121℃ for 15min) can obtain the maximum yield of phenylethanol ( Fig. 9 ), and by using the device described in Example 1 and the method described in Example 2 of the present invention, the maximum production rate of phenylethanol obtained by chemostating Kluyveromyces marxianus N2-2 with different growth rates was 0.166 g / L / h, which was 17.5 times higher than that of phenylethanol cultured in a shake flask (1 ml of second-generation activated 24-hour Kluyveromyces marxianus liquid was added to a 250 ml shake flask with 50 ml of culture medium, a sealing film was attached to the bottle mouth to prevent phenylethanol from volatilizing, and cultured at a shaking speed of 200 rpm for 24 hours), and the corresponding growth rate was 0.617 h -1 ( Fig.10 ).

[0046] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred arrangement scheme, a person skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A chemostat culture device for controlling the steady state of growth and metabolism of food microorganisms, characterized in that: The device comprises a reactor (1) and storage containers, wherein the storage containers are divided into a first storage container, a second storage container and a third storage container, wherein the first storage container (8) is used to store fresh culture medium, and the second storage container (9) and the third storage container (10) are used to store old culture medium; The first storage container (8) is connected to the reactor (1) via a pipeline, and a first peristaltic pump (5) is arranged on the pipeline to pump the fresh culture medium stored in the first storage container into the reactor; The reactor (1) is connected to the second and third storage containers through a pipeline and a three-way valve (7). A second peristaltic pump (6) is arranged on the pipeline to pump the old culture medium after fermentation in the reactor into the second storage container (9) and / or the third storage container (10).

2. The device according to claim 1, characterized in that The reactor (1) is provided with a stirring device (3), wherein a stirring paddle is arranged inside the reactor tank and is completely immersed in the culture liquid, and the stirring paddle is coaxially connected to a motor, which is arranged outside the reactor tank.

3. The device according to claim 1 or 2, characterized in that: The reactor (1) is also provided with a ventilation device (2) and an online OD detection device (4), which are respectively used to transport gases required for microbial growth, including nitrogen, carbon dioxide, and oxygen, into the reactor, and to monitor the OD value of the culture solution in real time.

4. A method for controlling the steady state of growth and metabolism of food microorganisms by using the device according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: First, the strain is inoculated into the culture medium in the reactor (1), and the ventilation device (2) and the stirring device (3) are turned on at the same time. After the strain grows to the late logarithmic growth phase or the plateau phase, the first peristaltic pump (5) is turned on to allow the fresh culture medium in the first storage container (8) to flow into the reactor. The second peristaltic pump (6) is turned on, and the three-way valve (7) is adjusted to allow the old culture medium to flow from the reactor into the second storage container (9). The flow rate of the peristaltic pump is adjusted according to the required growth rate. The flow rate of the first peristaltic pump is the same as that of the second peristaltic pump. The online OD detection device (4) is turned on to detect the absorbance of the chemostat system in the reactor (1) at 600 nm. When the absorbance value fluctuates within a very small range, it means that the chemostat system has reached a steady state. At this time, the three-way valve (7) is adjusted to allow the old culture medium to flow into the third storage container (10) for subsequent experimental analysis.

5. The chemostat culture method according to claim 4, characterized in that: The method is applicable to a variety of food microorganisms including Lactobacillus delbrueckii subsp. bulgaricus, Lactococcus lactis subsp. lactis and Kluyveromyces marxianus.

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