CN116751732B - Culture method for increasing number of living cells of cheese bacillus - Google Patents

Culture method for increasing number of living cells of cheese bacillus Download PDF

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CN116751732B
CN116751732B CN202310845631.2A CN202310845631A CN116751732B CN 116751732 B CN116751732 B CN 116751732B CN 202310845631 A CN202310845631 A CN 202310845631A CN 116751732 B CN116751732 B CN 116751732B
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姚国强
钟智
陈永福
于洁
白梅
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Inner Mongolia Agricultural University
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Abstract

The invention provides a culture method for improving the number of living cells of cheese bacillus, belonging to the technical field of microbial culture; the culture method comprises the following steps: fermenting and culturing the cheese bacillus; and adding an oxidant into the fermentation culture system. In the invention, the oxidation-reduction potential of the system can be reduced by adding the oxidant into the fermentation culture system, and the lower oxidation-reduction potential is beneficial to the improvement of the biomass of the cells of the cheese bacillus, so that the number of living cells of the cheese bacillus is increased, and the high-density culture of the cheese bacillus is realized.

Description

Culture method for increasing number of living cells of cheese bacillus
Technical Field
The invention belongs to the technical field of microbial culture, and particularly relates to a culture method for increasing the number of living cells of cheese bacillus.
Background
Lactic acid bacteria (lactic acidbacteria, LAB) are a generic term for a group of bacteria that can produce large amounts of lactic acid using fermentable carbohydrates. Lactic acid bacteria are ideal materials for research classification, biochemistry, genetics, molecular biology and genetic engineering (theoretically have important academic values), and have extremely high application values in important fields closely related to human life, such as industry, agriculture and animal husbandry, food, medicine and the like.
With the rapid development of the food industry, lactic acid bacteria are increasingly concerned and accepted by the international academic field and consumers due to the good probiotic characteristics, and products such as fermented dairy products, live lactobacillus-containing beverages, direct lactobacillus starter and probiotic composite bacteria are popular in the market. The lactic acid bacteria product needs extremely high basic viable count to realize the deep development of the lactic acid bacteria product, so that a high-density culture method is needed to obtain the lactic acid bacteria with high viable count.
Because different lactic acid bacteria have specific optimal culture conditions, the culture conditions in the high-density culture process of the specific lactic acid bacteria need to be reasonably set. There is currently no report on dynamic regulation of the high-density culture of cheese bacillus.
Disclosure of Invention
The invention aims to provide a culture method for increasing the number of living cells of cheese bacillus, which can realize high-density culture of cheese bacillus.
The invention provides a culture method for increasing the number of living cells of cheese bacillus, which comprises the following steps:
fermenting and culturing the cheese bacillus; and adding an oxidant into the fermentation culture system.
Preferably, the oxidizing agent comprises potassium ferricyanide.
Preferably, in the fermentation culture process, the oxidation-reduction potential of a fermentation culture system is less than or equal to 0mV.
Preferably, in the fermentation culture process, the oxidation-reduction potential of the fermentation culture system is-320-0 mV.
Preferably, in the fermentation culture process, the oxidation-reduction potential of the fermentation culture system is-300 mV to-100 mV.
Preferably, in the fermentation culture process, the concentration of the oxidant in a fermentation culture system is less than or equal to 3g/L.
Preferably, the pH of the fermentation culture system is 5.9 during the fermentation culture.
Preferably, the fermentation culture is performed under conditions of 0.05 MPa.
Preferably, the fermentation culture adopts nitrogen pressure maintaining or air pressure maintaining.
Preferably, the lactobacillus casei comprises lactobacillus casei Zhang.
The invention provides a culture method for increasing the number of living cells of cheese bacillus, which comprises the following steps: fermenting and culturing the cheese bacillus; and adding an oxidant into the fermentation culture system. During the culture of the cheese bacillus, oxidation-reduction potential (ORP) in a culture system gradually changes to a high reduction state, and the ORP changes rapidly due to higher cell activity during high-density culture. Therefore, the oxidation agent is dynamically added into the system for fermenting and culturing the cheese bacillus to reduce the oxidation-reduction potential of the system, and the lower oxidation-reduction potential is beneficial to the improvement of the biomass of the bacteria of the cheese bacillus, so that the number of living cells of the cheese bacillus is increased, and the high-density culture of the cheese bacillus is realized.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the growth curves of Lactobacillus casei Zhang in static culture systems with different aeration strategies;
FIG. 2 shows ORP variation in static culture systems with different aeration strategies;
FIG. 3 shows the change in ORP of aeration-regulated Lactobacillus casei Zhang high-density culture system;
FIG. 4 shows the change in ORP of an oxidant-regulated Lactobacillus casei Zhang high density culture system;
FIG. 5 is an ORP growth curve of a aeration-regulated cheese-forming Bacillus casei Zhang high-density culture system;
FIG. 6 is an ORP growth curve of potassium ferricyanide-controlled Lactobacillus casei Zhang high density culture system;
FIG. 7 shows the yield of cells from end-log dry cells under potassium ferricyanide control at different ORP conditions;
FIG. 8 shows the cell yields of end-of-log dry cells under different ORP conditions for aeration regulation;
FIG. 9 shows the effect of potassium ferricyanide concentration on the growth of Lactobacillus casei Zhang.
Detailed Description
The invention provides a culture method for increasing the number of living cells of cheese bacillus, which comprises the following steps:
fermenting and culturing the cheese bacillus; and adding an oxidant into the fermentation culture system.
In the present invention, the cheese bacillus is a facultative anaerobe; the lactobacillus casei preferably comprises lactobacillus casei Zhang; the cheese bacillus Zhang (Lactobacillus casei Zhang, L.casei Zhang) was isolated from the tin Lin Guole allied Zhenglan flag herder self-made mare's milk of inner Mongolian autonomous region in 2002, and is provided by the lactobacillus strain resource Library (LABCC) of the key laboratory of the university of inner Mongolian agricultural dairy biotechnology and engineering education department.
In the present invention, the lactobacillus casei is preferably activated to the third generation lactobacillus casei.
In the present invention, the fermentation culture of the cheese bacillus preferably includes fermentation culture by inoculating the cheese bacillus to a fermentation medium.
In the invention, the inoculation amount of the lactobacillus casei is preferably 5 percent, and the volume ratio of the lactobacillus casei bacterial liquid to the fermentation medium is 5:100; the effective viable count of the lactobacillus casei in the lactobacillus casei bacterial liquid is preferably more than 10 8 CFU/mL.
In the present invention, the fermentation medium is preferably double-added to the carbon source based on the general MRS medium. In the invention, the fermentation medium takes water as a solvent, and preferably comprises the following components in concentration: 10g/L bacteriological peptone, 8g/L beef extract powder, 4g/L yeast extract powder, 40g/L glucose, 1ml/L tween-80, 2g/L dipotassium hydrogen phosphate, 5g/L sodium acetate, 2g/L tri-ammonium citrate, 0.2g/L magnesium sulfate and 0.05g/L manganese sulfate.
The invention also preferably comprises the step of sequentially carrying out first-generation culture, second-generation culture and seed culture on the lactobacillus casei before the fermentation culture to obtain the lactobacillus casei for fermentation culture. The culture medium for the first and second generation culture and the seed culture is not particularly limited, and a conventional culture medium in the art can be used.
In the present invention, the oxidizing agent comprises potassium ferricyanide, which does not inhibit the growth of cheese bacillus. According to the invention, the oxidation agent is added into the system for fermenting and culturing the cheese bacillus to reduce the oxidation-reduction potential of the system, and the lower oxidation-reduction potential is beneficial to the improvement of the biomass of the bacteria of the cheese bacillus, so that the number of living cells of the cheese bacillus is increased, and the high-density culture of the cheese bacillus is realized. The oxidation-reduction value is influenced by oxygen, and the influence of oxygen on the cheese bacillus can be eliminated while the oxidation-reduction potential is regulated by the oxidant.
In the process of the fermentation culture, the concentration of the oxidant in a fermentation culture system is preferably less than or equal to 3g/L.
In the present invention, the fermentation culture is preferably performed in a smart fermenter.
In the present invention, the temperature of the fermentation culture is preferably 37 ℃; the pH of the initial fermentation system of the fermentation culture is preferably 6.5.
In the process of the fermentation culture, the oxidation-reduction potential of the fermentation culture system is preferably less than or equal to 0mV, more preferably, the oxidation-reduction potential of the fermentation culture system is between-320 and 0mV, even more preferably, the oxidation-reduction potential of the fermentation culture system is between-300 and-100 mV, most preferably, the oxidation-reduction potential of the fermentation culture system is between-200 and-250 mV. In the invention, the yield of the Lactobacillus casei Zhang thallus is continuously increased along with the gradual reduction of the ORP when the ORP of 0 to-300 mV is in a neutral and reduced state. The yield of wet thalli (2.198+/-0.059)% is obviously higher than that of thalli (2.099 +/-0.048)% (P < 0.05) when the pressure of nitrogen at the top of the tank is maintained (-390 mV) when the ORP is-300 mV. Thus, the maximum cell yield of wet cells of Lactobacillus casei Zhang at the end of log could be obtained in a culture environment of-300 mV. When the oxidation-reduction potential in the fermentation culture system reaches the lower limit of the set value, the flow rate of the oxidant is regulated to be high, and after the oxidation-reduction potential reaches the upper limit of the set value, the flow rate is regulated to be low, and the oxidation-reduction potential in the culture system is stabilized in the set range repeatedly. In the invention, the oxidation-reduction potential of the fermentation culture system is preferably monitored in real time by adopting the online analysis and measurement of a Metrele brand P14805-DPAS-SC-K8S/225 type ORP electrode.
In the present invention, the pH of the fermentation culture system is preferably 5.5 to 6 during the fermentation culture. The reagent for adjusting the pH of the fermentation culture system is preferably an aqueous ammonia solution having a volume concentration of 30%.
In the present invention, the fermentation culture is preferably performed under stirring conditions; the rotation speed of the stirring culture is preferably 400rmp.
In the present invention, the fermentation culture is preferably performed under a pressure of 0.03 to 0.05 MPa.
In the present invention, the fermentation culture is preferably maintained under nitrogen or air pressure, more preferably maintained under nitrogen.
In the present invention, the fermentation culture is performed for a period of time when the cell growth proceeds to the end of log (initial stage of stabilization).
In the present invention, the fermentation culture is preferably an anaerobic fermentation culture.
Before the fermentation culture, the invention preferably further comprises flushing the fermentation medium in a fermentation device by introducing sterile nitrogen; the flow rate of the sterile nitrogen is preferably 2.0L/min; the time for introducing the sterile nitrogen gas is preferably 1min.
For further explanation of the present invention, a method for increasing the number of living cells of Lactobacillus casei, which is provided by the present invention, will be described in detail with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1 Materials and methods
1.1 Materials
1.1.1 Test strains
Cheese bacillus Zhang (Lactobacillus casei Zhang, l.casei Zhang) was isolated in 2002 from the inner Mongolian municipality tin Lin Guole allied blue flag herder homemade fermented mare milk, offered by the inner Mongolian agricultural university (LABCC).
1.1.2 Major reagents and sources
Table 1 major reagents and Specification manufacturers
1.2 Instrumentation
TABLE 2 Main instrument name, model and manufacturer
2 Method
2.1 Activation of Strain
Placing Lactobacillus casei Zhang defatted milk strain tube preserved at-80deg.C in refrigerator, thawing, activating into MRS culture medium, culturing at 37deg.C for 24 hr for activating to one generation, and gram staining to determine bacteria shape and purity.
2.2 Medium formulation
2.2.1 First and second Generation Medium
10G/L bacteriological peptone, 8g/L beef extract powder, 4g/L yeast extract powder, 20g/L glucose, tween-801 ml/L, 2g/L dipotassium hydrogen phosphate, 5g/L sodium acetate, 2g/L tri-ammonium citrate, 0.2g/L magnesium sulfate and 0.05g/L manganese sulfate, adjusting pH to 6.5 by NaOH, sterilizing at 121 ℃ for 15min, and immediately cooling by ice water bath after sterilization.
2.2.2 Seed Medium
10G/L bacteriological peptone, 8g/L beef extract powder, 4g/L yeast extract powder, 40g/L glucose, tween-801 ml/L, 2g/L dipotassium hydrogen phosphate, 5g/L sodium acetate, 2g/L tri-ammonium citrate, 0.2g/L magnesium sulfate and 0.05g/L manganese sulfate, adjusting pH to 6.5 by NaOH, sterilizing at 121 ℃ for 15min, and immediately cooling by ice water bath after sterilization.
2.2.3 Fermentation Medium
10G/L bacteriological peptone, 8g/L beef extract powder, 4g/L yeast extract powder, 40g/L glucose, 801ml/L tween-L, 2g/L dipotassium hydrogen phosphate, 5g/L sodium acetate, 2g/L tri-ammonium citrate, 0.2g/L magnesium sulfate and 0.05g/L manganese sulfate.
1. And sterilizing the second-generation culture medium, glucose of the seed culture medium and other components together, sterilizing the glucose of the fermentation culture medium and the other components separately, sterilizing at 115 ℃ for 15min, and immediately cooling by ice water bath after sterilization.
The improvement of the fermentation medium is based on the following steps: when the MRS culture medium is used for culturing the strain of the three-generation cheese bacillus Zhang in the preliminary experiment, 2% of carbon sources in the common MRS culture medium cannot meet the nutrition requirement of the strain in constant pH batch culture, and the thalli are still in a rapid growth stage at 4-6 h after fermentation begins, but the carbon sources are insufficient to enter a stable stage, and the oxidation-reduction potential in a culture system under the monitoring of an ORP electrode does not reach the minimum ORP under the condition of the high-density culture of the cheese bacillus Zhang, so that double addition treatment is carried out on the carbon sources on the basis of the common MRS culture medium.
2.3 Culture method
Inoculating the first generation fermentation liquor to a second generation culture medium according to the proportion of 2%, carrying out aerobic culture for 12h to second generation at 37 ℃, inoculating the second generation fermentation liquor to a third generation culture medium (seed culture medium) according to the proportion of 2%, carrying out aerobic culture for 12h to third generation at 37 ℃, and inoculating the third generation strain to a 2L parallel bioreactor according to the inoculum size of 5%.
2.4PH measurement
The pH of the culture solution before sterilization is measured by a Metrele brand FE20 type precise instrument, and the pH monitoring during the culture process is measured by adopting a Metrele brand 405-DPAS-SC-K8S/225 type pH electrode on-line analysis.
2.5 Oxidation-reduction potential control method
ORP in the culture system gradually changes to a high reduction state in the process of culturing lactobacillus, and the ORP changes rapidly due to higher thallus activity in high-density culture, so that the purpose of balancing ORP is achieved by adding an oxidizing substance (oxidant). The ORP is regulated and controlled by intermittently introducing sterile air and adding an oxidant potassium ferricyanide solution, when the ORP in the culture system reaches the lower limit of a set value, the flow of the sterile air or the oxidant introduced into the tank is regulated to be high, and after the ORP reaches the upper limit of the set value, the flow is regulated to be low, and the operation is repeated, so that the ORP in the culture system of each experimental group is stabilized in the set range. ORP monitoring during the culture process is determined by online analysis of the Metrehler brand P14805-DPAS-SC-K8S/225 ORP electrode.
2.6 Measurement of microbial Mass
2.6.1 Dry weight method
Sampling the fermentation liquor of each group of different ORPs, sterilizing in water bath at 80 ℃ for 30min, cooling to room temperature, centrifuging (8000 Xg, 5 min), discarding supernatant, washing thalli for 2-3 times by distilled water, placing the thalli obtained by centrifugation in a drying oven for drying (105 ℃) to constant weight, weighing by an analytical balance, recording data, and obtaining the dry weight (DCW) of the thalli by the difference between the weight recorded after drying and the weight of the empty tube.
2.6.2 Determination of viable count
The measurement was performed by plate counting. The Lactobacillus casei Zhang fermentation broth is subjected to 10-fold incremental gradient dilution by using normal saline, 2-3 proper dilutions are selected, 1mL of the dilutions are respectively taken to be transferred into a sterile culture dish during the incremental dilution, the MRS solid culture medium is selected for carrying out a pouring plate method, two proper dilutions are selected, each dilution is performed in three parallels, namely six parallels are selected for each sample, the constant temperature aerobic culture (48+/-2) at 37 ℃ is carried out, and the average value and the standard deviation are calculated, so that the result is expressed as CFU/mL.
2.6.3 Determination of cell Density
The cell density of Lactobacillus casei Zhang was measured at 600nm using a multifunctional microplate detector from each sample of the fermentation broth. The value of the cell density is effective data when the cell density is 0.2-0.8, and if the cell density is more than 0.8, the dilution of the bacterial liquid is required, and the detection parameters are set to be 30s before detection, so that the oscillation speeds are consistent. 200 μl of fermentation broth was added in each well in sequence, and was blown down uniformly, three replicates were established for each sample (n=3), and the mean and standard deviation were recorded, with each data expressed as mean ± standard deviation.
2.4 Design
2.4.1 Static culture based on different aeration strategies
Inoculating activated cheese bacillus Zhang to the third generation into an intelligent fermentation tank filled with 2.0L of culture medium according to 5% of inoculation amount, introducing sterile nitrogen with the flow of 2.0L/min to the bottom of the fermentation tank after the fermentation tank is assembled to replace tank top gas for 1min, adjusting the initial ORP of each experimental group to the range of-20 mV to +20mV, then adjusting according to the ventilation strategy of each experimental group (2.0L/min air is fully introduced, 0.05MPa air pressure is maintained at the top of the tank, 0.05MPa nitrogen pressure is maintained at the top of the tank), setting the culture temperature to 37 ℃, setting the initial pH of the culture medium to 6.5, adding no neutralizing agent, stirring the culture medium at the rotation speed of 400rmp, and not regulating ORP and pH in the culture system in the culture process. The fermentation broth was sampled every 1 hour at intervals during the culture, and after sampling 20mL of each of the three tubes (5 mL for measurement of viable cell count, 5mL for measurement of cell density, 10mL for use), the sample was immediately placed in ice water to terminate the culture, and 50ml×6 was taken at the end of the log for measurement of cell dry weight and wet weight.
2.4.2 Changing redox status in batch culture System by aeration strategy
The value of the constant ORP during fermentation of Lactobacillus in the experimental group was determined from the limit value of the ORP of the Lactobacillus casei Zhang fermentation broth measured in the pre-experiment, and based on this group of ORP values, the following was developed. Inoculating activated cheese bacillus Zhang to the third generation into an intelligent fermentation tank filled with 2.0L of fermentation medium according to the inoculum size of 5%, introducing 2.0L/min sterile nitrogen to flush the medium for 1min after the fermentation tank is assembled, introducing sterile air into the medium after nitrogen flushing until the ORP is 0 (+ -10) mV, and introducing 2.0L/min air, -100 (+ -20) mV, -200 (+ -20) mV and-250 (+ -20) mV in the whole course according to the set value of an experimental group and the set value of a control group, wherein the set value of the control group is 0.05MPa air pressure maintaining on the tank top. When the pH in the culture system is reduced to 5.9, automatic alkali addition is started, and the constant pH is regulated and controlled to 5.9. When the ORP in the culture system reaches the lower limit of the set value, sterile air is introduced into the tank to reach the upper limit of the set value, and the steps are repeated to stabilize the ORP in the culture system of each experimental group within the set range. The culture temperature is 37 ℃, the initial pH is 6.5, the constant pH is 5.9, the neutralizer is 30% ammonia water solution, and the stirring speed is 400rmp. The sampling method is the same as that in 2.4.1.
2.4.3 Redox State in batch culture System by addition of an oxidant
Inoculating activated cheese bacillus Zhang of the third generation into an intelligent fermentation tank with 2L volume according to 5% inoculum size, wherein 2.0L fermentation medium is filled in the fermentation tank, the temperature of the fermentation tank is set to 37 ℃, the initial pH is 6.5, the constant pH is 5.9, the neutralizer is 30% ammonia water solution, the stirring rate is 400rmp, the tank bottom is filled with 2.0L/min sterile nitrogen to flush the culture medium for 1min after the fermentation equipment is assembled, the pressure is maintained to 0.05MPa of the tank top pressure after the nitrogen flushing, an air outlet valve is closed, potassium ferricyanide solution is filled into the tank to adjust the ORP to a set value, and the ORP of an experimental group is lower than the current ORP after the ORP is adjusted to 0mV, and then the ORP is adjusted and controlled after the ORP is reduced to the set value. The experimental group set values are +100 (+ -20) mV, 0 (+ -20) mV, -100 (+ -20) mV, -200 (+ -20) mV and-300 (+ -20) mV, and when the culture environment in the fermentation tank is reduced to the lower limit of the set value, the potassium ferricyanide solution is added into the fermentation tank, and the solution flows to the upper limit of the set value. When the pH in the culture system is reduced to 5.9, automatic alkali addition is started, and the constant pH is regulated and controlled to 5.9. The sampling method is the same as that in 2.4.1.
2.4.4 Effect of Potassium ferricyanide concentration on the growth of Lactobacillus casei Zhang
Inoculating the activated cheese bacillus Zhang to the third generation into a full-automatic growth curve instrument according to the inoculum size of 2%, carrying out aerobic culture for 24 hours, setting the concentration of potassium ferricyanide to be 1g/L, 2g/L, 3g/L, 4g/L and 5g/L, and blank control groups, and drawing a growth curve graph according to experimental results.
2.4.5 Data processing
Data statistics were performed using Excel and IBM SPSS STATISTICS, and data were analyzed for Tukey HSD significance differences using ANOVA. Graphics rendering is performed using origin 2021.
The technical effects are as follows:
3 results and analysis
3.1 Static culture of Lactobacillus casei Zhang
3.1.1 Cell Density and viable count construction of growth Curve
The growth rule of the cheese bacillus Zhang in each period of the culture process can be reflected by the growth curve, the growth condition of the cheese bacillus Zhang under a static culture system with different aeration conditions is mainly verified, and the experimental results are shown in fig. 1, table 3 and table 4.
Table 3 table of cell density (OD 600 nm) data for different aeration strategies (n=3,)
Table 4 the number of viable bacteria [ Log 10 (CFU/mL) ] data table (n=3,)
The results show the cell density and viable count of Lactobacillus casei Zhang in static culture systems under three different aeration conditions. The growth curve graph constructed by the thallus density (OD 600nm) of the fermentation liquid can reflect that the cheese bacillus Zhang cultured under two ventilation strategies of tank top air pressure maintaining and tank top nitrogen pressure maintaining enters a logarithmic growth period from 2 hours after starting culture, the delay period is shorter, the logarithmic growth period is 2-11 hours, the thallus density at the moment is extremely fast to increase, and the change condition of thallus density values under the two ventilation strategies of the logarithmic growth period is basically consistent; the whole process of introducing 2.0L/min of air into the Lactobacillus casei Zhang starts to enter the logarithmic growth phase after the beginning of culturing for 3 hours, compared with two experimental groups of air pressure maintaining and nitrogen pressure maintaining, the delay period is longer, and the continuous air introducing group can lead the starting of the Lactobacillus casei Zhang to be slowed due to the continuous air entering into the fermentation liquor, the logarithmic growth phase is 3-13 hours, and compared with the tank top air pressure maintaining group and the tank top nitrogen pressure maintaining group, the logarithmic growth phase is more than 1 hour. Because the oxygen in the fermentation tank is limited when the air pressure maintaining group carries out air pressure maintaining on the top of the fermentation tank, the oxygen is completely consumed in the process of culturing, namely, the oxygen is approximately the same as the nitrogen pressure maintaining group, and therefore, the growth states of the top air pressure maintaining group and the top nitrogen pressure maintaining group in the lag phase and the logarithmic phase are basically the same. The viable counts of the three groups of experiments at the end of log growth were finally: 2.0L/min air set (1.52+ -0.021). Times.10 9 CFU/mL, tank top air pressure maintaining set (1.76+ -0.033). Times.10 9 CFU/mL, tank top nitrogen pressure maintaining set (1.95+ -0.035). Times.10 9 CFU/mL are introduced. It is notable that the static fermentation strain is inhibited by low pH and enters the stationary phase in advance, so that the bacterial density (OD 600nm) and the number of viable bacteria are both increased slightly after entering the stationary phase, and the number of the three highest viable bacteria is (2.02+/-0.028) multiplied by 10 9 CFU/mL. The cell densities OD 600nm at the end of log growth for the three experiments were: the whole process is carried out by a 2.0L/min air group OD 600nm = 2.729 plus or minus 0.021, a tank top nitrogen pressure maintaining group OD 600nm = 3.1795 plus or minus 0.018 and a tank top air pressure maintaining group OD 600nm = 3.102 plus or minus 0.011.
As can be seen from FIG. 1, the OD 600nm of the continuous 2.0L/min air group still slowly increased the final maximum viable count after entering the stationary phase, almost consistent with the other two groups. The number of the two groups of the viable bacteria data can be mutually verified with the number of the viable bacteria data, the viable bacteria data of the air pressure maintaining and nitrogen pressure maintaining group begin to show a slow descending trend after the whole-process air-through group OD 600nm is still slightly increased after 32h, and enter the decay period, but at the moment, the viable bacteria number of the whole-process air-through group with 2.0L/min is kept stable from 32h to 43 h.
3.1.2 ORP Change in static culture System with different aeration strategies
ORP changes during the culture are shown in FIG. 2 and Table 5 for the three experimental groups.
TABLE 5 ORP (mV) data sheets in static culture System with different aeration strategies
In the continuous aeration experimental group, the generation of some reducing substances is slightly inhibited due to the low pH of the fermentation liquor, so that the ORP is slowly raised, but at the moment, due to continuous air introduction, continuous aerobic molecules are dissolved in the culture medium, the slow raising of the fermentation liquor is caused by insufficient neutralization of the continuously introduced oxygen molecules by the reducing substances generated by the bacteria, and the generation of the reducing substances is far lower than the dissolution speed of the oxygen molecules in the air into the culture medium due to the reduction of the activity of the bacteria or the change of metabolic pathways after 27 hours, so that the ORP is in a rapid raising state.
In the tank top air pressure maintaining experimental group, as part of oxygen exists in the tank top air at the initial stage of culture, the part of oxygen is dissolved in the culture medium to cause small-amplitude increase of the ORP of the culture medium, and some reducing substances generated by the metabolism of bacteria are accompanied in the process of the increase of the ORP, but the reducing substances generated by small number of living bacteria are insufficient to reduce the ORP in value, as the oxygen molecules in the culture medium are continuously consumed by the bacteria and the generation rate of the reducing substances are continuously increased, the ORP increases to reach the peak at 1h and starts to decrease rapidly, the end of logarithm reaches the lowest point to 192mV at the beginning of 11h of culture due to the influence of acid inhibition, at the same time, the bacterial growth starts to enter a stable period, the ORP gradually starts to rise, and returns to the initial ORP level of the fermentation liquid at 32 h.
The ORP of the tank top nitrogen pressure maintaining experimental group starts to drop to minus 314mV of 5h immediately under the combined action of the reducing gas of nitrogen, and then slowly drops to minus 319mV of 5-9 h, and slowly rises to minus 251mV of 43h at a rate of 3-6 mV/h after 9 h.
3.2 High Density culture of Lactobacillus casei Zhang
3.2.1 Aeration and Oxidation methods to regulate ORP in Zhang culture System
3.2.1.1 Regulation of ORP by aeration
As can be seen from FIGS. 3 and 6, as the incubation proceeds, ORP of all experimental groups reached the set range quickly, and the lower the set point, the longer the time to reach the set range. The ORP increased from the initial +12.3mV to +110.5mV after 1h from the beginning of the incubation in a full-air 2.0L/min group, and the ORP decreased slowly to +48mV on average at a rate of 5.1mV per hour for 1-15 h, remaining substantially constant after 15 h. The 100mV group reached the set range at 0.8h of initial incubation. The 200mV group reached the set range at 1.9h from the start of the incubation. Group 300mV reached the set range at 3.6h from the start of the incubation. The ORP of the air pressure maintaining group reaches-353.56 mV after the culture is started for 5.8 hours until the ORP is basically unchanged after the culture is ended.
TABLE 6 ORP (mV) data sheet in aeration-controlled Lactobacillus casei Zhang high density culture System
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3.2.1.2 Oxidation method for ORP Regulation
After nitrogen flushing, closing the air outlet valve, maintaining the pressure until the air pressure at the top of the tank is 0.05MPa, closing the air outlet valve, introducing 5g/L potassium ferricyanide solution into the tank to adjust the ORP to a set value, adjusting the ORP to 0mV after the ORP of the experimental group is lower than the current ORP, and then adjusting and controlling the ORP after the ORP is reduced to the set value. The experimental set points were +100mV, 0mV, -100 (+ -20) mV, -200 (+ -20) mV, -300 (+ -20) mV. As shown in FIG. 4, since lactobacillus is a continuous ORP decreasing process at the initial stage of fermentation, 5g/L potassium ferricyanide solution is required to be introduced at the beginning of fermentation in order to regulate and control the ORP of fermentation liquid to +100mV and 0mV, the value of ORP is observed in real time to regulate and control the flow rate of potassium ferricyanide solution, so that constant regulation and control of ORP are realized, and other groups are regulated and controlled by the method after ORP reaches a preset range except that a tank top nitrogen pressure maintaining experimental group does not need regulation and control.
The ORP of the tank top nitrogen pressure maintaining experimental group is kept unchanged within the range of-390 (+ -25) mV after 1.68h from the beginning of the cultivation, the time for the ORP of the cultivation system to reach-100 mV is 0.9h, the time for the ORP of the cultivation system to reach-200 mV is 1.2h, and the time for the ORP of the cultivation system to reach-300 mV is 1.51h.
3.2.2 Cell Density and viable count construction of growth Curve
3.2.2.1 Effect of aeration control ORP on Lactobacillus casei Zhang growth
As shown in FIG. 5, table 7 and Table 8, under five ORP conditions, lactobacillus casei Zhang rapidly enters the logarithmic growth phase after a short delay period.
Table 7 data table of ORP cell density (OD 600 nm) of aeration-conditioned high-density culture system (n=3,)
Table 8 data table of ORP viable count of aeration-controlled high-density culture system [ Log 10 (CFU/mL) ] (n=3,)/>
The density of the thalli under five ORP conditions after entering the logarithmic growth phase is rapidly increased, the relatively lower ORP (-200 mV, -250mV, air pressure maintaining) is more beneficial to the growth of the thalli than the relatively higher ORP (2.0L/min air, -100 mV) under the ventilation regulation and control, and the concentration of the thalli and the viable count are respectively the maximum and the minimum when the air pressure maintaining at the tank top and the 2.0L/min air are fully introduced; under the culture condition of tank top air pressure maintaining, the cheese bacillus Zhang enters a logarithmic growth phase, the density of thalli rapidly increases after entering the logarithmic growth phase, the logarithmic growth phase is continued until the end of 14 hours, at the moment, the density OD 600nm = 5.8245 +/-0.022 of the thalli is (1.76 +/-0.02) multiplied by 10 10 CFU/mL, the viable count enters a stationary phase after 14 hours, and the phenomenon that the thalli obviously enter a decay phase is not observed in the culture process of 32 hours; under the culture conditions of regulating and controlling the ORP of the fermentation liquor to enter a logarithmic growth phase for 2 hours under the culture conditions of-200 mV and-250 mV, the density of the bacteria and the number of the viable bacteria rapidly increase, the bacteria enter a stationary phase for 12 hours at the beginning of the culture, the density of the bacteria OD 600nm = 4.3745 +/-0.045 under the culture environment of-200 mV at the end of the logarithmic growth phase, the number of the viable bacteria is (9.43+/-0.25) multiplied by 10 9 CFU/mL, the density of the bacteria OD 600nm = 4.533 +/-0.033 under the condition of-250 mV, and the number of the viable bacteria is (9.98+/-0.012) multiplied by 10 9 CFU/mL; under the culture conditions that the ORP of the fermentation liquid is regulated and controlled to be at 100mV below zero and 2.0L/min air is introduced in the whole process, the change conditions of growth curves of the two culture conditions are basically consistent, the density of thalli entering the logarithmic phase and the number of viable bacteria rapidly increase in 3 hours, the logarithmic phase continues until 16 hours, at the moment, the density of thalli OD 600nm = 4.0173 +/-0.018 under the culture condition of 100mV below zero, the number of viable bacteria reaches (9.37+/-0.11) multiplied by 10 9 CFU/mL, the density of thalli OD 600nm = 3.9465 +/-0.019 under the condition of 2.0L/min air is introduced in the whole process, the viable count reaches (9.30+/-0.03) multiplied by 10 9 CFU/mL, and the viable count obviously decreases when the strain grows into the stationary phase under the culture condition of introducing 2.0L/min air in the whole process, and the viable count of each group of the other viable counts tends to be stable.
3.2.2.2 Effect of potassium ferricyanide Regulation of ORP on the growth of Lactobacillus casei Zhang
The regulation of the ORP of the strain in an anaerobic culture environment can be realized by adding an oxidant, which makes it possible to eliminate the influence of oxygen on the strain while regulating the ORP.
As shown in FIG. 6, lactobacillus casei Zhang enters the logarithmic growth phase after a short delay period of 1h under the five ORP conditions of anaerobic culture, and the delay period is shortened by 1h compared with that of aerobic culture. The highest density of the Lactobacillus casei Zhang cells cultured under-300 mV ORP conditions (OD 600nm = 6.301 + -0.018) in the six experimental groups, the 1-12 h log phase of the strain under this culture condition and the 10h log phase of the other five groups of strains, although there is a 1h extension of the log phase at-300 mV ORP, the OD 600nm is significantly higher (P < 0.05) than the second highest cell density tank top nitrogen pressure maintaining group OD 600nm in the six groups of experiments. In six groups of experiments, the cell density (OD 600nm) is-300 mV (OD 600nm = 6.301 +/-0.018) from high to low, and the tank top nitrogen pressure maintaining (OD600nm＝6.088±0.065),-200mV(OD600nm＝5.832±0.098),-100mV(OD600nm＝5.655±0.080),0mV(OD600nm＝5.404±0.037),+100mV(OD600nm＝4.601±0.042). is remarkable in that the cell density of each group is slowly reduced except that the cell density under the condition of-300 mV is not changed obviously when the cell growth enters the stable period.
The tendency of the viable count change at each growth period is approximately the same as that of the viable count change at different growth periods under different ORP conditions, but there is a large difference in the viable count values from the cheese-forming bacterium Zhang. As shown in FIG. 6, it was found from the measurement of the viable count of Lactobacillus casei Zhang for 20 hours that ORP was-300 mV, which had a higher viable count at the end of log phase and a longer logarithmic phase than the other experimental groups, indicating that the conditions of anaerobic culture of-300 mV were most suitable for the growth of Lactobacillus casei Zhang in the six experimental groups. The number of viable bacteria of the 300mV culture system is (2.38+/-0.46). Times.10 10 CFU/mL, the number of viable bacteria of the tank top nitrogen pressure maintaining culture system is (2.07+/-0.14). Times.10 10 CFU/mL, the number of viable bacteria of the 200mV culture system is (1.80+/-0.32). Times.10 10 CFU/mL, the number of viable bacteria of the 100mV culture system is (1.74+/-0.51). Times.10 10 CFU/mL, the number of viable bacteria of the 0mV culture system is (1.64+/-0.79). Times.10 10 CFU/mL, and the number of viable bacteria of the +100mV culture system is (1.37+/-0.23). Times.10 10 CFU/mL.
3.2.3 Effect of different high Density cultures ORP on Lactobacillus casei Zhang biomass
3.2.3.1 Determination of the Effect of Potassium ferricyanide Regulation ORP on the biomass of Lactobacillus casei Zhang by the dry weight method
The growth and proliferation of the cheese bacillus Zhang are affected by ORP in a culture system, the growth and metabolism conditions of the thalli are quite different under the culture environments of different ORP, in order to understand the relationship between the growth of the cheese bacillus Zhang and the ORP in the culture environment and research the optimal culture ORP suitable for the growth and proliferation of the cheese bacillus Zhang, different ORP culture environments are designed for research, as shown in FIG. 7 and Table 9, the yield of the dried thalli of the cheese bacillus Zhang is the lowest under the condition that the ORP value is in an oxidation state culture environment, and the yield of the dried thalli of the cheese bacillus Zhang is gradually increased along with the gradual reduction of the ORP until the biomass of the dried thalli reaches the maximum value at-300 mV. The results of the experiments were that the stronger the reducibility in the culture environment, the more suitable for the growth of Lactobacillus casei Zhang, the more strongly the reduction of-390 (+ -25) mV was compared with-300 mV, but the dry cell yield was significantly lower than-300 mV, the more 100mV was (0.2817.+ -. 0.002)%, the 0mV was (0.3167.+ -. 0.001)%, the 100mV was (0.334.+ -. 0.003)%, the 200mV was (0.3943.+ -. 0.003)%, the 300mV was (0.488.+ -. 0.002)%, and the top nitrogen pressure was (0.4172.+ -. 0.001)%.
Table 9 data table of dry cell yields (%) at different ORP conditions (n=3,)
3.2.3.2 Determination of the Effect of aeration control ORP on Lactobacillus casei Zhang biomass by the dry weight method
The cell yields of the Lactobacillus casei Zhang end-of-log dry cells in the culture environments with different ORPs by aeration control are shown in FIG. 8 and Table 10. The yield of dry thallus of the ORP of the aeration regulation culture system is the same as the change rule of the biomass reflected by the thallus density OD 600nm of the fermentation broth, namely the lower the ORP value in the culture system is, the lower the thallus density is, and the lower the thallus yield is.
Table 10 data sheet for yield (%) of dry cells at the end of log under conditions of ventilation control of different ORPs (n=3,)/>
The dry bacterial cell yields of different ORP conditions in the aeration method regulation cheese bacillus Zhang culture environment at the end of the logarithm are respectively, namely, an aeration group (0.2357 +/-0.003)%, a100 mV group (0.2482 +/-0.005)%, a 200mV group (0.2847 +/-0.002)%, a 250mV group (0.3227 +/-0.005)%, and an air pressure maintaining group (0.4097 +/-0.005)%, in the whole course.
3.3 Effect of Potassium ferricyanide concentration in culture System on the growth of Lactobacillus casei Zhang
In this example, potassium ferricyanide with different concentrations was added to the MRS medium, and a 24-hour growth curve was drawn by a full-automatic growth curve analyzer, and experimental results are shown in fig. 9 and table 11.
Table 11 table of cell density (OD 600 nm) at various potassium ferricyanide concentrations (n=3,)
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Under continuous culture for 24 hours, potassium ferricyanide with different initial concentrations does not inhibit the growth of cheese bacillus Zhang. In the continuous flow potassium ferricyanide constant control ORP experiment process of high density culture, the concentration accumulation of potassium ferricyanide in the fermentation tank is not more than 3g/L at maximum, so that the influence of toxic action of potassium ferricyanide on experiment results can be eliminated.
Discussion 4
The experiment is used for researching the biomass of the thallus which is cultured in high density under the two different ORP regulation modes by carrying out static culture on cheese bacillus.
4.1 Analysis of Experimental results differences during static culture
Cheese dry bacteria Zhang belongs to homolactic fermentation strains, glucose is consumed in the growth process to produce lactic acid to cause the reduction of pH of fermentation liquor, the low-pH culture environment can cause adverse effect on the growth of the strains, under the environment, the undissociated factor forms of H + and acid can strongly inhibit the strains, the undissociated factor forms of the acid and phospholipid molecules of cell membranes have high intersolubility, and the undissociated factor forms of the acid and the phospholipid molecules of the cell membranes can enter cells in a passive diffusion mode until the concentration of intracellular and extracellular acid molecules is kept consistent. The intracellular acids have an inhibitory effect on the active substances in the cells, so that an increase in ORP values at a pH value falling to a certain value is observed in 3.1.
The tolerance of the strain to pH values is also different due to different culture environments. Therefore, in the static culture experiment, the ORP of the culture system starts to rise at ph=5 in the culture environment with continuous aeration, starts to rise rapidly at ph=3.98 in the culture environment with pressure maintaining in the tank top air, and starts to rise slowly at ph=4.16 in the culture environment with pressure maintaining in the tank top nitrogen. The cheese-forming bacillus Zhang is a process of continuously producing reducing substances in the logarithmic growth phase of the culture, and the increase of ORP means the decrease of the proportion of the reducing substances in the culture environment, and can reflect the decrease of the metabolic capacity of the strain and the change of metabolic flow. Three groups of ventilation strategies in the static culture process are used for creating three different culture environments, and the ventilation strategy of ventilation in the whole process is used for leading the whole culture system to be in an oxidation state, namely ORP is positive value due to continuous addition of oxygen in the air; the ventilation strategy of the air pressure maintaining at the tank top enables the ORP of the culture system to show a trend of rising and then falling, and the ORP is in a reduction state for most of the time in the culture process; under the ventilation strategy of tank top nitrogen pressure maintaining, the ORP is immediately lowered to be stable at the initial stage of cultivation due to almost no participation of oxygen in the cultivation process, and a lower reduction state than tank top air pressure maintaining is created. From the experimental result of 3.1, it is known that the reduced culture environment created by nitrogen pressure maintaining and air pressure maintaining can obtain higher cell density at the end of the logarithm stage of static culture, but does not exclude the inhibition effect of dissolved oxygen on cell growth under the ventilation strategy of whole-course ventilation, but is not the culture environment in an oxidation state.
4.2 Comparison of the Effect of two ORP control methods on the high Density culture of Lactobacillus casei Zhang
In the experiment of the ventilation regulation and control culture system ORP, a large amount of dissolved oxygen exists in the fermentation liquor of a high ORP experiment group while the ORP is regulated and controlled, and the experiment forms two independent culture systems with the anaerobic environment created by potassium ferricyanide regulation and control ORP. This step was aimed at comparing the effect of different ORP modulation patterns on the biomass of Lactobacillus casei Zhang cells.
In a high-density culture experiment for regulating and controlling the ORP of a cheese bacillus Zhang culture environment by a ventilation method, the biomass of the cheese bacillus Zhang is in an increasing trend along with the reduction of the ORP, a tank top air pressure maintaining group with the lowest ORP in a logarithmic growth end-stage experiment group reaches the maximum biomass of the bacteria, the density OD 600nm = 5.8245 +/-0.022 of the bacteria is 1.76 +/-0.02) multiplied by 10 10 CFU/mL, the yield of the dry bacteria is 0.4097 +/-0.005%, the yield of the wet bacteria is 2.031 +/-0.047%, and various indexes are obviously higher than those of other groups (P < 0.05). The ORP in the aeration regulation culture system is due to the existence of dissolved oxygen in the fermentation broth, and the higher the set value of the ORP, the larger the aeration quantity, the more oxygen is introduced into the fermentation tank, and the optimal growth ORP of the cheese bacillus Zhang under the ORP condition of tank top air pressure maintaining is not proved.
In the high-density culture experiment using potassium ferricyanide to regulate and control the ORP of the cheese bacillus Zhang culture environment, the final-logarithmic phase bacterial biomass with the ORP of-300 mV in the culture environment is set to be the highest, the bacterial density OD 600nm = 6.301 +/-0.018, the viable count of the bacterial is (2.07+/-0.14) multiplied by 10 10 CFU/mL, the yield of dry bacterial is (0.488+/-0.002), and the yield of wet bacterial is (2.198+/-0.059). The ORP value built under the mode of pressure maintaining and ventilation of nitrogen at the tank top is lower than-300 mV, but each index of biomass of the thalli is obviously lower than-300 mV of experimental group. The combination of the two forms of regulation of ORP by aeration and regulation of ORP by potassium ferricyanide can lead to the conclusion that: the low oxidation-reduction potential is beneficial to the improvement of the biomass of the lactobacillus casei Zhang, and the ORP in a culture system can be controlled at-300 mV by using potassium ferricyanide under the anaerobic condition, so that the biomass of the lactobacillus casei Zhang is obviously improved.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (3)

1. A method of culturing for increasing the number of viable cells of lactobacillus casei (Lacticaseibacillus casei), comprising the steps of:
Fermenting and culturing the cheese bacillus; in the process of fermentation culture, the set value of the oxidation-reduction potential of a fermentation culture system is-300+/-20 mV, and when the oxidation-reduction potential of the culture environment in the fermentation tank is reduced to the lower limit of the set value, potassium ferricyanide solution is added into the fermentation tank and flows to the upper limit of the set value;
the fermentation culture adopts nitrogen pressure maintaining;
The cheese bacillus is cheese bacillus Zhang.
2. The method according to claim 1, wherein the pH of the fermentation culture system is 5.9 during the fermentation culture.
3. The method according to claim 1, wherein the fermentation culture is performed under a condition of 0.05 MPa.
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