CN112980719B - Oxygen-resistant animal bifidobacterium - Google Patents
Oxygen-resistant animal bifidobacterium Download PDFInfo
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Abstract
The invention provides an aerotolerant Bifidobacterium animalis (AR 668-R1 strain which is preserved in China general microbiological culture Collection center with the preservation number ofCGMCC No.20457. The strain can normally grow under the shake flask aerobic culture condition, and the viable count can reach 1.7 multiplied by 10 9 CFU/mL or more, 10.0mM H 2 O 2 The aerotolerant Bifidobacterium AR668-R1 has viable count and H under aerobic culture condition 2 O 2 The tolerance is obviously higher than that of the existing strain, and the strain has good oxygen resistance and hydrogen peroxide tolerance and has wide application prospect in the food industry.
Description
Technical Field
The invention belongs to the technical field of food science, and particularly relates to an oxygen-resistant animal bifidobacterium AR668-R1 strain.
Background
Bifidobacteria (bifidobacteria) play a crucial role in maintaining the intestinal microecological balance, being the most important group of probiotics in the human and animal intestines. The bifidobacterium plays an important role in the aspects of regulating intestinal flora, enhancing the immunity of organisms, relieving lactose intolerance, preventing infectious diarrhea and the like, and is widely applied to the fields of food, health-care products, medicines and the like. The bifidobacteria approved by the office of the Ministry of health of China to be applied to food comprise bifidobacterium animalis, bifidobacterium bifidum, bifidobacterium adolescentis, bifidobacterium infantis, bifidobacterium breve and bifidobacterium longum, and the bifidobacteria which can be used for infant food are only bifidobacterium animalis.
The bifidobacterium is widely applied to foods such as milk powder, yoghourt, ice cream, compound vegetable juice, beer, health-care drinks and the like. In order to better exert the probiotic effect of the bifidobacteria, the bacteria need to keep higher viable count and adhere to and colonize when passing through the gastrointestinal tract, but the oxygen stress is encountered in the actual production, processing, storage and packaging processes, so that the viable count and activity of the bifidobacteria are often insufficient, and the application of the bifidobacteria in functional food is limited. Therefore, the oxygen-resistant bifidobacterium is screened out, and a foundation is laid for solving the oxygen sensitivity problem of the bifidobacterium and industrial application thereof. However, the oxygen resistance and hydrogen peroxide resistance of the currently existing patent report strains are not strong, such as patents CN201110089493.7 and 202010739487.
Disclosure of Invention
The present invention has been made to solve the above problems, and an object of the present invention is to provide an oxygen-tolerant Bifidobacterium animalis strain.
The invention provides an aerotolerant Bifidobacterium animalis (Bifidobacterium animalis) AR668-R1 strain, which has the following characteristics: the aerotolerant bifidobacterium animalis AR668-R1 strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.20457.
Action and effects of the invention
According to the oxygen-resistant bifidobacterium animalis AR668-R1 strain, AR668-R1 can normally grow under the shake flask aerobic culture condition, and the viable count can reach 1.7 multiplied by 10 9 CFU/mL or more, 10.0mM H 2 O 2 The aerotolerant Bifidobacterium animalis AR668-R1 has viable count and H under aerobic culture condition 2 O 2 The tolerance is obviously higher than that of the existing strain, and the strain has good oxygen resistance and hydrogen peroxide tolerance and has wide application prospect in the food industry.
Drawings
FIG. 1 shows a process of acclimatizing aerotolerant Bifidobacterium animalis in an example of the present invention;
FIG. 2 is a comparison of the growth of Bifidobacterium animalis AR668 vs. Bifidobacterium aerotolerant AR668-R1 under anaerobic and aerobic conditions in an example of the invention;
FIG. 3 is the colony count of the Bifidobacterium broth after 24h incubation under anaerobic and aerobic conditions in an example of the invention.
Detailed Description
In order to make the technical means and functions of the present invention easy to understand, the present invention is specifically described below with reference to the embodiments and the accompanying drawings.
< example >
The Bifidobacterium animalis AR668-R1 strain of the embodiment has been deposited in China general microbiological culture Collection center (address: no. 3 of Xilu No. 1 of Beijing, chaoyang district, beijing) at 27.07.2020, and the deposit number is CGMCC No.20457.
The oxygen-resistant bifidobacterium animalis AR668-R1 strain is obtained by domesticating bifidobacterium animalis AR668 with oxygen resistance, and the specific process is as follows:
the materials used were: bifidobacterium animalis AR668 is derived from infant feces and is obtained by separation; bifidobacterium BS medium, qingdao haibo biotechnology limited.
Using the instruments and equipment: shaking table shaking incubator, anaerobic incubator, ultraviolet spectrophotometer, and sterilizing pot.
FIG. 1 shows a process of acclimatization of oxygen-tolerant Bifidobacterium animalis in an example of the present invention.
As shown in fig. 1, the acclimation process is as follows: activating a single clone of bifidobacterium animalis AR668 in a liquid BS culture medium, sequentially carrying out passage three times in the BS liquid culture medium containing 4mM, 5mM, 6mM and 8mM of hydrogen peroxide, adding 5% of seed liquid into 100mLBS liquid culture medium, carrying out anaerobic culture at 37 ℃ for 24h, inoculating 5% of seed liquid into a 250mL conical flask sealed by a sealing film, carrying out shaking culture at the rotating speed of 100rpm for 24h, filling the liquid into 100mL, carrying out anaerobic-aerobic circulation 10 times, then carrying out secondary domestication, increasing the rotating speed of a shaking table to 150rpm, and carrying out alternate culture circulation 10 times. Performing third acclimation with shaking table rotation speed of 200rpm, and alternately culturing for 10 times to obtain oxygen-resistant animal Bifidobacterium AR668-R1.
In this example, the growth of bifidobacterium animalis AR668 and bifidobacterium animalis AR668-R1 resistant to oxygen was compared under the same culture conditions, and the specific process was as follows:
AR668 and AR668-R1 were activated overnight and adjusted to the same OD with BS broth 600 . Inoculating into 100mL BS liquid culture medium at an inoculum size of 5%, performing sealed standing culture and shaking culture at 100rpm, 150rpm and 200rpm for 24h, respectively, and measuring at 600nm for 1 time every 2hAbsorbance, 3 replicates at a time.
FIG. 2 is a comparison of the growth of Bifidobacterium animalis AR668 compared to Bifidobacterium aerotolerant AR668-R1 under anaerobic and aerobic conditions in the examples of the invention, and FIG. 3 is the colony count of Bifidobacterium broth after 24h incubation under anaerobic and aerobic conditions in the examples of the invention.
As shown in FIG. 2, (a), (b), (c) and (d) in FIG. 2 are growth comparisons under anaerobic conditions, aerobic culture conditions with shaking tables at 100rpm, 150rpm and 200rpm, respectively, and AR668-R1 shows the maximum growth OD of the cells under the aerobic culture conditions with standing and shaking tables at 100rpm, 150rpm and 200rpm 600 1.44, 1.41, 1.32 and 1.22 respectively, it can be seen that the acclimatized AR668-R1 can grow normally under the aerobic condition of the shaking table, the maximum bacterial concentration is slightly reduced along with the increase of the rotation speed of the shaking table, as shown in figure 3, the viable count can reach 1.7 × 10 under all three aerobic conditions 9 Above CFU/mL, the highest viable count reaches 2 × 10 9 CFU/mL. In contrast, the starting strain AR668 was OD under anaerobic conditions 600 The highest viable count can only reach 1.08, and the viable count is 1.3 multiplied by 10 9 CFU/mL, its growth is also greatly inhibited under the aerobic condition of shaking table oscillation at different rotating speeds, and the growth OD after reaching the stable period 600 All are less than 0.2, and the viable count is less than 2 x 10 8 No significant changes in growth of AR668 were observed with CFU/mL and varying shaker rotation speed. Therefore, compared with AR668, the oxygen resistance of the strain AR668-R1 obtained by intermittent domestication is greatly improved, and the oxygen-resistant bifidobacterium animalis AR668-R1 of the embodiment grows obviously better than the starting strain under aerobic and anaerobic conditions.
In this example, the hydrogen peroxide tolerance of bifidobacterium animalis AR668 and bifidobacterium animalis AR668-R1 resistant to oxygen was also compared, and the specific process was as follows:
activating AR668 and AR668-R1 overnight, culturing Bifidobacterium overnight, and regulating bacterial liquid OD 600 =0.6 ± 0.05, so as not to contain H 2 O 2 The BS liquid medium of (1) is a blank control. At final concentrations of 3.5, 4.5, 6.0, 8.5, 10.0, 12.0mM H 2 O 2 In the case of inoculation, the strain is inoculated for 3 hours according to the proportion of 1 percent,bacterial dilution, plate coating, plate counting of bifidobacteria survival rate, each group repeat 3 times. Through H 2 O 2 After incubation, AR668 and AR668-R1 were subjected to plate coating method to determine H 2 O 2 The maximum lethal concentration, determined as shown in Table 1,
TABLE 1 Change in Hydrogen peroxide tolerance before and after Bifidobacterium acclimation
As shown in Table 1, AR668 at 6.0mM H 2 O 2 All deaths were observed at this concentration, but the survival rate of AR668-R1 did not decrease significantly, whereas AR668-R1 was at 10.0mM H 2 O 2 Survival at concentration, H of AR668-R1 2 O 2 The maximum lethal tolerance was 12.0mM, which is nearly two-fold higher than AR668, indicating that AR668-R1 has significantly increased hydrogen peroxide tolerance.
In summary, the oxygen-tolerant bifidobacterium animalis AR668-R1 screened by the embodiment can normally grow under the shake flask aerobic culture condition, and the viable count can reach 1.7 × 10 9 CFU/mL or more, and 10.0mM of H 2 O 2 Can survive in the concentration, and has excellent oxygen resistance and hydrogen peroxide tolerance.
Effects and effects of the embodiments
According to the oxygen-resistant bifidobacterium animalis AR668-R1 strain related by the embodiment, AR668-R1 can normally grow under the shake flask aerobic culture condition, and the viable count can reach 1.7 multiplied by 10 9 CFU/mL or more, 10.0mM H 2 O 2 The aerotolerant Bifidobacterium animalis AR668-R1 has viable count and H under aerobic culture condition 2 O 2 The tolerance is obviously higher than that of the existing strain, and the strain has good oxygen resistance and hydrogen peroxide tolerance and has wide application prospect in the food industry.
The above embodiments are preferred examples of the present invention, and are not intended to limit the scope of the present invention.
Claims (1)
1. A Bifidobacterium animalis (AR 668-R1) strain with oxygen tolerance is characterized in that: the aerotolerant bifidobacterium animalis AR668-R1 strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.20457.
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