CN110279056B - Application of bacillus coagulans ACCC10229 in inhibiting aspergillus flavus - Google Patents
Application of bacillus coagulans ACCC10229 in inhibiting aspergillus flavus Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
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Abstract
The invention provides an application of bacillus coagulans ACCC10229 in inhibiting aspergillus flavus, relates to the technical field of microorganisms, and aims to inhibit the growth of aspergillus flavus by a concentrate of a fermentation supernatant of the bacillus coagulans ACCC10229 shown in an aspergillus flavus bacteriostasis test recorded in a specific embodiment of the invention. The invention also provides an aspergillus flavus inhibitor of which the effective substance is composed of the concentrated solution of the fermentation supernatant of the bacillus coagulans ACCC10229, which is obtained by fermenting the bacillus coagulans ACCC10229 and is safe to people and environment. Therefore, the invention provides a safe and effective aspergillus flavus inhibitor.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to application of bacillus coagulans ACCC10229 in inhibiting aspergillus flavus.
Background
Aspergillus flavus is a spoilage microorganism with extremely strong vitality, can cause damage to people and animals when growing in animal feed, and is one of main pollution sources causing feed spoilage mildewing in the processes of feed storage and storage.
A great deal of research is carried out at home and abroad on how to inhibit the growth of the aspergillus flavus, such as physical methods of heat treatment, infrared treatment and the like, chemical methods of adding additives such as lactic acid, benzoic acid and the like, but with practical application, the traditional methods have not proved to be economical, effective and feasible. Research and development of green feed additive products have become a worldwide research topic.
At present, domestic and foreign researches show that part of strains have certain inhibition effect on the growth of aspergillus flavus and the formation of toxin; but no mature and efficient strain application for inhibiting aspergillus flavus exists.
Disclosure of Invention
The invention provides the application of bacillus coagulans ACCC10229 in inhibiting aspergillus flavus in order to overcome the defect that a preparation for effectively inhibiting aspergillus flavus is lacked in the prior art, and the concentrate of the fermentation supernatant of the bacillus coagulans ACCC10229 has the effect of inhibiting aspergillus flavus and is a safe and effective aspergillus flavus inhibitor.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of bacillus coagulans ACCC10229 in inhibiting aspergillus flavus.
Preferably, the pH value of the concentrated solution of the fermentation supernatant of the bacillus coagulans ACCC10229 is 4.2-5.5, and the viable count is 5.84-11.32 lgcfu g-1。
Preferably, the application of the concentrated solution of the fermentation supernatant of the bacillus coagulans ACCC10229 in inhibiting aspergillus flavus is provided.
Preferably, the preparation method of the concentrated solution of the fermentation supernatant of the bacillus coagulans ACCC10229 comprises the following steps:
inoculating bacillus coagulans ACCC10229 to a fermentation culture medium, fermenting for 20-30 h at 32-37 ℃, and performing solid-liquid separation to obtain a fermentation supernatant; and concentrating the fermentation supernatant by 8-15 times to obtain a concentrated solution of the fermentation supernatant of the bacillus coagulans ACCC 10229.
Preferably, the bacterial suspension OD of the bacillus coagulans600The value is 0.3 to 4.8.
Preferably, the inoculation amount of the bacillus coagulans ACCC10229 is 8-15% of the volume of the fermentation medium.
Preferably, the fermentation medium is selected from MRS medium.
Preferably, the fermentation is accompanied by stirring, and the stirring speed is 100-160 rpm.
The invention also provides an aspergillus flavus inhibitor, the effective component of which is composed of the concentrated solution of the fermentation supernatant of bacillus coagulans ACCC 10229.
Preferably, the formulations of the aspergillus flavus inhibitor comprise suspending agent, emulsifiable concentrate, solution, effervescent agent and powder.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides application of bacillus coagulans ACCC10229 in inhibiting aspergillus flavus. As shown in the Aspergillus flavus bacteriostasis test recorded in the specific embodiment of the invention, the concentrate of the fermentation supernatant of the Bacillus coagulans ACCC10229 has an inhibiting effect on the growth of Aspergillus flavus. The invention also provides an aspergillus flavus inhibitor of which the effective substance is composed of the concentrated solution of the fermentation supernatant of the bacillus coagulans ACCC10229, which is obtained by fermenting the bacillus coagulans ACCC10229 and is safe to people and environment. Therefore, the invention provides a safe and effective aspergillus flavus inhibitor.
Drawings
FIG. 1 is a growth curve of Lactobacillus plantarum;
FIG. 2 is a growth curve of Pediococcus acidilactici;
FIG. 3 is a growth curve of Bacillus coagulans;
FIG. 4 is a growth curve of mixed culture of Lactobacillus plantarum, Pediococcus acidilactici and Bacillus coagulans;
FIG. 5 is a graph showing the comparison of growth of Aspergillus flavus spores at different concentrations (48 h);
FIG. 6 is a diagram showing the bacteriostatic effect of the supernatant concentrate after fermentation of the strains.
Detailed Description
The invention provides application of Bacillus coagulans (Bacillus coagulans) ACCC10229 in inhibiting aspergillus flavus. Preferably, the concentrated solution of the fermentation supernatant of the bacillus coagulans ACCC10229 is applied to inhibiting aspergillus flavus. As shown in the specific implementation mode of the invention, the product fermented by the bacillus coagulans ACCC10229 can effectively inhibit aspergillus flavus. In the invention, the bacillus coagulans ACCC10229 is purchased from China agricultural microbial culture collection management center.
In the invention, the pH value of the concentrated solution of the fermentation supernatant of the bacillus coagulans ACCC10229 is preferably 4.2-5.5, and more preferably 4.25-4.4; the number of viable bacteria in the concentrated solution of the fermentation supernatant of the Bacillus coagulans ACCC10229 is preferably 5.84-11.69 lgcfu g-1. More preferably 11.30-11.701 gcfu/g.
In the present invention, the method for preparing the concentrated solution of the fermentation supernatant of bacillus coagulans ACCC10229 preferably comprises the following steps:
inoculating bacillus coagulans ACCC10229 to a fermentation culture medium, fermenting for 20-30 h at 32-37 ℃, and performing solid-liquid separation to obtain a fermentation supernatant; and concentrating the fermentation supernatant by 8-15 times to obtain a concentrated solution of the fermentation supernatant of the bacillus coagulans ACCC 10229.
In the invention, the inoculated Bacillus coagulans ACCC10229 is bacterial suspension, and OD of the bacterial suspension600The value is 0.3 to 4.8. The preparation method of the bacterial suspension of the bacillus coagulans ACCC10229 is not particularly limited.
In the invention, the inoculation amount of the bacillus coagulans ACCC10229 is preferably 8-15% of the volume of a fermentation medium, and more preferably 10%. In the present invention, the fermentation medium includes, but is not limited to, MRS medium.
In the present invention, the time for the fermentation is preferably 24 hours; the temperature of the fermentation is preferably 35 ℃; the fermentation is preferably accompanied by stirring, and the stirring speed is preferably 100-160 rpm, more preferably 120 rpm.
The solid-liquid separation method is not particularly limited in the present invention, and a method known in the art, such as centrifugation or filtration, may be used. The present invention is not particularly limited as to the manner of concentration of the fermentation supernatant, and a concentration method known in the art, such as rotary evaporation, may be employed. In the present invention, the concentration is preferably 10 times.
The invention also provides an aspergillus flavus inhibitor, the effective component of which is composed of the concentrated solution of the fermentation supernatant of bacillus coagulans ACCC 10229. In the invention, the aspergillus flavus inhibitor can also comprise pharmaceutically acceptable auxiliary materials. Specifically, the aspergillus flavus inhibitor can be prepared into various formulations known in the art, including but not limited to suspending agents, emulsifiable concentrates, solutions, effervescent agents, powders and the like.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1 materials and methods
Test materials
Test strains: lactobacillus plantarum ACCC11016 is purchased from China agricultural microbial culture collection management center; pediococcus acidilactici CGMCC 1.4 is purchased from China general microbiological culture Collection center; bacillus coagulans ACCC10229 is purchased from China agricultural microorganism culture collection management center; aspergillus flavus CICC No.2219 is purchased from China center for culture Collection of Industrial microorganisms.
Culture medium: MRS Medium (Modified MRS Medium Base, MRS) was purchased from Hippobo Biotech, Inc., a high-tech industrial park in Qingdao; potato Dextrose (PDA) solid medium: 200g of potatoes and 8g of agar are dissolved in 1L of deionized water, sterilized at 121 ℃ for 20min and prepared according to the formula described in Zhanghua (Zhanghua, research on the flora structure and dominant species metabolism in the traditional wheaten food leavening agent in different regions [ D ]. Zhejiang university, 2014.).
The test instrument: LRH-250A biochemical incubator, produced by Shaoguan City Taihong medical instruments Limited; an HNTC-2027 constant temperature culture shaker, manufactured by Tianjin Ono instruments GmbH; SBA-40E biosensing analyzer, produced by institute of biological sciences of Shandong province academy of sciences; UV-6100 ultraviolet visible spectrophotometer, produced by Shanghai Mei spectral instruments, Inc.; conventional reagents and instrumentation are provided by the laboratory.
1.2 test methods
1.2.1 activation of the bacterial species
Taking out Lactobacillus plantarum, Pediococcus acidilactici and Bacillus coagulans from the cryopreservation tube by using an aseptic inoculating loop, respectively marking lines on an MRS solid inclined plane, culturing in an incubator at 35 ℃ until colonies appear, activating for 2 times in this way, selecting a single colony, inoculating in an MRS liquid culture medium, culturing at 35 ℃ and 120rpm overnight for later use.
1.2.2 measurement of growth Curve, pH value and residual glucose amount (RG) in fermentation broth
Respectively activating Lactobacillus plantarum, Pediococcus acidilactici and Bacillus coagulans to obtain OD600Inoculating 10% (v: v) of mixed bacteria with nm value of 1:1:1 in MRS liquid culture medium, culturing at 35 deg.C and 120rpm for 24 hr, and periodically sampling during culture period to determine OD of bacteria liquid600And (3) drawing a growth curve, a pH change curve and an RG change curve of each strain according to the nm absorbance, the pH value and the RG value so as to observe the growth change of the independent culture and the mixed culture of each strain, wherein each group of experiments is repeated for 3 times.
1.2.3 plate count of strains
The activated mixed culture broth was diluted to 10% with 0.85% (w: v) sterile PBS buffer-7、10-8、10-9、10-10Spreading was then performed, incubated overnight at 35 ℃ and colony counts were performed, with 3 replicates per set of experiments.
1.2.4 bacteriostatic test
1.2.4.1 preparation of Aspergillus flavus spore suspension and determination of concentration of indicator bacterium
Inoculating aspergillus flavus on a PDA (PDA dextrose agar) planar medium, culturing for 4 days in an incubator at 30 ℃, adding a certain amount of sterile PBS (phosphate buffer solution) to scrape spores on a slope, washing the aspergillus flavus spores, collecting the washed aspergillus flavus spores in a centrifuge tube, washing the centrifuge tube for 3 times by using the sterile PBS, filtering the centrifuge tube for 3 times by using sterile gauze to remove hyphae of the aspergillus flavus spores, and counting by using a blood counting chamber. Adjusting spore suspension concentration to 103Spore/ml, 104Spore/ml, 105Spore/ml, 106Spore/ml, 107Spores/ml, spread on PDA plates and cultured at 30 ℃.
1.2.4.2 preparation of fermentation supernatant concentrate
Inoculating the activated mixed culture strain into MRS liquid culture medium, culturing at 35 deg.C and 120rpm for 24 hr, measuring pH, centrifuging at 4 deg.C and 10000rpm for 15min, collecting supernatant, measuring pH with acidimeter, and concentrating the supernatant by 10 times.
1.2.4.3 determination of bacteriostatic Activity
The bacteriostasis experiment is measured by an agar diffusion method, and 100 mul of indicator bacterial spore suspension (each plate contains about 10 percent of indicator bacterial spore suspension)6Spore) and a PDA semisolid culture medium which is melted and kept at about 45 ℃ are mixed uniformly and poured onto a flat plate, a puncher with the inner diameter of 8mm is used for punching after solidification, 100 mu l of supernatant concentrated solution obtained by mixed culture of lactobacillus plantarum, pediococcus acidilactici and bacillus coagulans is injected into a hole, pre-diffusion is horizontally carried out at 4 ℃ for 24h, then culture is carried out at 30 ℃ for 12h, the diameter of a bacteriostatic circle is measured, and 3 experiments in each group are repeated.
1.3 data processing and analysis
Data analysis and charting were done by IBM SPSS static 20.0 and Microsoft Excel 2010, respectively, and data are expressed as "mean. + -. standard deviation".
In a bacteriostatic test, the supernatant concentrated solution obtained by mixed culture of pediococcus acidilactici, lactobacillus plantarum and bacillus coagulans has an inhibiting effect on the growth of aspergillus flavus; the bacteriostatic diameter of the supernatant concentrated solution obtained by mixed culture of pediococcus acidilactici, lactobacillus plantarum and bacillus coagulans is 15.173 + -0.032 mm.
Experimental result data:
2 results
2.1 growth curves of strains
In FIG. 1, it can be seen from the growth curve of Lactobacillus plantarum that the logarithmic growth phase started after a lag phase of about 2 hours and reached the maximum OD at 22 hours600The nm value is 3.482 +/-0.003; the initial pH value is 5.40, the pH value rapidly decreases after 2 hours, and the minimum value is 4.16 +/-0.021 after 22 hours; as can be seen from the curve of the residual glucose level in the fermentation broth, the residual glucose level in the fermentation broth decreased in a decreasing trend of approximately 1.0g/L per 2 hours, the RG value was the lowest at 22 hours, 7.58. + -. 0.144, which was 8.25g/L lower than the RG value of the starting fermentation broth, the OD600nm value,The change curves of the pH value and the RG value are consistent.
As can be seen from the growth curve (FIG. 2) of Pediococcus acidilactici, the logarithmic growth phase started after a lag phase of about 2h and reached the maximum OD at 20h600The nm value is 4.686 +/-0.006; the pH value of the solution is reduced to the minimum value of 4.21 from the initial 5.37 after 22 h; different from lactobacillus plantarum, the RG value of the fermentation liquor of the pediococcus acidilactici is lower and is 4.42 +/-0.144 (P) at 24h<0.05); OD of Pediococcus acidilactici compared to Lactobacillus plantarum600Higher value of nm (P)<0.05)。
Unlike the two strains, Bacillus coagulans starts to enter logarithmic phase only after about 6 hours and has a short logarithmic phase, OD being about 20 hours, as shown in FIG. 3600The value of nm reaches the maximum of 4.094 +/-0.010, and the value is between that of lactobacillus plantarum and pediococcus acidilactici (P)<0.01); the pH value reaches the minimum value of 4.26 +/-0.025 after 12 hours, and the pH value is not changed greatly and is close to stable from 12 hours to 24 hours; the RG value of the fermentation liquid is 7.00 + -0.250 at the lowest in 22h, which is between Lactobacillus plantarum and Pediococcus acidilactici (P)<0.05)。
As shown in FIG. 4, the three bacteria were OD600The mixed culture solution inoculated with the nm value of 1:1:1 enters the logarithmic growth phase within about 2 hours, and the OD is between 2 and 8 hours600The change of the nm value is fast, the increase period is slow between 8h and 20h, and the 20h reaches the maximum value of 3.741 +/-0.003; the pH value of the fermentation liquor changes in two stages, the change trend and the OD600The nm values are consistent, and the lowest value is 4.24 +/-0.006 at 22 h; the minimum RG value of the fermentation liquor is 7.67 +/-0.289 at 24h, 6-8 h and 14-18 h, and the RG value has two obvious changes (P)<0.05)。
2.2 viable count of the Strain
TABLE 1 determination of viable bacteria fermented by the strains (lgcfu. g)-1)
The data in the same column with capital letters different in shoulder mark indicates significant difference (P <0.05), the data with the same capital letters indicates insignificant difference (P >0.05)
The difference of the capital letters of the shoulder marks of the data in the same column indicates that the difference is obvious (P <0.05), the capital letters containing the same indicate that the difference is not obvious (P >0.05)
As can be seen from the data in the same row in Table 1, the viable count difference of the lactobacillus plantarum culture solution is obvious (P is less than 0.05) between 4h and 24h, 4h to 16h are in logarithmic growth phase, and 16h to 24h are in decline phase; the lactic acid pediococcus culture solution is in logarithmic growth phase for 4-16 h, the difference of viable count is obvious (P <0.05), the viable count is in stable phase for 16-24 h, and the difference of viable count is not obvious (P > 0.05); the difference of viable count of the bacillus coagulans culture solution is obvious (P is less than 0.05) between 4h and 24h, the bacillus coagulans culture solution is in logarithmic growth phase for 4h to 10h, and is in decline phase for 10h to 24 h; the difference of viable count of the mixed culture solution is obvious between 4h and 24h (P is less than 0.05), 4h to 16h are in logarithmic growth phase, 16h to 24h are in recession phase, and the viable count change curve of the three bacteria and the mixed culture solution thereof has the same trend with the change curve of OD600nm value.
As can be seen from the data in the same column in Table 1, only three groups of data have no significant difference in the viable count of 12h pediococcus acidilactici and the mixed culture solution, 16h pediococcus acidilactici and the Bacillus coagulans culture solution, and 24h Lactobacillus plantarum and the Bacillus coagulans culture solution (P >0.05), and the significant difference in the viable count of the culture solutions among other different strains at the same time (P < 0.05). In the process of culturing the three bacteria in a mixed way for 24 hours, the viable count of the lactobacillus plantarum, the pediococcus acidilactici, the bacillus coagulans and the mixed culture solution thereof reaches the maximum value about 16 hours, 12 hours and 16 hours respectively.
2.3 determination of the concentration of the indicator Aspergillus flavus spore suspension
FIG. 5 is a graph showing comparison of Aspergillus flavus spore suspensions of different concentrations at a spore concentration of 10 after 48h growth3Spore/ml and 104The growth time of the aspergillus flavus needs 5-7 days for a flat plate with spores/ml, and the experimental period is too long. Spore suspension concentration is 107The spore/ml plate has over high spore concentration, and Aspergillus flavus hypha growth and spore germination are too fast, so that the result of the bacteriostasis experiment is not easy to observe, and the concentration is 105Spore/ml and 106The spore/ml is selected from 10 for easy observation and growth status6Bacteriostatic experiments were carried out on spore/ml bacterial suspensions.
2.4 bacteriostatic diameter of the supernatant concentrate of the fermentation of the strain
FIG. 6 is a graph showing the effect of Aspergillus flavus inhibition of the supernatant concentrate of the three types of bacteria and mixed culture thereof. As can be seen from Table 2, the concentration is 106Six groups of data with spores/ml Aspergillus flavus as indicator bacteria, wherein the bacteriostatic diameter of Lactobacillus plantarum and Pediococcus acidilactici is the largest (P)<0.05)。
The results of measuring the diameter of the zone of inhibition of the supernatant concentrate after fermentation of the strain are shown in table 2:
TABLE 2 measurement results of the diameter of the zone of inhibition of the supernatant concentrate after fermentation of the strain
Note: the same row data with different capital letters indicates significant difference (P <0.05), and the same data with lower case letters indicates insignificant difference (P > 0.05).
Discussion of 3
According to the invention, the growth curve, the pH value, the RG value, the viable count and the bacteriostatic diameter of the mixed culture solution are combined for the independent culture of lactobacillus plantarum, pediococcus acidilactici and bacillus coagulans, the OD600nm value, the pH value, the RG value and the maximum value of the viable count of the mixed culture solution are all between three bacteria in the fermentation process, the bacteriostatic diameter of the supernatant concentrated solution after 24h fermentation is between the three bacteria, the logarithmic growth period of the mixed culture is obviously prolonged, the strain growth of the three bacteria in the mixed culture process is presumed to have a sequential order, and the reason is to be further researched.
The test result of the invention shows that the concentrated solution of the fermentation supernatant obtained by single or mixed fermentation of lactobacillus plantarum, pediococcus acidilactici and bacillus coagulans can inhibit the growth of aspergillus flavus.
4 conclusion
In a bacteriostatic test, the pediococcus acidilactici, the lactobacillus plantarum, the bacillus coagulans and a supernatant concentrated solution obtained by mixed culture of the pediococcus acidilactici, the lactobacillus plantarum and the bacillus coagulans have an inhibiting effect on the growth of aspergillus flavus; the bacteriostatic diameters of the supernatant concentrated solution obtained by mixed culture of pediococcus acidilactici, lactobacillus plantarum, bacillus coagulans and the three are 15.860 + -0.050 mm, 15.737 + -0.155 mm, 14.287 + -0.096 mm and 15.173 + -0.032 mm respectively; wherein the bacteriostatic diameter of Lactobacillus plantarum and Pediococcus acidilactici is the largest (P < 0.05).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. The application of the concentrated solution of the fermentation supernatant of the bacillus coagulans ACCC10229 in inhibiting aspergillus flavus;
the preparation method of the concentrated solution of the fermentation supernatant of the bacillus coagulans ACCC10229 comprises the following steps: inoculating bacillus coagulans ACCC10229 to a fermentation culture medium, fermenting for 20-30 h at 32-37 ℃, and performing solid-liquid separation to obtain a fermentation supernatant; and concentrating the fermentation supernatant by 8-15 times to obtain a concentrated solution of the fermentation supernatant of the bacillus coagulans ACCC 10229.
2. The use according to claim 1, wherein the pH value of the concentrated solution of the fermentation supernatant of Bacillus coagulans ACCC10229 is 4.2-5.5, and the viable count is 5.84-11.70 lg cfu-g-1。
3. The use of claim 1, wherein the bacillus coagulans ACCC10229 is inoculated in an amount of 8-15% by volume of the fermentation medium.
4. The use according to claim 1, wherein the bacillus coagulans bacterial suspension has an OD600 value of 0.3-4.8.
5. Use according to claim 1, wherein the fermentation medium is selected from MRS medium.
6. The use of claim 1, wherein the fermentation is accompanied by stirring at a rate of 100 to 160 rpm.
7. An Aspergillus flavus inhibitor is characterized in that the effective component is a concentrated solution of fermentation supernatant of Bacillus coagulans ACCC 10229.
8. The Aspergillus flavus inhibitor according to claim 7, wherein the formulation of the Aspergillus flavus inhibitor comprises a suspension agent, an emulsifiable concentrate, a solution agent, an effervescent agent and a powder agent.
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