A method for preparing a mixed bacterial agent based on plant endophytes

Abstract

The present application relates to the field of biotechnology, and specifically to a method for preparing a mixed bacterial agent based on plant endophytes, the method comprising: culturing Bacillus polymyxa and Bacillus firmus in an activation medium, inoculating the activated two types of Bacillus single colonies into seed culture medium respectively, shaking and culturing in a shake bottle, and inoculating two types of Bacillus seed liquid respectively for fermentation; when the fermentation monitors the maximum bacterial concentration, stopping the feeding, adjusting the ventilation, dissolved oxygen and rotation speed, and continuing the fermentation, during the fermentation process of the mixed bacterial agent of the two types of Bacillus, collecting the mixed bacterial agent images and fermentation monitoring data at each moment to adjust the fermentation time, mixing the two types of Bacillus mixed fermentation liquid with a carrier and a freeze-drying protective agent, centrifuging, precipitating, precooling, and freeze-drying operations to obtain a mixed bacterial agent. The present application aims to adjust the fermentation time of the mixed bacterial agent and improve the stability and stress resistance of the mixed bacterial agent.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for preparing a mixed bacterial agent based on plant endophytes. Background Art

[0002] Plant endophytes refer to fungi or bacteria that live inside the tissues and organs of plants at a certain stage or all stages. These microorganisms are commonly found in higher plants, and different plant endophytes have different effects. Among plant endophytes, Paenibacillus polymyxa and Bacillus firmus are gram-positive bacteria of the genus Bacillus. Paenibacillus polymyxa can secrete a variety of metabolic active substances such as polypeptide antibiotics and antagonistic proteins, and has important functions such as promoting plant growth and inducing plant immune resistance, but has poor resistance to crop pests; Bacillus firmus can secrete antibacterial protein active products, which can strongly kill a variety of plant pests through antibacterial effects and spore germination, but does not have the effect of promoting plant growth. Therefore, Paenibacillus polymyxa and Bacillus firmus are used as mixed bacterial agents for complementation, which has significant synergistic and complementary effects in promoting plant growth and preventing and controlling pests.

[0003] In the process of preparing the mixed inoculant of Bacillus polymyxa and Bacillus firmus, the fermentation time needs to be strictly controlled. If the fermentation time is too long, it may lead to the formation of harmful by-products, resulting in a decrease in the activity of the target bacteria, thus affecting the safety and application effect of the mixed inoculant; if the fermentation time is too short, it may lead to the bacteria content failing to meet the standard, resulting in incomplete spore formation, affecting the stability and stress resistance of the inoculant. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of this application is to provide a method for preparing a mixed bacterial agent based on plant endophytes, and the technical scheme adopted is as follows:

[0005] The present invention provides a method for preparing a mixed bacterial agent based on plant endophytes, the method comprising the following steps:

[0006] (1) heat-shock Paenibacillus polymyxa and Bacillus firmus in a water bath, and then culture them in an activation medium to obtain activated Paenibacillus polymyxa and Bacillus firmus single colonies;

[0007] (2) inoculating the activated single colonies of Paenibacillus polymyxa and Bacillus firmus into seed culture medium respectively, and culturing them in a shaking flask to obtain Paenibacillus polymyxa and Bacillus firmus seed liquid respectively;

[0008] (3) inoculating a certain amount of Bacillus polymyxa and Bacillus firmus seed solution into a fermentation medium containing a dried distiller's grains hydrolyzate; during the fermentation process, maintaining the dissolved oxygen content, controlling the pH, and maintaining the glucose concentration in the fermentation liquid;

[0009] (4) When the fermentation monitor reaches the maximum bacterial concentration OD 620 After that, stop feeding, adjust ventilation, dissolved oxygen and rotation speed, and continue fermentation; in the fermentation process of the mixed inoculum of Bacillus polymyxa and Bacillus firmus, collect the mixed inoculum image at each moment and the mixed inoculum fermentation monitoring data; the mixed inoculum fermentation monitoring data includes but is not limited to the effective live bacteria count of Bacillus polymyxa and Bacillus firmus;

[0010] (4.1) obtaining the spore matching index of each pixel point according to the local edge distribution and gray value distribution of the pixel points of the mixed inoculum image at each moment;

[0011] (4.2) according to the distribution of the spore matching index of all pixels in the mixed inoculum image at each moment and combining the segmentation algorithm, the spore development maturity index of the mixed inoculum image at each moment is obtained;

[0012] (4.3) adjusting the fermentation time according to the spore development maturity index of the mixed inoculum image at consecutive moments and the mixed inoculum fermentation monitoring data combined with trend test to obtain a mixed fermentation liquid of Paenibacillus polymyxa and Bacillus firmus;

[0013] (5) The mixed fermentation liquid of Paenibacillus polymyxa and Bacillus firmus is mixed with a carrier and a freeze-drying protective agent, and then centrifuged, precipitated, precooled, and freeze-dried to obtain a mixed bacterial agent.

[0014] Preferably, the culture temperature in the activation medium is 28° C. and the culture time is 24 hours.

[0015] Preferably, the shaking culture temperature in the shake flask is 28° C., the rotation speed is 150 rpm, and the culture time is 24 hours.

[0016] Preferably, the maintenance of dissolved oxygen content is specifically as follows: maintaining a ventilation volume of 1.5 vvm by an air pump, controlling the bubble diameter to 0.5 mm using a gas disperser, and maintaining an oxygen capacity of 40%.

[0017] Preferably, the pH is controlled to be 7.0 by adding 25% ammonia aqueous solution.

[0018] Preferably, the specific operation of maintaining the glucose concentration in the fermentation broth is: when the glucose concentration drops to 2 g / L, feeding feed medium is added to maintain the glucose concentration in the fermentation broth not higher than 2 g / L.

[0019] Preferably, the ventilation, dissolved oxygen content and rotation speed are adjusted to 0.8 vvm, the dissolved oxygen content is maintained at 12.5%, and the rotation speed is 100 rpm.

[0020] Preferably, the spore matching index of each pixel point is specifically:

[0021] The edge pixel points of the mixed bacterial agent image at each moment are obtained by using an edge detection algorithm on the mixed bacterial agent image at each moment;

[0022] For the mixed inoculant image at each moment; taking each pixel point as the center, a spore monitoring window of each pixel point is established with a preset side length; a linear fitting algorithm is used for the edge pixel points in the spore monitoring window of each pixel point to obtain the goodness of fit of each pixel point;

[0023] For each pixel point, when the number of edge pixels in the spore monitoring window of the pixel point is greater than a preset value, the minimum grayscale value of all pixels in the spore monitoring window of the pixel point is obtained, the absolute value of the difference between the grayscale value of each pixel in the spore detection window of the pixel point and the minimum grayscale value is calculated, the reciprocal of the sum of the absolute value of the difference and a preset coordination factor greater than zero is calculated, the mean of the reciprocals of all pixels in the spore detection window of the pixel point is calculated, and the product of the mean and the goodness of fit of the pixel point is used as the spore growth line matching index of each pixel point; otherwise, 0 is used as the spore growth line matching index of each pixel point;

[0024] According to the spore growth line matching index, a clustering algorithm is used to obtain each cluster for all pixel points in the mixed inoculum image at each moment, and the mean of the spore growth line matching index of all pixel points in each cluster is calculated, and the cluster with the largest mean is taken as the spore growth cluster; the pixel points in the spore growth cluster are subjected to connected domain analysis and linear fitting, and the inclination angle is obtained by inverse trigonometric function;

[0025] For each pixel point, obtain the edge pixel points that are the first and second closest to the pixel point, connect them respectively, and use the slopes of the perpendicular lines connecting the first and second closest edge pixel points as the first slope and second slope of the pixel point respectively;

[0026] When a pixel point belongs to a spore growth cluster, half of the sum of the first slope and the second slope of the pixel point is calculated; the half result is used as the independent variable of the inverse tangent function; the absolute value of the difference between the calculation result of the inverse tangent function and the inclination angle is used as the inclination angle difference, and the inverse of the sum of the inclination angle difference and a preset coordination factor greater than zero is used as the spore matching index of the pixel point; otherwise, 1 is used as the spore matching index of the pixel point.

[0027] Preferably, the spore development maturity index of the mixed inoculum image at each moment is specifically:

[0028] The spore matching index of all pixels in the mixed inoculum image at each moment is used as the input of the region growing algorithm, and each region is output;

[0029] Calculate the mean of the horizontal and vertical coordinates of all pixels in each region in the pixel coordinate system, and use the point corresponding to the mean of the horizontal and vertical coordinates as the center point of each region; use the mean of the Euclidean distance from the center point to each boundary point of the corresponding region as the growth mean of each region;

[0030] The spore development maturity index of the mixed inoculum image at each moment is expressed as:

[0031]

[0032] In the formula, H t Represents the mixed bacterial agent image F t The spore development maturity index, m t Represents the mixed bacterial agent image F t The number of growth regions in the t Represents the mixed bacterial agent image F t The mean of the growth means of all growth regions in , f t (d 1 ), f t (d 2 ) represent the mixed bacterial agent images F t Middle d 1 d 2 The growth mean in the growth area, δ represents the coordination factor that is preset to be greater than zero, F t Represents the mixed inoculum image at the tth moment.

[0033] Preferably, the mixed fermentation liquid of Paenibacillus polymyxa and Bacillus firmus is obtained by:

[0034] For the mixed inoculum image at each moment, a preset number of mixed inoculum images are taken to form a spore development atlas at each moment, and the spore development maturity indexes of the mixed inoculum images at all moments in the spore development atlas at each moment are combined into a spore development maturity index sequence according to the time sequence, and a trend test is performed on the spore development maturity index sequence to obtain a p value;

[0035] The expression of spore termination judgment index at each time is:

[0036]

[0037] In the formula, Q t represents the sporulation termination judgment index at the tth moment, P t represents the p-value of the hypothesis test of the spore development and maturity index sequence at the t-th moment, U t 、V t respectively represent the effective viable counts of Bacillus polymyxa and Bacillus firmus at the tth moment, U 0 、V 0represent the target required number of effective live bacteria of Paenibacillus polymyxa and Bacillus firmus respectively, and α and β represent coordination factors preset to be greater than zero;

[0038] The normalized value of the spore termination judgment index at each moment is obtained, and a termination judgment threshold is set; when the normalized value at each moment is greater than or equal to the termination judgment threshold, the spores are mature; otherwise, the spores are not mature; when the spores are mature for a preset number of consecutive moments, the fermentation is stopped to obtain a mixed fermentation liquid of Bacillus polymyxa and Bacillus firmus.

[0039] This application has at least the following beneficial effects:

[0040] The present application constructs a spore matching index by analyzing the shape characteristics of spores in the mixed inoculum during the fermentation process, reflecting the degree to which each pixel in the mixed inoculum image at each time point conforms to the spore shape characteristics, thereby improving the accuracy of identifying spores; constructs a spore development and maturity index to reflect the spore maturity characteristics in the mixed inoculum image at each time point, thereby effectively judging whether the spores are mature; based on the spore development and maturity index at each time point, the change characteristics of spore growth and development over time in the mixed inoculum image are analyzed, and a spore termination judgment index is constructed in combination with the effective live bacterial count of the strain, which is used to judge whether the fermentation should be terminated so that the effective live bacterial count of the strain at the end of the fermentation reaches the standard, while ensuring that the spores grow and develop well and can all mature, thereby improving the stability and stress resistance of the mixed inoculum. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions and advantages in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 A flowchart of a method for preparing a mixed bacterial agent based on plant endophytes provided in one embodiment of the present application;

[0043] Figure 2 This is a schematic diagram of the spore shape in the mixed bacterial agent;

[0044] Figure 3 Schematic diagram of the positional relationship between the pixels in the spore growth cluster and other edge pixels;

[0045] Figure 4 Adjust the flow chart for fermentation time. DETAILED DESCRIPTION

[0046] In order to further explain the technical means and effects adopted by the present application to achieve the predetermined invention purpose, the following is a detailed description of the method for preparing a mixed bacterial agent based on plant endophytes proposed in the present application, its specific implementation method, structure, characteristics and effects, in combination with the accompanying drawings and preferred embodiments. In the following description, different "one embodiment" or "another embodiment" does not necessarily refer to the same embodiment. In addition, specific features, structures or characteristics in one or more embodiments may be combined in any suitable form.

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0048] The specific scheme of the method for preparing a mixed bacterial agent based on plant endophytes provided in the present application is described in detail below with reference to the accompanying drawings.

[0049] See also Figure 1 , which shows a flow chart of the steps of a method for preparing a mixed bacterial agent based on plant endophytes provided in one embodiment of the present application, the method comprising the following steps:

[0050] (1) After heat shocking at 80°C in a water bath for 5 minutes, Paenibacillus polymyxa and Bacillus firmus were respectively applied to an activation culture medium at 28°C for 24 hours to obtain activated Paenibacillus polymyxa and Bacillus firmus single colonies. The pH value of the activation culture medium was 7.5, and the main components and contents were shown in Table 1 below:

[0051] Table 1: Main components and contents of activation medium

[0052]

[0053] (2) A 500 mL triangular flask was filled with 100 mL of seed culture medium, and the single colonies of Paenibacillus polymyxa and Bacillus firmus activated in the previous step were inoculated into the seed culture medium, respectively, and cultured in a shaking flask at a temperature of 28° C. and a rotation speed of 150 rpm for 24 hours to obtain Paenibacillus polymyxa and Bacillus firmus seed solutions, respectively, wherein the pH of the seed culture medium was 7.5, and the main components and contents were shown in Table 2 below:

[0054] Table 2: Main components and contents of seed culture medium

[0055]

[0056] (3) 1L of fermentation medium was placed in a 3L fermentation tank, and the seed liquids of Bacillus polymyxa and Bacillus firmus in S2 were inoculated at an inoculum rate of 2.5% (v / v), respectively, with a stirring speed of 150 rpm and a fermentation temperature of 28° C. The pH value of the fermentation medium of the dried distiller's grains hydrolyzate was 7.5, and the main components and contents were shown in Table 3 below:

[0057] Table 3: Main components and contents of fermentation medium containing dried distiller's grains hydrolysate

[0058]

[0059] The preparation process of the dry distiller's grains enzymatic hydrolyzate is as follows: the dry distiller's grains and distilled water are uniformly mixed at a mass ratio of 1:20, the pH is adjusted to 10 with 10% sodium hydroxide solution, treated in a water bath at 80°C for 60 minutes, alkaline protease is added at 200U / g of the dry distiller's grains at 50°C for enzymatic hydrolysis for 24 hours, and the supernatant is collected by centrifugation to obtain the dry distiller's grains enzymatic hydrolyzate.

[0060] During the fermentation process, the ventilation volume was maintained at 1.5 vvm by an air pump, and the bubble diameter was controlled to 0.5 mm by a gas disperser, thereby maintaining the dissolved oxygen content at 40%; the pH was controlled to 7.0 by adding 25% ammonia solution; when the glucose concentration dropped to 2 g / L, 25 mL of feed medium was added to maintain the glucose concentration in the fermentation broth not higher than 2 g / L, wherein the main components and contents of the feed medium were: 500 g / L glucose, 50 g / L glycerol, and the rest was distilled water;

[0061] (4) When the fermentation monitor reaches the maximum bacterial concentration OD 620 After that, stop feeding, adjust the ventilation volume to 0.8vvm, maintain the dissolved oxygen content at 12.5%, the rotation speed is 100rpm, and continue fermentation for 1 to 4 hours. During the fermentation process of the mixed inoculum of polymyxa and bacillus firmus, the mixed inoculum image in the fermentation process is continuously collected at intervals T by a high-definition camera, and the collected image type is RGB type. The collected mixed inoculum RGB image is converted into a grayscale image by grayscale averaging method; Green Fluorescent Protein (GFP) is used as a specific fluorescent marker of bacillus, and the effective viable counts of polymyxa and bacillus firmus are collected by a flow cytometer and a high-definition camera at the same frequency, respectively, and the effective viable counts of polymyxa and bacillus firmus are recorded as mixed inoculum fermentation monitoring data. The collection interval is recorded as T, and T is 5s in this embodiment.

[0062] Since the collected images may contain noise due to environmental interference and other factors during the image collection process, in order to avoid the influence of noise on the subsequent analysis process, this embodiment uses a bilateral filtering algorithm to denoise the collected images, and at the same time, in order to make the details in the image clearer, this embodiment uses an adaptive contrast enhancement algorithm to enhance the denoised images. The bilateral filtering algorithm and the adaptive contrast enhancement algorithm are well-known technologies and will not be described in detail in this embodiment.

[0063] The preprocessing results of each mixed bacterial agent image are denoted as F 1 、F 2 ,…,F n , where F 1 、F 2 、F n They represent the mixed bacterial agent images at the 1st, 2nd, and nth moments respectively.

[0064] In the process of collecting mixed bacterial fermentation monitoring data, the collected data may be affected by environmental interference and other factors during data transmission, resulting in abnormalities such as missing values. In order to avoid the missing values ​​affecting the subsequent calculation results, this embodiment uses a regression filling method to fill the missing values. The regression filling method is a well-known technology, and the specific process is not repeated in this embodiment.

[0065] At this point, the mixed bacteria image and mixed bacteria fermentation monitoring data during the mixed bacteria fermentation process are obtained.

[0066] For the convenience of description, this embodiment uses the mixed bacterial agent image F at the tth moment t As an example, it is described. During the fermentation process of the mixed bacterial agent, when the bacteria form spores, elliptical or circular spores can be observed in the central position of the bacteria. At the same time, because the spores grow outward from the central area, there is a line on the edge of the spore that points to the center of the circle. It is recorded as the spore growth line. The direction of the spore growth line should be consistent with the direction of the arc segment of the spore edge pointing to the center of the circle. At the same time, from the outside to the inside, the color of the spore gradually changes from light yellow to white. Among them, the schematic diagram of the spore shape in the mixed bacterial agent is shown in the figure below. Figure 2 shown.

[0067] The spore matching index is constructed to reflect the mixed inoculant image F t Specifically, the mixed inoculant image F t As the input of Canny edge detection algorithm, the output is the mixed bacterial agent binary map F t ′ , that is, the mixed bacterial agent image F is obtained t The edge pixels in the mixed bacterial agent image F tThe square window with the ath pixel as the center and the side length of b is recorded as the spore monitoring window of the ath pixel, and the coordinates of the edge pixels in the spore monitoring window in the pixel coordinate system are linearly fitted using the least squares method to obtain the goodness of fit, where the value of b in this embodiment is 7, the Canny edge detection algorithm and the least squares method are well-known technologies, and the specific process is not repeated in this embodiment. Then the spore growth line matching index can be calculated, and the calculation formula is as follows:

[0068]

[0069] Where Y t (a) represents the mixed bacterial agent image F t The spore growth line matching index of the a-th pixel in n t (a) represents the mixed bacterial agent image F t The number of edge pixels in the spore monitoring window of the a-th pixel, R t (a) represents the mixed bacterial agent image F t The goodness of fit after linear fitting of the edge pixels in the spore monitoring window of the a-th pixel in the image, F t (c) represents the mixed bacterial agent image F t The gray value of the cth edge pixel in the spore monitoring window of the ath pixel in F t (c,min) represents the mixed bacterial agent image F t The minimum value of the grayscale value of the pixel points in the eight-neighborhood of the cth edge pixel point in the spore monitoring window of the ath pixel point, ∈ represents a coordination factor preset greater than zero, which is used to avoid the denominator being zero and causing the calculation to be impossible. In this embodiment, ∈ is taken as 1.

[0070] In the spore monitoring window of the mixed inoculant image, the better the linear fitting of the edge pixels in the window, that is, R t (a) is larger, indicating that the edge pixel is closer to a straight line and more consistent with the shape characteristics of the spore growth line. At the same time, due to the large color difference between the two ends of the circular or elliptical boundary of the spore, the difference between the edge pixel and the minimum gray value of the pixel in the eight neighborhoods is smaller, that is, |F t (c)-F t The smaller (c,min)| is, the less likely the edge pixel is to be on the boundary of the spore. Therefore, the edge pixel is more likely to be inside the spore and the more likely it is to be the spore growth line. Therefore, the larger the spore growth line matching index is.

[0071] The spore growth line matching index of each pixel point is used as the input of the K-means clustering algorithm. In this embodiment, K is set to 3, and each cluster is output. The mean of the spore growth line matching index of all pixels in each cluster is used as the central representative value of each cluster. The three clusters are arranged in descending order according to the central representative value and recorded as a spore growth cluster, a spore boundary cluster, and a non-spore cluster respectively; the pixel points in the spore growth cluster in the mixed inoculant image are analyzed for the connected domain, and the pixel points in the connected domain are linearly fitted using the least squares method to obtain the slope after fitting, and the inclination angle θ corresponding to the slope is calculated by the inverse tangent function a , where the positional relationship between the pixels in the spore growth cluster and other edge pixels is shown in the figure Figure 3 The K-means clustering algorithm and connected domain analysis are well-known technologies, and the specific process will not be described in detail in this embodiment.

[0072] Therefore, the spore matching index is calculated as follows:

[0073]

[0074] In the formula, Z t (a) represents the mixed bacterial agent image F t The spore matching index of the a-th pixel in the image, arctan() represents the inverse tangent function, l a1 , l a2 Represent the mixed bacterial agent image F t The first slope and second slope of the a-th pixel in , θ a Represents the mixed bacterial agent image F t The tilt angle after linear fitting of the pixel points in the spore growth cluster where the a-th pixel point is located, S 1 represents the spore growth cluster, a represents the pixel number, and ε represents a coordination factor preset to be greater than zero, which is used to avoid the denominator being zero and causing calculation failure. In this embodiment, the value is 1. t The method for obtaining the first slope and the second slope of the a-th pixel in is: connect the two edge points closest to the a-th pixel respectively, and use the slope of the vertical line connecting the a-th pixel and its first and second near edge pixels as the first slope and the second slope of the a-th pixel. as the tilt angle difference.

[0075] In the mixed inoculant image, the smaller the difference between the inclination angle corresponding to the mean of the first slope and the second slope of the a-th pixel and the inclination angle after fitting the pixel points in the spore growth cluster where the a-th pixel is located, the better the The smaller it is, the more the a-th pixel point conforms to the circular or elliptical features of the spore boundary, so the calculated spore matching index is larger.

[0076] Using mixed bacterial agent image F t The mixed inoculum images at each time point were processed in the same way to obtain the spore matching index of each pixel in the mixed inoculum images at all time points.

[0077] The mixed bacterial agent image F t The spore matching index of all pixels in the image is used as the input of the region growing method, and the mixed inoculant image F t The edge pixel points are used as seed points, the growth criterion is that the ratio of the absolute value of the difference between the spore matching indexes of two adjacent pixels to the maximum value of the spore matching indexes of the two pixels is less than or equal to 0.2, and the output is a number of regions after the pixel points grow. The mean values ​​of the horizontal and vertical coordinates of the pixels in each region in the pixel coordinate system are calculated respectively, and the obtained coordinates are used as the center points of each region respectively, and the mean value of the Euclidean distance from the center point to each boundary point of the region is calculated, which is recorded as the growth mean. The regional growth method is a well-known technology, and the specific process is not repeated in this embodiment. Then the spore development maturity index can be calculated, and the calculation formula is as follows:

[0078]

[0079] In the formula, H t Represents the mixed bacterial agent image F t The spore development maturity index, m t Represents the mixed bacterial agent image F t The number of growth regions in the t Represents the mixed bacterial agent image F t The mean of the growth means of all growth regions in , f t (d 1 ), f t (d 2 ) represent the mixed bacterial agent images F t Middle d 1 d 2 The growth mean value in the growth area, δ represents a coordination factor preset to be greater than zero, which is used to avoid the denominator being zero and causing calculation failure. In this embodiment, the value is 1.

[0080] In the mixed inoculum image F t The smaller the difference in the growth mean of different growth areas, the smaller the difference in the growth mean of different growth areas, that is, |f t (d 1 )-f t (d 2 )| is smaller, indicating that the mixed bacterial agent image F t The sizes of different spores are close, and the larger the mean of the growth mean of all growth areas, that is, f t The larger the value, the larger the spore size, i.e. the mixed inoculant image F tThe more mature the comprehensive development of the spores, the larger the calculated spore development maturity index.

[0081] At this point, the spore development maturity index of the mixed inoculum image at different times can be obtained.

[0082] The spore development maturity index at each time point reflects the maturity characteristics of the spores in the mixed inoculant images at each time point. When collecting images during the fermentation process of the mixed inoculant, the positions of the high-definition camera and the mixed inoculant did not change, that is, the position of the center point of the spore growth was close in different images, that is, the coordinates of the spore center positions in different images in the pixel coordinate system should be close, and the slower the speed at which the outer edge of the spore grows outward in a circular shape, the more mature the spore development is.

[0083] Taking the tth moment as an example, the set of mixed inoculant images taken at k time points before the tth moment in chronological order is recorded as the spore development image set of the tth moment. The value of k in this embodiment is 9. If the number of time points before the tth moment is less than k, the mixed inoculant images at the existing time points are used as the spore development image set, and the sequence of spore development maturity indexes of the mixed inoculant images in the spore development image set in chronological order is recorded as the spore development maturity index sequence. The above sequence is used as the input of the Wilcoxon trend test. The null hypothesis of the hypothesis test is that there is a trend in the spore development maturity index sequence. The output is the p-value of the hypothesis test, which reflects the reliability of the null hypothesis. The Wilcoxon trend test is a well-known technology, and the specific process will not be repeated in this embodiment. Calculate the sporulation termination judgment index, the calculation formula is as follows:

[0084]

[0085] In the formula, Q t represents the sporulation termination judgment index at the tth moment, P t represents the p-value of the hypothesis test of the spore development and maturity index sequence at the t-th moment, U t 、V t respectively represent the effective viable counts of Bacillus polymyxa and Bacillus firmus at the tth moment, U 0 、V 0 represents the target number of effective viable bacteria of Bacillus polymyxa and Bacillus firmus, respectively. In this embodiment, the values ​​are 1.2×10 12 CFU / mL, 8.0×10 9 CFU / mL, α and β represent coordination factors preset to be greater than zero, which are used to avoid calculation failure due to the denominator being zero. In this embodiment, both values ​​are 1.

[0086] The larger the p-value of the hypothesis test at the t-th moment, that is, P tThe larger the value is, the higher the degree of maturity of all spores in the mixed inoculant at the tth moment. That is, as the culture time increases, the spores do not continue to develop. At the same time, the difference between the target number of effective live bacteria of Bacillus polymyxa and that of Bacillus firmus at the tth moment is smaller, that is, |U t -U 0 | and |V t -V 0 The smaller | is, the more the spore concentration in the mixed inoculant at the tth moment meets the preparation requirements. The fermentation should be ended as soon as possible to avoid the influence of too long fermentation time on the spore activity. Therefore, the larger the spore termination judgment index is.

[0087] At this point, the sporulation termination determination index at each time point was obtained.

[0088] The sporulation termination judgment index at each time point is normalized using the Z-score method to obtain a normalized sporulation termination judgment index. If the normalized sporulation termination judgment index at the t-th moment is greater than or equal to the termination judgment threshold, it indicates that the spores at time point t have matured; if the normalized sporulation termination judgment index at time point t is less than the termination judgment threshold, it indicates that the spores at time point t have not yet matured. In order to avoid misjudgment caused by accidental events, in this embodiment, the sporulation termination judgment index at k consecutive moments is greater than or equal to the termination judgment threshold, then the fermentation can be stopped, wherein in this embodiment, k is taken as 10, and the termination judgment threshold is taken as 0.8. The Z-score normalization method is a well-known technology, and the specific process will not be repeated in this embodiment. Among them, the fermentation time adjustment flow chart is as follows: Figure 4 shown.

[0089] When the effective viable count of Paenibacillus polymyxa was 1.2×10 10 CFU / mL, effective viable count of Bacillus firmus was 8.0×10 9 CFU / mL, the fermentation was stopped to obtain a mixed fermentation liquid of Paenibacillus polymyxa and Bacillus firmus.

[0090] (5) The mixed fermentation liquid obtained in the previous step is mixed with the carrier and the freeze-drying protective agent, and then subjected to high-speed centrifugation. The precipitate is collected and pre-cooled at -40°C for 6 hours, and then placed in a vacuum freeze dryer for lyophilization for 36 hours to obtain a mixed bacterial agent.

[0091] At this point, a method for preparing a mixed bacterial agent based on plant endophytes has been completed.

[0092] In summary, the embodiment of the present application constructs a spore matching index by analyzing the shape characteristics of spores in the mixed inoculant during the fermentation process, reflecting the degree to which each pixel in the mixed inoculant image at each time point conforms to the spore shape characteristics, thereby improving the accuracy of identifying spores; constructs a spore development and maturity index to reflect the spore maturity characteristics in the mixed inoculant image at each time point, effectively judging whether the spores are mature; based on the spore development and maturity index at each time point, the change characteristics of spore growth and development over time in the mixed inoculant image are analyzed, and a spore termination judgment index is constructed in combination with the effective live bacterial count of the strain, which is used to judge whether the fermentation should be terminated so that the effective live bacterial count of the strain at the end of the fermentation reaches the standard, while ensuring that the growth and development of the spores are good and can all mature, thereby improving the stability and stress resistance of the mixed inoculant.

[0093] It should be noted that the above sequence of the embodiments of the present application is for description only and does not represent the advantages and disadvantages of the embodiments. The above is a description of a specific embodiment of this specification. In addition, the processes depicted in the accompanying drawings do not necessarily require the specific order or continuous order shown to achieve the desired results. In some embodiments, multitasking and parallel processing are also possible or may be advantageous.

[0094] The various embodiments in this specification are described in a progressive manner, and the same or similar parts between the various embodiments can be referenced to each other, and each embodiment focuses on the differences from other embodiments.

[0095] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, improvements, etc. made within the principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing a mixed bacterial agent based on plant endophytes, characterized in that: The method comprises the following steps: (1) heat-shock Paenibacillus polymyxa and Bacillus firmus in a water bath, and then culture them in an activation medium to obtain activated Paenibacillus polymyxa and Bacillus firmus single colonies; (2) inoculating the activated single colonies of Paenibacillus polymyxa and Bacillus firmus into seed culture medium respectively, and culturing them in a shaking flask to obtain Paenibacillus polymyxa and Bacillus firmus seed liquid respectively; (3) inoculating a certain amount of Bacillus polymyxa and Bacillus firmus seed solution into a fermentation medium containing a dried distiller's grains hydrolyzate; during the fermentation process, maintaining the dissolved oxygen content, controlling the pH, and maintaining the glucose concentration in the fermentation liquid; (4) When the fermentation monitor reaches the maximum bacterial concentration OD 620 After that, stop feeding, adjust ventilation, dissolved oxygen and rotation speed, and continue fermentation; in the fermentation process of the mixed inoculum of Bacillus polymyxa and Bacillus firmus, collect the mixed inoculum image at each moment and the mixed inoculum fermentation monitoring data; the mixed inoculum fermentation monitoring data includes but is not limited to the effective live bacteria count of Bacillus polymyxa and Bacillus firmus; (4.1) obtaining the spore matching index of each pixel point according to the local edge distribution and gray value distribution of the pixel points of the mixed inoculum image at each moment; (4.2) according to the distribution of the spore matching index of all pixels in the mixed inoculum image at each moment and combining the segmentation algorithm, the spore development maturity index of the mixed inoculum image at each moment is obtained; (4.3) adjusting the fermentation time according to the spore development maturity index of the mixed inoculum image at consecutive moments and the mixed inoculum fermentation monitoring data combined with trend test to obtain a mixed fermentation liquid of Paenibacillus polymyxa and Bacillus firmus; (5) mixing the mixed fermentation liquid of Paenibacillus polymyxa and Bacillus firmus with the carrier and the freeze-drying protective agent, centrifuging, precipitating, precooling, and freeze-drying to obtain a mixed bacterial agent; The spore matching index of each pixel point is specifically: The edge pixel points of the mixed bacterial agent image at each moment are obtained by using an edge detection algorithm on the mixed bacterial agent image at each moment; For the mixed inoculant image at each moment; taking each pixel point as the center, a spore monitoring window of each pixel point is established with a preset side length; a linear fitting algorithm is used for the edge pixel points in the spore monitoring window of each pixel point to obtain the goodness of fit of each pixel point; For each pixel point, when the number of edge pixels in the spore monitoring window of the pixel point is greater than a preset value, the minimum grayscale value of all pixels in the spore monitoring window of the pixel point is obtained, the absolute value of the difference between the grayscale value of each pixel in the spore detection window of the pixel point and the minimum grayscale value is calculated, the reciprocal of the sum of the absolute value of the difference and a preset coordination factor greater than zero is calculated, the mean of the reciprocals of all pixels in the spore detection window of the pixel point is calculated, and the product of the mean and the goodness of fit of the pixel point is used as the spore growth line matching index of each pixel point; otherwise, 0 is used as the spore growth line matching index of each pixel point; According to the spore growth line matching index, a clustering algorithm is used to obtain each cluster for all pixel points in the mixed inoculum image at each moment, and the mean of the spore growth line matching index of all pixel points in each cluster is calculated, and the cluster with the largest mean is taken as the spore growth cluster; the pixel points in the spore growth cluster are subjected to connected domain analysis and linear fitting, and the inclination angle is obtained by inverse trigonometric function; For each pixel point, obtain the edge pixel points that are the first and second closest to the pixel point, connect them respectively, and use the slopes of the perpendicular lines connecting the first and second closest edge pixel points as the first slope and second slope of the pixel point respectively; When a pixel point belongs to a spore growth cluster, half of the sum of the first slope and the second slope of the pixel point is calculated; the half result is used as the independent variable of the inverse tangent function; the absolute value of the difference between the calculation result of the inverse tangent function and the inclination angle is used as the inclination angle difference, and the inverse of the sum of the inclination angle difference and a preset coordination factor greater than zero is used as the spore matching index of the pixel point; otherwise, 1 is used as the spore matching index of the pixel point.

2. The method for preparing a mixed bacterial agent based on plant endophytes according to claim 1, characterized in that: The culture temperature in the activation medium is 28° C., and the culture time is 24 hours.

3. The method for preparing a mixed bacterial agent based on plant endophytes according to claim 1, characterized in that: The shaking culture temperature in the shake flask is 28° C., the rotation speed is 150 rpm, and the culture time is 24 hours.

4. The method for preparing a mixed bacterial agent based on plant endophytes according to claim 1, characterized in that: The maintenance of dissolved oxygen content is specifically as follows: maintaining a ventilation volume of 1.5 vvm by an air pump, controlling the bubble diameter to 0.5 mm by a gas disperser, and maintaining an oxygen capacity of 40%.

5. The method for preparing a mixed bacterial agent based on plant endophytes according to claim 1, characterized in that: The pH is controlled to be 7.0 by adding 25% ammonia aqueous solution.

6. The method for preparing a mixed bacterial agent based on plant endophytes according to claim 1, characterized in that: The specific operation of maintaining the glucose concentration in the fermentation broth is: when the glucose concentration drops to 2 g / L, feed medium is added to maintain the glucose concentration in the fermentation broth not higher than 2 g / L.

7. The method for preparing a mixed bacterial agent based on plant endophytes according to claim 1, characterized in that: The ventilation, dissolved oxygen content and rotation speed are specifically adjusted to 0.8 vvm, the dissolved oxygen content is maintained at 12.5%, and the rotation speed is 100 rpm.

8. The method for preparing a mixed bacterial agent based on plant endophytes according to claim 1, characterized in that: The spore development maturity index of the mixed inoculum image at each moment is specifically: The spore matching index of all pixels in the mixed inoculum image at each moment is used as the input of the region growing algorithm, and each region is output; Calculate the mean of the horizontal and vertical coordinates of all pixels in each region in the pixel coordinate system, and use the point corresponding to the mean of the horizontal and vertical coordinates as the center point of each region; use the mean of the Euclidean distance from the center point to each boundary point of the corresponding region as the growth mean of each region; The spore development maturity index of the mixed inoculum image at each moment is expressed as: In the formula, H t Represents the mixed bacterial agent image F t The spore development maturity index, m t Represents the mixed bacterial agent image F t The number of growth regions in the t Represents the mixed bacterial agent image F t The mean of the growth means of all growth regions in , f t (d1), f t (d2) represent the mixed bacterial agent image F t The growth mean in the d1th and d2th growth regions, δ represents the coordination factor preset to be greater than zero, F t Represents the mixed inoculum image at the tth moment.

9. The method for preparing a mixed bacterial agent based on plant endophytes according to claim 1, characterized in that: The mixed fermentation liquid of Paenibacillus polymyxa and Bacillus firmus is specifically: For the mixed inoculum image at each moment, a preset number of mixed inoculum images are taken to form a spore development atlas at each moment, and the spore development maturity indexes of the mixed inoculum images at all moments in the spore development atlas at each moment are combined into a spore development maturity index sequence according to the time sequence, and a trend test is performed on the spore development maturity index sequence to obtain a p value; The expression of spore termination judgment index at each time is: In the formula, Q t represents the sporulation termination judgment index at the tth moment, P t represents the p-value of the hypothesis test of the spore development and maturity index sequence at the t-th moment, U t 、V t respectively represent the effective viable counts of Paenibacillus polymyxa and Bacillus firmus at the t-th moment, U0 and V0 respectively represent the target required counts of the effective viable counts of Paenibacillus polymyxa and Bacillus firmus, and α and β represent coordination factors that are preset to be greater than zero; Obtain the normalized value of the spore termination judgment index at each moment and set the termination judgment threshold; When the normalized value at each moment is greater than or equal to the termination judgment threshold, the spores are mature; Otherwise, the spores do not mature; When the spores mature for a preset number of times in a row, the fermentation is stopped to obtain a mixed fermentation liquid of Bacillus polymyxa and Bacillus firmus.

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