Nisin titer detection method based on microwell plate method

Abstract

The invention discloses a nisin titer detection method based on a microwell plate method. According to the growth inhibition effect of nisin on indicator bacteria, a plant lactobacillus L30 with a good strain growth amount-nisin titer curve is screened from existing strains, the plant lactobacillus L30 is applied to a microwell plate method, and a nisin titer determination method which is high in accuracy, convenient and economical is developed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of potency detection of polypeptide antibacterial agents, in particular to a method for detecting the potency of nisin based on a microplate method. BACKGROUND

[0002] With the increasing demand for green, healthy and environmentally friendly food, more and more foods are developing towards natural and healthy, and free of chemical preservatives. Natural antibacterial agents have become a hot spot in the research and development of various foods. Nisin, as a natural bacteriostatic agent, is widely used in food and has been approved for use in about 80 countries, including the European Union, the United States, China, etc. The GB 2760-2024 "Standard for the Use of Food Additives" issued by China makes provisions for the addition of nisin in foods such as milk and dairy products, instant rice and flour products, cooked meat products, etc.

[0003] The biological potency of nisin is an important parameter for measuring its bacteriostatic ability. The activity content of commercially available nisin products varies, and the activity content determines the potency. The thermal stability of nisin is easily affected by temperature, pH, matrix, etc., which in turn limits the application of nisin in food thermal processing to some extent and affects the final bacteriostatic effect. The methods for determining the potency of nisin mainly include agar diffusion method, chemical immunization method, biological fluorescence method, high performance liquid chromatography method, turbidity method, etc.

[0004] The potency detection method in Appendix A of GB 1886.231-2023 Nisin Product Standard is the agar diffusion method. The agar diffusion method is simple and low in cost. Although the accuracy and sensitivity have been greatly improved after years of improvement and improvement, they are still limited. In addition, factors such as strain activity, bacterial suspension concentration, agar thickness and agar concentration will affect the final experimental results. Moreover, the method is complex to operate and has poor repeatability of results in actual detection process.

[0005] The colorimetric comparison method mainly adds resazurin indicator. The more live bacteria in the culture medium, the easier it is to form an anaerobic environment, causing resazurin to change from blue to purple and then to pink. Comparing the color of the sample with the color of the standard curve to convert the results. The detection error is large and the operation is complicated. In the turbidity method, the growth of Micrococcus luteus is slow, resulting in a long experimental period and poor accuracy of experimental results.

[0006] Therefore, there is still an unmet need for developing a simple and stable method for determining the biological potency of nisin. SUMMARY

[0007] In order to make up for the shortage of the prior art, the present application screens a plantarum L30 with a good growth amount-streptococcal nisin titer curve from existing strains according to the growth inhibition of nisin on the indicator bacteria, and applies it to a microplate method, aiming to develop a nisin titer determination method which is high in accuracy, convenient and economical.

[0008] In one aspect, the present application provides a microplate method-based nisin titer determination method, which comprises the following steps:

[0009] S1: Establishing a standard curve of nisin titer concentration-indicator bacteria growth condition;

[0010] S2: Preparing a nisin sample into a sample diluent;

[0011] S3: Detecting the growth condition of the indicator bacteria mixed and cultured with the sample diluent, and substituting into the standard curve to calculate the nisin titer in the nisin sample;

[0012] Wherein, the growth condition of the indicator bacteria is characterized by absorbance.

[0013] In one or more embodiments, the indicator bacteria is Lactiplantibacillus plantarum CICC 6076.

[0014] In one or more embodiments, the detection wavelength of the absorbance is 500-650 nm.

[0015] Preferably, the detection wavelength of the absorbance is 550 nm.

[0016] In one or more embodiments, the indicator bacteria is stored in the form of a glycerol tube.

[0017] In one or more embodiments, in the step S1, the concentration range of the titer in the standard curve is 10-50 IU / mg.

[0018] In one or more embodiments, in the step S3, the initial concentration of the mixed and cultured indicator bacteria is 1x10 4 -4x10 4 CFU / mL.

[0019] Preferably, in the step S3, the initial concentration of the mixed and cultured indicator bacteria is 2x10 4 -3x10 4 CFU / mL.

[0020] In one or more embodiments, the step S2 comprises aseptically weighing the streptozocin sample, fully dissolving and constant volume with aseptic hydrochloric acid, and diluting the sample diluent with aseptic hydrochloric acid by an appropriate multiple.

[0021] In one or more embodiments, the concentration of the aseptic hydrochloric acid is 0.01-0.03 mol / L.

[0022] Preferably, the concentration of the aseptic hydrochloric acid is 0.015-0.025 mol / L. More preferably, the concentration of the aseptic hydrochloric acid is 0.02 mol / L.

[0023] In another aspect, the present application provides the use of Lactiplantibacillus plantarum CICC 6076 in detecting the streptozocin titer.

[0024] In one or more embodiments, the detection of the streptozocin titer is based on the microplate method.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] (1) Compared with the conventional agar diffusion method, the method for determining the streptozocin titer based on the microplate method of the present application can effectively avoid the influencing factors such as the thickness, concentration, punching aperture, and pre-diffusion of the plate of the agar; the agar diffusion method needs to elute the medium slope with physiological saline to obtain a bacterial suspension, and the concentration and diluent composition of the bacterial suspension obtained each time have uncertainty, while in the present application, the bacterial liquid is preserved and activated in a glycerol tube, which can ensure the high consistency of the growth state (including the bacterial age and the inoculation amount) of the indicator bacteria within a long period of time (6 months); the present application uses an enzyme label instrument to read, which is more accurate than the manual measurement of the diameter of the inhibition zone in the agar diffusion method.

[0027] (2) Based on Lactiplantibacillus plantarum L30 and the microplate method, the present application establishes a method for determining the streptozocin titer based on the microplate method on the basis of the preferred preservation method, the inoculation amount of the indicator bacteria, the range of the standard curve, and the aseptic treatment method of the sample, and the fitting correlation coefficient r of the method in the value range reaches 0.99604.

[0028] (3) Through methodological investigation, it can be known that the intermediate precision RSD and the repeatability determination RSD of the method for determining the streptozocin titer based on the microplate method of the present application are both lower than 5%, the standard addition recovery rate is between 95% and 105%; the RSD of the determination results of the method and the national standard method is 0.18%-8.80%. The method of the present application has high accuracy and reliability, and provides a convenient and economical scheme for the detection of the streptozocin titer. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is the growth of Micrococcus flavus in water and 0.02 mol / L sterile hydrochloric acid.

[0030] Figure 2 is the growth of each strain in water and 0.02 mol / L sterile hydrochloric acid.

[0031] Figure 3 is the relationship curve between the titer concentration of nisin in the culture solution and the growth of each strain.

[0032] Figure 4 is the statistical graph of the colony number of L30 glycerol tube stock strain after subculture under different frozen storage time.

[0033] Figure 5 is the relationship curve between the titer concentration of nisin in the culture solution and the growth of L30 when the concentration of bacterial suspension is 10 8 CFU / mL.

[0034] Figure 6 is the relationship curve between the titer concentration of nisin in the culture solution and the growth of L30 when the concentration of bacterial suspension is 10 7 CFU / mL.

[0035] Figure 7 is the relationship curve between the titer concentration of nisin in the culture solution and the growth of L30 when the concentration of bacterial suspension is 10 6 CFU / mL.

[0036] Figure 8 is the relationship curve between the titer concentration of nisin in the culture solution and the growth of L30 when the concentration of bacterial suspension is 10 7 CFU / mL in the titer concentration range of 10-50 IU / mL. DETAILED DESCRIPTION

[0037] In the present application, the method for determining the titer of nisin by microplate method is established based on colorimetric comparison method and turbidimetry. In a certain concentration range, the proliferation activity of Lactobacillus plantarum is negatively correlated with the titer concentration of added nisin, so the proliferation activity of Lactobacillus plantarum can be characterized according to the change of absorbance, and then the standard curve of absorbance and titer concentration is established, and the titer of nisin in the sample can be calculated by substituting the corresponding absorbance of the sample.

[0038] In the following examples, all experimental materials or strains are commercially available unless otherwise specified. The nisin standard (1178 IU / mg, CAS: 1414-45-5) is purchased from TOKU-E company.

[0039] In the following examples, the experimental equipment or instruments mainly include: SMI12 biochemical incubator (purchased from SHELLAB Company, USA), LA2-6A1 biological safety cabinet (double person) (purchased from Esco Company, Singapore), HXC-608 culture medium storage cabinet (refrigerated) (purchased from Qingdao Haier Company), MS304S electronic balance (purchased from Mettler Toledo Company, Switzerland), SX700 autoclave (purchased from TOMY Company, Japan), M200 full-wavelength multifunctional microplate analysis system (purchased from TECAN Company, Switzerland), FE20K pH meter (purchased from Mettler Toledo Company, Switzerland), 5415D centrifuge (purchased from Eppendorf Company, Germany), ReasearchPlus 20-200 μL pipette (purchased from Eppendorf Company, Germany), Reasearch Plus 100-1000 μL pipette (purchased from Eppendorf Company, Germany).

[0040] In the following examples, the preparation method of 0.02 mol / L sterile hydrochloric acid solution is as follows: 1.68 mL of concentrated hydrochloric acid is measured, water is added to 1 L, and a 0.22 μm aperture needle filter is used to filter and sterilize under sterile conditions.

[0041] In the following examples, the preparation method of S1 medium (S1 plate) is as follows: 0.8% (w / v) of tryptone, 0.5% (w / v) of yeast extract, 0.5% (w / v) of glucose, 0.5% (w / v) of sodium chloride, 0.2% (w / v) of disodium hydrogen phosphate, and 1.2%-1.5% (w / v) of agar powder are weighed according to a total volume of 1 L, water is added to 1 L, pH is adjusted to 6.8-7.0, and sterilization is performed at 121°C for 20 min to prepare a stock solution; double S1 medium is prepared by doubling the contents of tryptone, yeast extract, glucose, sodium chloride, and disodium hydrogen phosphate in the above S1 medium.

[0042] In the following examples, the preparation method of S1 liquid medium is as follows: 0.8% (w / v) of tryptone, 0.5% (w / v) of yeast extract, 0.5% (w / v) of glucose, 0.5% (w / v) of sodium chloride, and 0.2% (w / v) of disodium hydrogen phosphate are weighed according to a total volume of 1 L, water is added to 1 L, pH is adjusted to 6.8-7.0, and sterilization is performed at 121°C for 20 min to prepare a stock solution; double S1 liquid medium is prepared by doubling the contents of tryptone, yeast extract, glucose, sodium chloride, and disodium hydrogen phosphate in the above S1 liquid medium.

[0043] In the following examples, the activation method of the strain is as follows:

[0044] Micrococcus luteus (NCIMB 8166) activation: Take one loop of Micrococcus luteus from the freeze-dried tube with a sterile inoculation loop, inoculate on a sterile S1 plate, perform streak isolation, select full and smooth edge colonies, inoculate on S1 test tube slant, and incubate in a 30°C incubator for 24 h, then store in a 0-4°C refrigerator for standby.

[0045] Test strain activation: After the freeze-dried tube is taken out, it is restored to room temperature. Take one magnetic bead from the freeze-dried tube with a sterile inoculation needle, transfer it to MRS broth culture medium, and incubate in a 36°C incubator for 24 h, then inoculate on an MRS test tube slant, and incubate in a 36°C incubator for 24 h, then store in a 0-4°C refrigerator for standby.

[0046] In the following examples, the preparation of the glycerol tube of the test strain and the activation method of the stock strain are as follows:

[0047] S1: Inoculate the strain from the MRS test tube slant culture medium into MRS broth culture medium with a sterile inoculation loop, incubate at 36°C for 24 h, mix 150 μL of bacterial suspension with 150 μL of sterile 50% (v / v) glycerol water solution to prepare a glycerol tube, and store it at -30°C as a stock strain, which can be stored for 6 months.

[0048] S2: Take the glycerol tube of the stock strain out of the refrigerator and restore it to room temperature, take an appropriate amount of bacterial liquid and inoculate it into 5 mL of sterilized MRS broth culture medium, and incubate at 36°C for 20 h to complete the activation of the stock strain of the test strain, and the activated bacterial liquid is ready for use.

[0049] In the following examples, the preparation method of the test bacterial suspension is as follows:

[0050] Centrifuge the bacterial suspension after activation of the stock strain of the test strain at 3500 rpm for 5 min, and discard the supernatant. Resuspend the bacterial cells with sterile normal saline, centrifuge at 3500 rpm for 5 min, discard the supernatant, and repeat the step; after resuspending and centrifuging the bacterial cells for a total of 3 times, resuspend the bacterial cells with 5 mL of sterile normal saline, dilute the bacterial suspension with sterile normal saline as appropriate to adjust the concentration, and take 50 μL of the diluted bacterial suspension into 10 mL of double MRS broth culture medium as the test bacterial suspension.

[0051] Example 1: Test of strain acid tolerance

[0052] The present application learns from the treatment method of nisin in GB 1886.231-2023, that is, dissolving the nisin sample in a 0.02 mol / L hydrochloric acid solution. Unlike the agar diffusion method, the microplate method used by the present application is a liquid culture system. When determining the titer, the entire culture system is in a strongly acidic state. Therefore, the microplate method has a higher requirement for the acid tolerance of the indicator bacteria, and the growth of the indicator bacteria still needs to have a certain correlation with the concentration of nisin. In order to establish a method for determining the titer of nisin by the microplate method, the present embodiment aims to screen a strain that is sensitive to nisin and acid-tolerant.

[0053] Micrococcus flavus acid tolerance experiment

[0054] The activated yellow micrococcus (NCIMB 8166) bacterial solution was inoculated into 5 mL of S1 liquid culture medium and cultured at 30°C for 20 h. The culture solution was washed by centrifuging at 3500 rpm for 5 min to discard the supernatant and resuspended with sterile normal saline, a total of 3 times. Then, 5 mL of sterile normal saline was used for resuspension, and 50 μL of the resuspension was taken into 10 mL of double S1 liquid culture medium as a test bacterial suspension.

[0055] In a sterile 96-well microplate, 150 μL of 0.02 mol / L sterile hydrochloric acid and 150 μL of the test bacterial suspension of yellow micrococcus were added; another control group was set up, in which 150 μL of sterile water and 150 μL of the test bacterial suspension of yellow micrococcus were added to the microplate. Each treatment was repeated 8 times. The absorbance at 550 nm wavelength was measured after 24 h and 48 h of culture at 30°C.

[0056] The growth of yellow micrococcus in water and 0.02 mol / L sterile hydrochloric acid is shown in Figure 1 As can be seen from Figure 1 , when the yellow micrococcus grows for 24 h with the addition of hydrochloric acid, the absorbance is only 0.0821, and when it grows for 48 h, the absorbance does not increase significantly; in contrast, in the control group without the addition of hydrochloric acid, the absorbance is 0.2452 when it grows for 24 h, and the absorbance is 0.3782 when it grows for 48 h. It can be seen that the growth of yellow micrococcus NCIMB 8166 in a strongly acidic environment is significantly inhibited, and in addition, the absorbance is 0.3782 when it grows in sterile water for 48 h, which is at a relatively low level. Therefore, it is speculated that the liquid culture system may not be suitable for the growth of this strain and the determination of the titer of nisin.

[0057] Test strain acid tolerance experiment

[0058] Each strain listed in Table 1 was activated and inoculated into 5 mL of Lactobacillus broth, and cultured at 36°C for 20 h. The culture medium was then centrifuged at 3500 rpm for 5 min, the supernatant was discarded, and the culture was resuspended in sterile physiological saline. This washing was repeated three times. After resuspending in 5 mL of sterile physiological saline, 50 μL of the resuspended solution was transferred to 10 mL of double-strength Lactobacillus broth as the test suspension.

[0059] Add 150 μL of 0.02 mol / L sterile hydrochloric acid and 150 μL of the test bacterial suspension of the above-mentioned experimental strain to a sterile 96-well microplate; set up a control group, adding 150 μL of sterile water and 150 μL of the test bacterial suspension of the experimental strain to a microplate. Each treatment was repeated 8 times. Incubate at 36℃ for 24 h, and measure the absorbance at 550 nm.

[0060] Table 1: Information on test strains

[0061]

[0062] Comparison of the growth of each test strain in water and in 0.02 mol / L sterile hydrochloric acid. Figure 2 As shown. By Figure 2 As can be seen, after 24 hours of cultivation, the absorbance (A550) of each tested strain was generally greater than 0.5, significantly higher than that of *Micrococcus faecalis* NCIMB 8166. While there were some differences in acid tolerance among the tested strains, the growth of each strain in water and in 0.02 mol / L sterile hydrochloric acid showed little difference. Therefore, this experiment demonstrates that the selected lactic acid bacteria strains used in the experiment possess good acid tolerance, and their acid tolerance is stronger than that of *Micrococcus faecalis*.

[0063] Example 2: Growth of various test strains under the inhibition of different concentrations of nisin

[0064] The growth curves of each test strain in different concentrations of nisin culture medium are shown below. Figure 3 .Depend on Figure 3 It can be seen that the growth of each strain decreased as the potency of nisin increased; however, the absorbance-potency curves of each strain were not identical.

[0065] Strains L9 and L27 showed slight inhibition with increasing nisin concentration, indicating that these two strains were less sensitive to nisin in the range of nisin concentration. Strains L8, L31, L32 and L37 were completely inhibited by nisin when the nisin concentration was greater than 0 IU / mL, indicating that these strains were too sensitive to nisin. Strains L7, L16, L26, L28 and L45 also showed a dramatic increase in growth inhibition when the nisin concentration reached a certain range, and the marginal increase in inhibition was too strong. Relatively speaking, strain L30 (Lactiplantibacillus plantarum CICC6076) showed a slow decrease in growth with increasing nisin concentration in the range of 10-50 IU / mL, and could well present an "S" type curve - low concentration of strain growth was not affected, and high concentration of strain growth was inhibited, indicating that the strain was potentially suitable for the determination of nisin titer by microplate method.

[0066] Example 3: Preparation of absorbance-nisin titer standard curve

[0067] Preparation of nisin standard working solution

[0068] According to the purity of the nisin standard, the corresponding 100000 IU activity of nisin standard (accurate to 0.0001 g) was accurately weighed, dissolved with 0.02 mol / L sterile hydrochloric acid, and diluted to 100 mL to obtain 1000 IU / mL of nisin standard solution.

[0069] The nisin standard solution (1000 IU / mL) was diluted by 10 times gradient twice with 0.02 mol / L sterile hydrochloric acid to obtain nisin standard working solution with titer concentrations of 100 IU / mL and 10 IU / mL, respectively.

[0070] Preparation of standard curve series solution

[0071] Prepare the standard curve series solutions S1-S7 according to Table 1. Three sets of standard curve series solutions were prepared. Among them, the nisin titer concentrations of S1-S7 were 10 IU / mL, 15 IU / mL, 20 IU / mL, 25 IU / mL, 30 IU / mL, 40 IU / mL and 50 IU / mL, respectively. In addition, 0.02 mol / mL sterile hydrochloric acid was prepared for UN and IN groups, respectively. UN represents unseeded blank centrifuge tube (0 IU / mL), and IN represents seeded blank centrifuge tube (0 IU / mL).

[0072] Table 1: Standard curve series solutions

[0073] Centrifuge tube number UN IN S1 S2 S3 S4 S5 S6 S7 0.02 mol / mL sterile hydrochloric acid / μL 500 500 0 560 400 600 560 300 250 10 IU / mL standard working solution / μL 0 0 500 0 0 0 0 0 0 100 IU / mL standard working solution / μL 0 0 0 100 100 200 240 200 250

[0074] Activity determination and standard curve establishment

[0075] In each well of a sterile microplate, 150 μL of the test bacterial suspension (50 μL of 10 7 CFU / mL L30 bacterial suspension mixed with 10 mL of double-strength lactobacillus broth) was added, followed by the addition of 150 μL of the 0.02 mol / mL sterile hydrochloric acid (group IN) or the solutions of S1-S7, respectively. Another group of wells, UN, was prepared by first adding 150 μL of double-strength MRS broth medium to each well of a sterile microplate, followed by the addition of 150 μL of 0.02 mol / mL sterile hydrochloric acid. After the addition of the samples to the wells, the microplate was incubated in an incubator at 36°C for 20 h.

[0076] After the incubation, each well was visually inspected. The culture medium in the un-inoculated blank (UN) was clear, and the turbidity of the culture medium in the standard curve wells showed a certain gradient difference. The microplate was covered with a film, mixed well, and placed in a microplate reader to detect the absorbance of each well at 550 nm. The absorbance and the titer concentration were used as coordinates to establish a standard curve, which was then fitted using software.

[0077] Example 4: Detection of the streptozocin titer in a sample

[0078] A sample of 0.1 g (m) was accurately weighed (to 0.0001 g) under sterile conditions, dissolved with 0.02 mol / mL sterile hydrochloric acid, and diluted to 100 mL (V). The sample was diluted by an appropriate factor (f) using 0.02 mol / mL sterile hydrochloric acid, based on the estimated streptozocin titer concentration, to obtain a sample dilution, so that the streptozocin titer concentration in the sample dilution was 10-50 IU / mL.

[0079] In each well of a sterile microplate, 150 μL of the test bacterial suspension (50 μL of 10 7 CFU / mL L30 bacterial suspension mixed with 10 mL of double-strength lactobacillus broth) was added, followed by the addition of 150 μL of the 0.02 mol / mL sterile hydrochloric acid (group IN) or the solutions of S1-S7, respectively. Another group of wells, UN, was prepared by first adding 150 μL of double-strength MRS broth medium to each well of a sterile microplate, followed by the addition of 150 μL of 0.02 mol / mL sterile hydrochloric acid. After the addition of the samples to the wells, the microplate was incubated in an incubator at 36°C for 20 h.

[0080] After the incubation, each well was visually inspected. The culture medium in the un-inoculated blank (UN) was clear, and the turbidity of the culture medium in the standard curve wells showed a certain gradient difference. The microplate was covered with a film, mixed well, and placed in a microplate reader to detect the absorbance of each well at 550 nm. The absorbance and the titer concentration were used as coordinates to establish a standard curve, which was then fitted using software.

[0081]

[0082] In the formula:

[0083] X – Potency of the sample, in International Units per milligram (IU / mg); C – Potency concentration of the sample solution obtained by substituting the absorbance into the standard curve, in International Units per milliliter (IU / mL); V – Volume of the sample solution prepared, in milliliters (mL); m – Mass of the sample, in grams (g); f – Dilution factor of the sample solution; 1000 – Conversion factor.

[0084] Example 5: Validation of Glycerol-containing Bacterial Activity

[0085] At 0, 0.5, 1, 2, 4, and 6 months after the L30 glycerol tubes were prepared and stored, the tubes were removed and brought to room temperature. 50 μL of the bacterial suspension from the glycerol tubes was transferred to 5 mL of sterile MRS broth and incubated at 36°C for 20 h. The culture was then serially diluted 10-fold with sterile physiological saline, with 100 μL of each dilution added to two sterile Petri dishes. MRS broth was poured into each dish, and the dishes were incubated at 36°C for 72 h. The colony counts on the plates were then recorded, and the viability of the reserve strains (based on the total colony count) was statistically analyzed. The results are shown below. Figure 4 As shown.

[0086] Depend on Figure 4 It can be seen that, in the 6th month, the total number of colonies in the bacterial suspension after subculturing in glycerol tubes of the reserve strain only increased from the original 3.2 × 10⁻⁶. 8 CFU / mL decreased to 2.1×10 8 CFU / mL remained at 10 8 A concentration of CFU / mL or higher indicates that the stock strain of glycerol tubes can maintain good bacterial activity during 6 months of frozen storage.

[0087] Compared to GB 1886.231-2023, which requires eluting the culture medium slant with physiological saline to obtain the bacterial suspension, the concentration and dilution method of the bacterial suspension obtained each time are uncertain. Glycerol tube preservation can ensure that the growth status of the indicator bacteria, including the bacterial age and inoculation amount, is relatively consistent in each test within 6 months after preservation.

[0088] Example 6: Determination of indicator bacteria inoculum size and standard curve range

[0089] The amount of indicator bacteria in the test bacterial suspension directly affects the correlation in the titer standard curve of the microplate method and the accuracy of the titer results.

[0090] Adjust the bacterial suspension concentration to 10. 8 CFU / mL, 10 7 CFU / mL and 10 6CFU / mL, take 50 μL into 10 ml of double MRS broth medium, and then establish a standard curve according to the method in Example 3 to evaluate the appropriate inoculum amount of indicator bacteria; it can be seen that 10 8 CFU / mL, 10 7 CFU / mL and 10 6 The bacterial suspension concentration of CFU / mL corresponds to approximately 2.5 × 10⁻⁶. 5 CFU / mL, 2.5 × 10 4 CFU / mL and 2.5×10 3 CFU / mL initial concentration in culture medium.

[0091] Figure 5-7 The inoculum size of the indicator bacteria, which was fitted by the software, was 10. 8 CFU / mL, 10 7 CFU / mL and 10 6 Standard curve of nisin potency concentration versus growth at CFU / mL. (The text appears to be incomplete and requires further context.) 8 CFU / mL, 10 7 CFU / mL and 10 6 The potency standard curves at CFU / mL all fit according to the four-parameter equation, with correlation coefficients r of 0.98536, 0.99723, and 0.99611, respectively. Regarding the correlation coefficient r, the bacterial suspension concentration was 10... 7 CFU / mL and 10 6 The potency standard curve fits better at CFU / mL.

[0092] The bacterial suspension concentration was 10. 6 CFU / mL potency standard curve ( Figure 7 In the standard curve, when the titer concentration reached 30 IU / mL, the absorbance was only 0.1882, indicating that the indicator bacteria were almost completely inhibited. Therefore, the concentration window for indicating titer in this standard curve is only 10–30 IU / mL. Meanwhile, at a bacterial suspension concentration of 10… 7 CFU / mL potency standard curve ( Figure 6 In this standard curve, the indicator bacteria are in a completely inhibited state at a titer concentration of 50 IU / mL. The concentration window for indicating titer in this standard curve is 10–50 IU / mL, which is a relatively wider range. Furthermore, the requirements for estimating sample concentration and dilution are relatively less stringent compared to a 10-10 standard. 6 The bacterial suspension concentration was lower at CFU / mL. Therefore, the bacterial suspension concentration was adjusted to 10. 7 The method for preparing the bacterial suspension for testing is as follows: take 50 μL of the CFU / mL solution and mix it with 10 mL of double-strength MRS broth.

[0093] The bacterial suspension concentration is 107 The standard curve of titer (CFU / mL) is shown in Figure 1. Figure 6 As shown in Figure 1, when the titer concentration is 0 IU / mL, 5 IU / mL and 10 IU / mL, the absorbance of the three points is almost the same, indicating that the indicator bacteria are basically in a state of complete non-inhibition. Therefore, the 5 IU / mL standard point is meaningless, and 0 IU / mL is retained as the blank inoculation. The titer concentration of 10 IU / mL is used as the first point of the standard curve. When the titer concentration is ≥50 IU / mL, the indicator bacteria are basically in a state of complete inhibition, so the titer concentration points greater than 50 IU / mL are discarded. In this way, the standard curve is redrawn as shown in Figure 2. When the titer concentration is 10-50 IU / mL, the fitted standard curve is y = 0.14687 + 0.6287 / [1 + (x / 21.94426)], with a correlation coefficient r = 0.99604, and the fitting degree of the standard curve is good. Figure 8 3.89887

[0094] Example 7: Selection of sample sterilization method

[0095] In this experiment, three samples of streptozyme with estimated titers of 3000 IU / mg, 2000 IU / mg and 1200 IU / mg were selected. Different sample sterilization methods were used in the titer detection process, i.e. 1) Streptozyme sample was weighed aseptically, dissolved with 0.02 mol / mL aseptic hydrochloric acid and diluted to volume (without filtration), or 2) The sample was weighed and dissolved with 0.02 mol / mL hydrochloric acid, filtered with a 0.22 μm pore size filter to remove bacteria after dilution to volume (filtration), and then the titer of streptozyme was detected according to the method of Example 4, and the titer results detected by the national standard method were compared to select a more suitable sample sterilization method. The titer results and relative errors of the three samples are shown in Table 2.

[0096] Table 2: Titer determination results and relative errors of streptozyme under different sample sterilization methods

[0097]

[0098]

[0099] ​​As can be seen from Table 2, the titer results obtained after dissolving and filtering the streptozocin are generally lower than those obtained by aseptic weighing and aseptic dissolving, and the relative errors of the two samples other than sample 1 are greater than 10% compared with the national standard method, indicating that the filtering operation may cause the titer results to differ greatly from the national standard method. It is speculated that since streptozocin is essentially a polypeptide substance, the filter material may adsorb part of the streptozocin, resulting in a decrease in the content of the filtered sample, thereby showing a low titer. Subsequently, the sample to be tested was prepared by aseptically weighing the streptozocin sample, fully dissolving it with 0.02 mol / mL aseptic hydrochloric acid, and diluting to volume.

[0100] Example 8: Methodology Investigation

[0101] Precision investigation

[0102] An accurate amount of 1 part of the streptozocin sample (sample 2) 0.1362 g was weighed, and the titer concentration was detected according to the method of Example 4. The titer was determined 6 times in succession, and the average titer and RSD value were calculated to investigate the precision of the instrument. The results are shown in Table 3. As can be seen from Table 3, the precision RSD of the 6 titer determination results is 2.16%, which is lower than 5%, indicating that the precision of the instrument is good.

[0103] Table 3: Precision investigation of the microplate method for determining the titer of streptozocin

[0104]

[0105] Intermediate precision investigation

[0106] According to the method of Example 4, 3 times of titer concentration of the same streptozocin sample (sample 2) were detected by 2 different experimenters in 2 different time periods, and the average titer and RSD value were calculated to investigate the intermediate precision of the method. The results are shown in Table 4. As can be seen from Table 4, the intermediate precision RSD is 4.39%, which is lower than 5%, indicating that the influence of different experimenters on the titer results determined by the microplate method is small, and the intermediate precision of the method is good.

[0107] Table 4: Investigation of the intermediate precision of the microplate method for determining the titer of streptozocin

[0108]

[0109]

[0110] Reproducibility investigation

[0111] The same sample of Streptozyme (sample 2) was accurately weighed 6 times, the titer concentration was detected according to the method of Example 4, and the average titer and RSD value were calculated to investigate the repeatability of the method. The sample weight and titer determination results of the 6 samples are shown in Table 5. As can be seen from Table 5, the repeatability determination result RSD is 4.91%, which is lower than 5%, indicating that the repeatability of the method is good.

[0112] Table 5: Repeatability investigation of Streptozyme titer determination by microplate method

[0113]

[0114] Investigation of the recovery rate of the standard addition

[0115] Six Streptozyme samples (previously detected, titer concentration about 1000 IU / mg) were accurately weighed, and each Streptozyme sample was weighed 4 times. Among them, 3 were added with low, medium and high levels of Streptozyme standard, and the low, medium and high levels of Streptozyme standard were 0.5 times (500 IU / mg), 1.0 times (1000 IU / mg) and 2.0 times (2000 IU / mg) of the sample titer estimate, respectively, as three kinds of samples; the other one was blank control without adding Streptozyme standard. The titer concentration of each group of samples was detected according to the method of Example 4, and the recovery rate of each sample at low, medium and high levels was calculated, and the results are shown in Table 6. The calculation formula is recovery rate of adding standard Where P0 is the titer concentration of Streptozyme in the sample, P1 is the titer concentration of Streptozyme in the added standard, and P2 is the total titer concentration of Streptozyme measured.

[0116] The sample recovery rate is an important indicator to verify the accuracy and reliability of the analysis method. As can be seen from Table 6, the recovery rate determination result is 99.3% to 101.1%, which is located between 95% to 105%, indicating that the recovery rate of the method is good.

[0117] Table 6: Investigation of Streptozyme titer determination by microplate method

[0118]

[0119] Example 9: Comparison and analysis of Streptozyme titer determination results by national standard method and microplate method

[0120] In this example, 20 Streptozyme samples sent from January to July 2025 were selected, and the microplate method and the national standard agar diffusion method were used for analysis and detection, and the results are shown in Table 7.

[0121] Table 7: Titer results of different Streptozyme determined by national standard method and microplate method

[0122]

[0123] From Table 7, it can be seen that the titers of most of the nisin samples are between 1000 IU / mg and 5000 IU / mg. For different batches of nisin produced by the same manufacturer and nisin produced by different manufacturers with similar antibacterial activity, the titer determination results of the microplate method are within the range of 90% to 110% of the estimated value. At the same time, among the measurement results of each sample, the minimum relative standard deviation RSD between the titer results of the microplate method and the national standard method is 0.18%, and the maximum is 8.80%, both of which are less than 10%, indicating that the titer determination results of the microplate method and the national standard method are similar and have good accuracy.

Claims

1. A method for detecting nisin potency based on microplate method, characterized in that, The method includes the following steps: S1: Establishment of a standard curve for nisin potency concentration versus indicator bacteria growth; S2: Nisin sample is prepared as a sample dilution solution; S3: Detect the growth of indicator bacteria when mixed with the sample dilution and culturing, and calculate the potency of nisin in the lactic acid nisin sample by substituting into the standard curve; The growth of the indicator bacteria is characterized by absorbance.

2. The method as described in claim 1, characterized in that, The indicator bacterium is Lactiplantibacillus plantarum CICC 6076.

3. The method as described in claim 1, characterized in that, The detection wavelength for the absorbance is 500–650 nm.

4. The method as described in claim 1, characterized in that, The indicator bacteria are stored in glycerol tubes.

5. The method as described in claim 1, characterized in that, In step S1, the concentration range of the potency in the standard curve is 10–50 IU / mg.

6. The method as described in claim 1, characterized in that, In step S3, the initial concentration of the indicator bacteria in the mixed culture is 1×10⁻⁶. 4 ~4×10 4 CFU / mL.

7. The method as described in claim 1, characterized in that, Step S2 includes aseptically weighing the nisin sample, fully dissolving and bringing the volume to a final volume with sterile hydrochloric acid, and then diluting it with sterile hydrochloric acid by an appropriate factor to obtain a sample dilution.

8. The method as described in claim 7, characterized in that, The concentration of the sterile hydrochloric acid is 0.01–0.03 mol / L.

9. Application of Lactiplantibacillus plantarum (CICC 6076) in the detection of nisin titers.

10. The method as described in claim 9, characterized in that, The determination of the lactic acid nisin potency was based on the microplate method.

Images