Method for detecting mould in feed by ATP (adenosine triphosphate) method

Abstract

The invention relates to a method for detecting mould in feed by an ATP (adenosine triphosphate) method, which comprises the following steps: S1, sequentially carrying out first mixing and extraction treatment on a feed sample and normal saline, and collecting supernate as an extracting solution; s2, the extracting solution and a benzalkonium bromide solution with the concentration being 0.10%-0.20% are sequentially subjected to second mixing and cracking treatment, and a cracking solution is obtained; s3, carrying out third mixing on the lysis solution and an ATP reaction reagent to obtain a solution to be detected; and S4, measuring the RLU value of the to-be-measured liquid by using a handheld ATP (adenosine triphosphate) luminoscope. The method has the advantages of high detection speed and high accuracy, and can timely detect feed mildew at the early stage and realize feed mildew prevention and control.

Description

Technical Field

[0001] This disclosure relates to the field of mold detection technology, and more specifically, to a method for detecting mold in feed using the ATP method. Background Technology

[0002] Formulated feed and feed ingredients often become moldy due to environmental factors such as storage conditions. This is especially true for grain-based feed ingredients, which are prone to mold growth due to increased temperature and humidity during storage. The mycotoxins produced by moldy fungi are extremely harmful to human and animal health; therefore, moldy feed is inedible and causes significant losses to crops and livestock farming. Therefore, farms, feed suppliers, and individuals must detect and control mold growth, especially in the early stages.

[0003] Currently, common methods for assessing feed mold in the feed and livestock industry include: ① Sensory assessment, which relies on experience. Olfactory odor is used as the criterion, while visual inspection focuses on whether the feed or feed ingredients are clumped, have mold spots, or mycelia. This method has low sensitivity, making it difficult for non-long-term practitioners to accurately identify the odor and visual appearance of moldy feed, and easily missing early mold without visible mycelia; ② Total mold count method (GB / T 13092), which requires careful sampling and 2-3 days of incubation on specific plate media before counting. However, this method can introduce significant errors in the final colony count due to sampling and inoculation inaccuracies, and the long waiting time makes it ineffective for mold detection; ③ Carbon dioxide monitoring method, which uses the total amount of carbon dioxide produced by mold metabolism in the detection system to correspond to the total amount of mold. However, gas detection often introduces significant errors due to instability, and this method requires specific equipment, making it inconvenient for on-site operation and flexible sampling.

[0004] Currently, existing technologies cannot simultaneously guarantee detection speed and accuracy, making it difficult to control feed mold. Therefore, there is an urgent need for a method that is fast, enables real-time detection, and has high accuracy, so as to detect feed mold in its early stages and control it. Summary of the Invention

[0005] The purpose of this disclosure is to provide an ATP method for detecting mold in feed. This method is fast, accurate, and can detect mold in the early stages of feed spoilage, thereby enabling the prevention and control of feed mold.

[0006] To achieve the above objectives, this disclosure provides a method for detecting molds in feed using the ATP method, the method comprising: S1. The feed sample is mixed and extracted with physiological saline in sequence, and the supernatant is collected as the extract. S2. The extract and a 0.1-0.2% benzalkonium bromide solution are subjected to a second mixing and pyrolysis treatment to obtain a pyrolysis solution; S3. The lysis buffer and ATP reaction reagent are mixed in a third step to obtain the test solution; S4. Use a handheld ATP luminescence analyzer to determine the RLU value of the test solution.

[0007] Optionally, in step S1, the ratio of the weight of the feed sample to the volume of the physiological saline is 1:7-10.

[0008] Optionally, the first mixing includes: vortexing at a speed of 1000-1500 rpm for 30-60 seconds; The extraction process includes: standing for 3-8 minutes.

[0009] Optionally, in step S2, the volume ratio of the extract to the benzalkonium bromide solution is 1:1-2.

[0010] Optionally, the second mixing includes: repeatedly pipetting and aspirating 5-10 times using a pipette; The pyrolysis process includes incubation for 3-15 minutes.

[0011] Optionally, in step S3, the volume ratio of the lysis buffer to the ATP reaction reagent is 1:1-2; The ATP reaction reagent includes a substrate containing Luciferase and a phosphate buffer.

[0012] Optionally, the third mixing includes: repeatedly pipetting and aspirating 5-10 times using a pipette.

[0013] Optionally, the RLU value of the test solution is measured using a handheld ATP luminescence analyzer within 5 minutes of the test solution preparation.

[0014] Optionally, if the RLU value is greater than 50, it indicates that the feed sample contains mold.

[0015] Optionally, the mold includes Aspergillus chevalieri, Fusarium verticillatum, and Penicillium oxalate.

[0016] The present disclosure provides a method for detecting molds in feed using the ATP method, based on the above technical solution. This method has the following advantages: (1) The method is simple and the detection speed is fast. The detection can be completed in 30 minutes, which significantly improves the timeliness of feed mold detection; (2) It has high sensitivity and can detect mold in feed faster and earlier than the traditional plate coating method, providing timely and effective basis for the prevention and control of feed mold. (3) The method disclosed herein does not require a professional experimental platform, has good safety and high reliability, supports rapid on-site testing, and is more suitable for promotion and application in actual feed production and preservation.

[0017] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Attached Figure Description

[0018] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings: Figure 1 The effect of different extraction solutions on ATP measurement; Figure 2 The effect of different lysis buffers on ATP assay; Figure 3 The effect of different concentrations of BAB on ATP measurement; Figure 4 These are the feed mold growth curves detected by different methods over 28 days; Figure 5 The method disclosed in this paper was used to obtain mold growth curves at four different addition levels; Figure 6 The growth of mold in each group was obtained using the plate coating counting method. Figure 7 The method disclosed herein is used to obtain the growth status of molds in each group. Detailed Implementation

[0019] The specific embodiments of this disclosure will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit this disclosure.

[0020] This disclosure provides a method for detecting molds in feed using the ATP method, the method comprising: S1. The feed sample is mixed and extracted with physiological saline in sequence, and the supernatant is collected as the extract. S2. The extract and a 0.1-0.2% benzalkonium bromide solution are subjected to a second mixing and pyrolysis treatment to obtain a pyrolysis solution; S3. The lysis buffer and ATP reaction reagent are mixed in a third step to obtain the test solution; S4. Use a handheld ATP luminescence analyzer to determine the RLU value of the test solution.

[0021] The method disclosed herein is simple and fast, with detection completed in 30 minutes, significantly improving the timeliness of feed mold detection. It also has high sensitivity, enabling faster and earlier detection of mold in feed compared to the traditional plate coating method, providing timely and effective evidence for feed mold control. In addition, the method disclosed herein does not require a professional experimental platform, has good safety and high reliability, supports rapid on-site detection, and is more suitable for promotion and application in actual feed production and preservation.

[0022] To ensure the complete extraction of mold cells from the feed sample, this disclosure uses physiological saline to extract the mold. In one embodiment, in step S1, the weight ratio of the feed sample to the volume of the physiological saline is 1:7-10, preferably 1:9. The physiological saline used in this disclosure has a concentration of 0.9%, meaning 1 mL of water contains 9 mg of NaCl.

[0023] To further ensure the full extraction of mold cells, in one embodiment, the first mixing includes: vortexing at a speed of 1000-1500 rpm for 30-60 seconds; preferably, the first mixing may include: vortexing at a speed of 1000 rpm for 30 seconds; the extraction process includes: standing for 3-8 minutes, preferably standing for 5 minutes.

[0024] To ensure sufficient lysis of the fungal cells in the extract, this disclosure employs a benzalkonium bromide solution to lyse the cells in the extract, particularly a benzalkonium bromide solution with a concentration of 0.10-0.20%, for example, the concentration can be 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, or 0.20%, and any value between them; preferably 0.10%; wherein, a 0.10-0.20% benzalkonium bromide solution means that 1-2 mg of benzalkonium bromide (BAB) is contained in 1 mL of water.

[0025] To further ensure the complete lysis of fungal cells, in step S2, the volume ratio of the extract to the benzalkonium bromide solution is 1:1-2, preferably 1:1.

[0026] To ensure that the extract and benzalkonium bromide solution are mixed evenly and that the fungal cells are fully lysed, the second mixing includes: repeatedly pipetting and aspirating 5-10 times; for example, 5, 6, 7, 8, 9 or 10 times; the lysis treatment includes: incubation for 3-15 minutes, preferably 5 minutes.

[0027] To ensure the accuracy of RLU values ​​measured by the handheld ATP chemiluminescence analyzer, in step S3, the volume ratio of the lysis buffer to the ATP reaction reagent is 1:1-2; wherein, the ATP reaction reagent can be any commonly used by those skilled in the art; the ATP reaction reagent includes a substrate containing Luciferase and a phosphate buffer. For example, the BacTiter-Glo™ Microbial Cell Viability Assay Kit can be selected.

[0028] To further ensure that the lysis buffer and ATP reaction reagent are mixed evenly, in one embodiment, the third mixing includes: repeatedly pipetting and aspirating 5-10 times, for example, 5, 6, 7, 8, 9 or 10 times.

[0029] To ensure the accuracy of mold detection, in one embodiment, the RLU value of the test solution is measured using a handheld ATP luminescence analyzer within 5 minutes of the test solution preparation.

[0030] In this disclosure, the lysis buffer and ATP reaction reagent are mixed in a third step in a PCR tube, which avoids the use of expensive commercial swabs and also avoids the problem of inaccurate sample volume that may occur when using commercial swabs for sampling.

[0031] In one implementation, if the RLU value is greater than 50, it indicates that the feed sample contains mold.

[0032] In this disclosure, the mold includes Aspergillus chevalieri ( Aspergillus chevalieri ), Fusarium verticillata ( Fusarium verticillioides ) and Penicillium oxalate ( Penicillium oxalicum ).

[0033] The present disclosure will be further illustrated by the following examples, but the present disclosure is not limited thereto.

[0034] All raw materials used in the embodiments can be obtained through commercial purchase.

[0035] Example 1 This embodiment illustrates the ATP method for detecting molds in feed: S1. Mix 1g of feed sample with 9mL of 0.9% physiological saline and vortex at 1000rpm for 30s; then let stand for 5min; collect the supernatant as the extract. S2. Mix 50 μL of extract with 50 μL of 0.1% benzalkonium bromide solution and pipette repeatedly 6 times, then incubate at room temperature for 5 min to obtain the lysis buffer; S3. The ATP reaction reagent was prepared using the BacTiter-Glo™ Microbial Cell Viability Assay Kit (purchased from Promega). The Luciferase substrate was dissolved in phosphate buffer to obtain the ATP reaction reagent. 50 μL of lysis buffer and 50 μL of ATP reaction reagent were mixed in a PCR tube and pipette was used to repeatedly pipette 6 times to obtain the test solution. S4. Use a handheld ATP luminescence analyzer (Hygiena SystemSURE PLUS ATP rapid fluorescence detector) to determine the RLU value of the test solution.

[0036] Example 2 The method used is the same as in Example 1, except that the feed sample is replaced with a wastewater sample, diluted to different concentrations, wherein the high concentration has a total mold content of 10. 9 CFU / mL, with a medium concentration of total mold content of 10. 8 CFU / mL, with a low concentration of total mold content of 10. 7 CFU / mL; the results are shown in Table 1.

[0037] Comparative Example 1 Unlike Example 1, this comparative example uses commercially available swabs (Haijingna UltraSnap® model), and samples were collected and processed according to the instructions; the specific steps are as follows: S1. Mix 1g of feed sample with 9mL of 0.9% physiological saline and vortex at 1000rpm for 30s; then let stand for 5min; collect the supernatant as the extract. S2. Dip the swab into the sample to be tested, and ensure that the swab is immersed in the sample (i.e. the extract in step S1) for 5 seconds. Quickly put the swab back into the sleeve, break and squeeze the swab head to fully release the Luciferase reaction solution inside the swab. Then shake it manually 6 times and put it into the handheld ATP luminescence analyzer for reading. The test results are shown in Table 1.

[0038] Table 1

[0039] As shown in Table 1, the lysis reagents used in Example 2 and Comparative Example 1 are different. Using the method of Comparative Example 1, the RLU values ​​measured for the same sample varied significantly. While commercially available swabs have high sensitivity (i.e., high measured RLU values), the results for water samples with low pollution levels are unstable, with a CV exceeding 50% in three repeated experiments. Furthermore, there was no significant difference between water samples with medium and low pollution levels. Compared to Comparative Example 1, Example 2, using the method disclosed herein, exhibits better repeatability and can effectively distinguish water samples with different pollution levels.

[0040] The ATP reaction reagent and lysis buffer of this invention are mixed in a PCR tube, which avoids the problem of difficult-to-control sample volume in commercial swabs, and the test results are stable; it can effectively distinguish samples with low degree of contamination and has high accuracy.

[0041] Example 3 This example illustrates the optimization of mold extraction conditions in moldy feed samples: Effective extraction of mold from moldy samples is crucial for assessing the degree of mold growth, while the extract should not significantly inhibit luciferin-luciferase. Furthermore, the ATP reaction reagent in the BacTiter-Glo™ Microbial Cell Viability Assay Kit only has a lytic effect on bacteria and certain yeasts, thus requiring additional mold lysis buffer to release ATP. Therefore, to ensure effective extraction of mold and complete lysis of mold cells from moldy samples, the effectiveness of four mold extracts and four mold lysis buffers was compared.

[0042] 1. Screening of mold extracts: Take naturally moldy feed samples and determine the RLU value according to the steps in Example 1, with the difference being... The following were selected from the mold extracts: (1) 0.9% physiological saline; (2) Sterile water; (3) 0.01% Tween 80; (4) PBS buffer; The results are as follows Figure 1 As shown, based on the RLU values, the extraction effects of the four mold extracts differed significantly, and their order was physiological saline > sterile water > 0.01% Tween 80 > PBS buffer.

[0043] 2. Screening of mold lysate: Take naturally moldy feed samples and determine the RLU value according to the steps in Example 1, with the difference being... The following were selected as the mold lysis buffers: (1) 0.10% BAB; (2) 0.1% BAC (benzalkonium chloride); (3) 1% CATB (hexadecyltrimethylammonium bromide); (4) 1% Tritium X-100; The results are as follows Figure 2 As shown, among the four lysis buffers, 0.10% BAB showed the best performance.

[0044] Example 4 To further verify the optimal concentration of the lysis buffer, the following experiment was conducted: Take naturally moldy feed samples and determine the RLU value according to the steps in Example 1, with the difference being... The concentrations of BAB in the mold lysis buffer were selected as follows: 0.05%, 0.10%, 0.20%, 0.30%, 0.40%, and 0.50%; the results are as follows. Figure 3 As shown, the ATP luminescence response (RLU) reading first increased and then decreased with increasing BAB concentration, indicating that while BAB, as a surfactant, can lyse mold, it also inhibits the ATP luminescence reaction. Data showed that the RLU values ​​of samples under 0.10% and 0.20% BAB conditions were both high. To minimize the influence of external factors on the ATP luminescence reaction, a lower concentration of 0.10% BAB was chosen as the lysis buffer.

[0045] Example 5 ATP molecules contain three phosphate groups and two high-energy phosphate bonds, making their chemical structure unstable and prone to degradation. To ensure complete ATP reaction in samples without significant degradation, the effects of BAB lysis time and ATP reaction time on ATP detection were analyzed.

[0046] Take naturally moldy feed samples and determine the RLU value according to the steps in Example 1, with the difference being... (1) The lysis times of sample 1 were 3, 5 and 15 min, respectively; the reaction times of the lysis buffer with ATP were 0 and 5 min, respectively; (2) The lysis times of sample 2 were 3, 5 and 15 min, respectively; the reaction times of the lysate with ATP were 5 and 10 min, respectively; The results are shown in Table 2: Table 2

[0047] As shown in Table 2, (1) the ATP luminescence value decreased significantly as the BAB lysis time increased to 15 min, and a BAB lysis time of 5 min was more suitable. (2) The ATP reaction time refers to the time after the bacterial culture and ATP reaction reagent are mixed and left to stand. When the ATP reaction time is within 5 min, the luminescence value does not decrease significantly, but when it is extended to 10 min, the luminescence value decreases by as much as 60%. Therefore, the ATP detection should be completed within 5 min.

[0048] Example 6 This example illustrates the stability and linearity analysis of moldy sample detection: Using the method of Example 1, five parallel reactions were carried out on two different naturally moldy corn flour samples, and the RLU determination results are shown in Table 3.

[0049] Table 3

[0050] The five reactions were performed at 1-minute intervals, and the RLU values ​​fluctuated slightly. The data fluctuation was measured by the mean RLU value. For sample 1, the fluctuation ranged from 5.6% to 11.9%, and for sample 2, it ranged from 1.0% to 9.4%. The coefficients of variation for both groups were less than 10%, demonstrating that the reaction exhibits good stability for detecting molds.

[0051] Table 4

[0052] After the measurement was completed, a linear regression analysis was performed on the logarithm of the bacterial concentration and RLU, that is, a linear regression analysis was performed on lg (bacterial concentration) and lg (RLU) in Table 4 to obtain the coefficient of determination R. 2 =0.9838, which proves that the bacterial concentration and RLU value have a good linear correlation. Therefore, the ATP determination of moldy samples can reflect the number of molds to a certain extent and can be used as a reference indicator for evaluating antifungal agents.

[0053] Example 7 Long-term monitoring of mold growth in feed: Corn flour and soybean meal were ground to 40 mesh and mixed in a 7:3 ratio to form a 100g feed sample (moisture content 11-12%). The sample was placed in a container, sealed with a breathable membrane, and placed in a constant temperature chamber at 28℃ and approximately 75% humidity. The first day of storage was recorded as day 0. Samples were taken and tested every 7 days using both the national standard plate coating method and the method described in Example 1 of this disclosure. Data from 28 days were summarized as follows: Figure 4The results showed that, combined with sensory evaluation, no clumping or mold was observed in the first four weeks. Clumping appeared in the feed on day 28, but the plate coating results on day 28 did not show an increase in total mold count. However, detection using the method of Example 1 of this disclosure showed that the RLU value had increased by 50% from day 7 to day 14 and continued to rise until day 28. These phenomena indicate that the method of this disclosure can detect feed mold trends earlier than the traditional plate coating method, which is more conducive to early prevention and control.

[0054] Example 8 This example is used to evaluate the dosage of a certain feed mold inhibitor product: Four replicate samples were prepared using the same feed formulation as in Example 7. Each sample was supplemented with 0.25 kg / t, 0.5 kg / t, 1.0 kg / t, and 2.0 kg / t of a certain feed antifungal agent (containing 50% propionic acid, a product independently developed by Adisseo), respectively. The culture conditions and sampling methods were the same as in Example 7. The mold growth curves obtained using the method of Example 1 of this disclosure within 28 days are shown below. Figure 5 As shown. Theoretically, the efficacy of feed mold inhibitors is directly proportional to the amount added. Figure 5 The results on day 28 also showed that the higher the dosage, the better the anti-mold effect. It was also observed that from day 14 to day 21, the anti-mold effect decreased in the 0.25 kg / t dosage group, while the anti-mold effect in the 0.5 kg / t dosage group decreased slightly but remained better than the former. Furthermore, the 1.0 kg / t and 2.0 kg / t dosage groups still exhibited strong anti-mold effects on day 28. Based on these conclusions, the method disclosed herein can guide the design of feed anti-mold agent dosages during feed storage.

[0055] Example 9 This embodiment evaluates the anti-mold effects of 10 different anti-mold agents. The substrate is the same as the feed sample in Example 7. However, since the natural mold growth process is slow, an accelerated method is used in this embodiment. The culture conditions are the same as in Example 7, except that the feed moisture content is increased to 18% to accelerate mold growth. Eleven replicate samples were prepared under the above conditions. Ten of these replicate samples were supplemented with 10 different feed anti-mold agents at a dosage of 0.5 kg / t. Anti-mold agent 1 was a solid anti-mold agent containing 51% propionic acid; anti-mold agent 2 was a solid anti-mold agent containing 42% propionic acid and 12% ammonium propionate; anti-mold agent 3 was a liquid anti-mold agent containing 75% propionic acid; anti-mold agent 4 was a liquid anti-mold agent containing 82% propionic acid; anti-mold agent 5 was a solid anti-mold agent containing 8% propionic acid and 10% ammonium propionate; anti-mold agent 6 was a solid anti-mold agent containing 47% propionic acid; and anti-mold agent 7 was a solid anti-mold agent containing 40% propionic acid. Solid antifungal agents containing 10% propionic acid and 10% ammonium propionate were used. Antifungal agent 8 contained 20% propionic acid and 15% sodium diacetate. Antifungal agent 9 contained 50% propionic acid and 1% acetic acid. Antifungal agent 10 contained 60% propionic acid and 10% sodium propionate. The remaining group served as a blank control. Starting from day 0, the blank control group was observed daily for signs of mold growth. By day 7, mold growth was observed in all groups. Samples were taken and tested on that day. The results were statistically analyzed using both the flat-coated plate counting method and the method of Example 1 of this disclosure. Figure 6 and Figure 7 As shown.

[0056] Depend on Figure 6 and 7 It can be seen that the ATP reading results and the plate coating method results are highly consistent in trend, proving the reliability of the method disclosed herein. However, it is also easy to observe that, in the statistical results of the plate coating method, the total number of molds in all experimental groups was lower than that in the control group, meaning that all mold inhibitors had a mold-preventing effect; however, in the statistical results of the method disclosed herein, only the RLU of mold inhibitors 1, 2, and 10 was lower than that of the control group, meaning that only these three mold inhibitors produced a mold-preventing effect. Therefore, it can be concluded that the method disclosed herein is more discriminative in evaluating the efficacy of mold inhibitors and can more rigorously screen out feed mold inhibitors with better mold-preventing effects.

[0057] The preferred embodiments of this disclosure have been described in detail above with reference to the accompanying drawings. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure. It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0058] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. A method for detecting molds in feed using the ATP method, characterized in that, The method includes: S1. The feed sample is mixed and extracted with physiological saline in sequence, and the supernatant is collected as the extract. S2. The extract and a benzalkonium bromide solution with a concentration of 0.10-0.20% are subjected to a second mixing and pyrolysis treatment in sequence to obtain a pyrolysis solution; S3. The lysis buffer and ATP reaction reagent are mixed in a third step to obtain the test solution; S4. Use a handheld ATP luminescence analyzer to determine the RLU value of the test solution.

2. The method according to claim 1, wherein, In step S1, the ratio of the weight of the feed sample to the volume of the physiological saline is 1:7-10.

3. The method according to claim 2, wherein, The first mixing includes: vortexing at a speed of 1000-1500 rpm for 30-60 seconds; The extraction process includes: standing for 3-8 minutes.

4. The method according to claim 1, wherein, In step S2, the volume ratio of the extract to the benzalkonium bromide solution is 1:1-2.

5. The method according to claim 4, wherein, The second mixing process involves repeatedly pipetting and aspirating 5-10 times. The pyrolysis process includes incubation for 3-15 minutes.

6. The method according to claim 1, wherein, In step S3, the volume ratio of the lysis buffer to the ATP reaction reagent is 1:1-2; The ATP reaction reagent includes a substrate containing Luciferase and a phosphate buffer.

7. The method according to claim 6, wherein, The third mixing process includes repeatedly aspirating and purging with a pipette 5-10 times.

8. The method according to claim 1, wherein, The RLU value of the test solution was measured using a handheld ATP luminescence analyzer within 5 minutes of the test solution preparation.

9. The method according to claim 1, wherein, If the RLU value is greater than 50, it means that the feed sample contains mold.

10. The method according to claim 9, wherein, The molds include Aspergillus chevalieri, Fusarium verticillatum, and Penicillium oxalate.

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