Rapid detection method for yeast pollution of fruit grain milk

Abstract

The invention discloses a method for rapidly detecting yeast pollution of fruit granule milk. The method comprises the following steps: S1, treating a fruit granule milk sample by adopting a normal saline pretreatment solution containing protease K, EDTA (Ethylene Diamine Tetraacetic Acid) and polyethylene glycol 8000; s2, filtering and centrifugally removing impurities from the sample treated in the step S1; and S3, taking the total DNA of the sample, and detecting the yeast in the fruit grain milk by adopting a fluorescent quantitative PCR (Polymerase Chain Reaction) method. According to the method, the sterile normal saline containing protease K, EDTA and polyethylene glycol 8000 is adopted to pretreat the fruit grain milk sample, and then a double impurity removal method is adopted to remove impurity components in the fruit grain milk, so that physical interferents are effectively removed, target bacteria are concentrated, the DNA extraction purity is improved, and meanwhile, the detection rate of a low-pollution-degree sample is also improved. Moreover, the detection time is greatly shortened, the whole process from the sample to the result can be completed within 4 hours, which is far better than the traditional process of 3-5 days, so that the real'rapid detection 'is realized, and the detection sensitivity and accuracy are remarkably improved.

Description

Technical Field

[0001] This invention relates to the detection of microorganisms in food, specifically to a rapid, efficient, and highly sensitive method for detecting yeast in fermented milk containing fruit particles, exhibiting high viscosity, and being susceptible to yeast contamination. Background Technology

[0002] Fruit-flavored yogurt and other milk and fruit-based beverages are popular due to their rich nutrition and excellent taste. However, the sugar, fruit pieces, and suitable pH level in fruit-flavored milk products provide favorable conditions for yeast growth, leading to problems such as spoilage, bloating, and lid swelling. Yeast is widely distributed in nature and has both aerobic and anaerobic metabolism, capable of breaking down carbohydrates to produce alcohol and carbon dioxide. Spoilage yeast growth can be detected under high sugar, acidic, and high temperature environments. Studies have shown that the bloating and swelling problems commonly found in fermented milk are related to yeast contamination, with *Candida intermedius* being the main contaminating yeast in bloated products. Candida intermedia ), Portuguese yeast ( Clavispora lusitaniae ), Candida utilis ( Candidautilis ), Tropical Candida ( Candida tropicalis Yeast contamination generally occurs through two main pathways: introduction of yeast when adding fruit, honey, or other non-dairy ingredients to yogurt; and poor hygiene in the yogurt production and packaging environment. Therefore, rapid detection of yeast in fermented milk is crucial for quality control. Consequently, a rapid and accurate yeast contamination detection technology is urgently needed to ensure the safety, quality control, and shelf-life extension of fruit-flavored yogurt.

[0003] Currently, traditional detection methods mainly rely on plate culture (such as Bengal red agar). Although this method is considered the "gold standard," it has significant drawbacks: (1) Long detection cycle: Conventional culture requires 3 to 5 days, which cannot meet the needs of modern food industry for rapid response.

[0004] (2) Difficulty in pretreatment: Fruit particles and colloids in fruit milk are prone to encapsulating microorganisms, resulting in uneven sampling; high viscosity also affects dilution and coating operations, causing large deviations in results.

[0005] (3) Limited sensitivity: For low-level initial contamination, it may take longer to detect.

[0006] (4) Although molecular biology methods (such as qPCR) are fast, they face challenges when applied directly to fruit milk: mainly because fat, protein, polysaccharide and stabilizer in the sample are all strong PCR inhibitors that can seriously interfere with DNA amplification and lead to false negative results.

[0007] (5) Low DNA extraction purity: Conventional DNA extraction methods are difficult to efficiently extract pure yeast DNA from fruit milk with complex composition.

[0008] Therefore, there is an urgent need in this field for a new method that can overcome the interference of complex matrices in fruit milk and achieve rapid and accurate detection of yeast contamination. Summary of the Invention

[0009] To address the issues of long detection cycles and low sensitivity in fruit-flavored milk yeast contamination detection, this invention provides a rapid detection method for fruit-flavored milk yeast contamination. This method features a short detection cycle, high sensitivity, strong anti-interference ability, and simple operation.

[0010] To achieve the above objectives, the present invention provides a rapid detection method for yeast contamination in fruit-flavored milk, comprising the following steps: S1. Fruit milk samples were pretreated with physiological saline containing proteinase K, EDTA and polyethylene glycol 8000. S2. Filter and centrifuge the sample after step S1 to remove impurities. S3. Collect total DNA from the sample and use real-time PCR to detect yeast in the fruit milk.

[0011] This invention uses sterile physiological saline containing proteinase K, EDTA and polyethylene glycol 8000 to pretreat fruit-flavored yogurt samples, and then uses a dual impurity removal method to remove impurities from the fruit-flavored yogurt. This effectively removes physical interferences, achieves the purpose of enriching bacterial cells, improves the purity of DNA extraction, and also increases the detection rate of low-contamination samples.

[0012] Specifically, in step S1, the pretreatment solution is a sterile mixture containing 70-150 μg / mL proteinase K, 1-2% EDTA and 3-5% polyethylene glycol 8000 physiological saline.

[0013] Specifically, in step S1, the fruit milk sample is treated with a pretreatment solution at 37~42℃ for 15~30 min.

[0014] Preferably, in step S2, a 200~700 μm filter screen is used for filtration.

[0015] Specifically, in step S2, centrifugation includes: first centrifuging at 500~1000 ×g for 3~5 min to remove impurities, and then centrifuging at 6000~8000 ×g for 5~8 min.

[0016] Preferably, in step S3, during DNA extraction, 0.5%~2% (w / v) of polyvinylpyrrolidone (PVP) is added to the lysis system. Adding PVP to the DNA extraction lysis buffer efficiently removes PCR inhibitors (such as phenolic substances and pigments) from complex matrices, ensuring the efficiency and reliability of the qPCR reaction and greatly reducing the risk of false negatives.

[0017] Specifically, in step S3, the specific primer sequences used for quantitative real-time PCR are: Forward primer Primer 1: 3'-CAGACACGGTTTTACCGGGC -5' and Reverse primer: 3'-GCTTGCAACCATTACGCCAG -5'.

[0018] Specifically, yeast was absolutely quantified by plotting a standard curve, with a Cp value greater than 35 defined as negative and otherwise as positive.

[0019] Through the above technical solution, the present invention achieves the following beneficial effects: 1. This invention pretreats fruit milk samples with sterile physiological saline containing proteinase K, EDTA, and polyethylene glycol 8000. Then, a dual impurity removal method is used to remove impurities from the fruit milk, effectively removing physical interferences and concentrating the target bacteria, thus improving DNA extraction purity and the detection rate of low-contamination samples. It also significantly shortens the detection time; the entire process from sample to result can be completed within 4 hours, far superior to the traditional 3-5 days, achieving true "rapid detection" and significantly improving detection sensitivity and accuracy.

[0020] 2. Adding PVP to the DNA extraction lysis buffer can efficiently remove PCR inhibitors (such as phenolic substances, pigments, and other PCR inhibitors) in complex matrices, ensuring the efficiency and reliability of qPCR reactions and greatly reducing the risk of false negatives.

[0021] 3. This method is specifically designed for complex food systems containing high viscosity and particles, such as fruit milk and yogurt, solving the core challenges in sample pretreatment for these types of products. This method is easily adaptable to automated workstations and is suitable for enterprises to conduct rapid screening of large batches of samples. Attached Figure Description

[0022] Figure 1 The colonies (A) and cell morphology (B) of the strain isolated in Example 1 are shown. Figure 2 This is an analysis of the gas production capacity of the isolated strain in Example 1; Figure 3 It is a phylogenetic tree based on the 26S rDNA sequence; Figure 4 These are the qPCR amplification curve and melting curve from Example 1; Figure 5 These are the sensitivity and standard curve from Example 1; Figure 6 This is a comparison of the stability test results of the method of this invention and conventional methods; Figure 7 This is a comparison of the detection sensitivity of the present invention with conventional extraction techniques. Detailed Implementation

[0023] The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0024] Example 1: Construction of a yeast standard curve 1. Isolation of contaminating yeast from drum-top fruit milk Single colonies were selected for further purification based on colony morphology and growth rate. Crystal violet staining was used for cell staining, and cell morphology was observed under a microscope. Based on a combination of cell morphology, colony morphology, and size, nine yeast strains were isolated from three samples of yogurt with bulging caps: blueberry, mango, and yellow peach. These strains were numbered YN-1, YN-2, YN-5, YN-6, YN-7, YN-8, YN-9, YN-11, and YN-12. Their colony and cell morphologies are shown in the figure. Figure 1 As shown in the figure, the isolated colonies mostly exhibit typical yeast colony morphology. YN-1, YN-2, YN-7, YN-8, and YN-9 colonies are milky white, with a rounded surface and a raised center. YN-5, YN-6, YN-11, and YN-12 colonies are yellowish, with a waxy surface and regular, round shape. YN-5 colonies are round, with a slightly dry, wrinkled surface and serrated edges. In terms of cell morphology, most cells are oval or rod-shaped and reproduce through budding.

[0025] 2. Analysis of the gas-producing capacity of isolated yeast in yogurt with fruit pieces in a drum lid When yeast is introduced into fruit-flavored yogurt during production, the yeast grows and metabolizes in the nutrient-rich yogurt. Some yeast consumes sugars and produces CO2 as a metabolic byproduct. When CO2 accumulates to a certain level, it causes the yogurt product to bulge or swell, leading to spoilage. Therefore, the gas-producing capacity of yeast in bulging fruit-flavored yogurt was measured. The results... Figure 2As shown in the figure, different yeast strains have varying abilities to produce CO2. YN-1, YN-8, YN-9, and YN-11 exhibited the strongest gas production capacity, completely filling their Durham tubes under the same culture conditions. YN-2 and YN-5 followed, with gas occupying more than half the volume of their Durham tubes. YN-12 produced only a small amount of gas in its Durham tube, while YN-6 and YN-7 showed the weakest gas production capacity, with no gas present in their Durham tubes during the observation period. Furthermore, the liquid test tubes also revealed that during yeast growth, YN-5 and YN-6 formed a membrane-like substance on the liquid surface, while no membrane-like substance was observed on the surfaces of the other yeast strains.

[0026] 3. Identification of yeast isolated from yogurt with fruit pieces in a drum lid Yeast rDNA contains highly conserved sequences interspersed with regions of significant variability. These regions are highly conserved within a species but show significant interspecies differences, making them useful for identifying yeast species. This invention selects the D1 / D2 region sequence of the 26S rRNA gene for species identification. Using the total genomic DNA of isolated strains YN-1, YN-2, YN-5, YN-6, YN-7, YN-8, YN-9, YN-11, and YN-12 as templates, PCR amplification was performed using universal primers for the D1 / D2 region of the 26S rRNA gene, yielding approximately 500 bp of specific amplification products. The amplification products were sequenced by Shanghai Bioengineering Co., Ltd. The obtained sequences were compared for homology using the BLAST homology search tool in the NCBI database. Sequences with similar homology were downloaded, and MEGA software was used for comparative analysis of the determined sequences and homologous sequences. A molecular phylogenetic tree was constructed using the Neighbor-Joining method. The results are shown in [Figure 1]. Figure 3 As can be seen from the figure, YN-5 and abnormal Wickham yeast ( Wickerhamomyces anomalus They naturally clustered into one, with a development value of 99. YN-7 and YN-12 are similar to common Candida albicans ( Pichia inconspicua ) naturally gather into one branch, YN-2, YN-11, YN-1, YN-8 and YN-9 and Clavispora lusitaniae However, looking at the branches, YN-2 and YN-11 cluster more closely, while YN-6 and... Pichia cactophila The isolated strain naturally clustered into a single strain, exhibiting over 99% sequence similarity in 26S rDNA D1 / D2 to the known type strain. Currently, the internationally accepted method for yeast identification primarily relies on the sequence composition of the D1 / D2 region of the 26S rRNA gene. Differences in the D1 / D2 sequence between different strains within the same species should be within 1%. If the base difference in this sequence exceeds 1%, the strains can generally be identified as different species. Therefore, YN-5 was identified as *Saccharomyces cerevisiae* (abnormal Wickham yeast). Wickerhamomyces anomalus YN-7 and YN-12 were identified as Candida albicans ( Pichia inconspicua YN-2, YN-11, YN-1, YN-8, and YN-9 were identified as *Cercospora lucida* (Portunus lucida). Clavispora lusitaniae YN-6 was identified as Pichia pastoris (a type of yeast). Pichia cactophila ).

[0027] 4. Establishment of qPCR method and characteristics of detection primers Nine yeast strains derived from bagged fruit-flavored yogurt, along with four yeast strains previously isolated from yogurt, were used. These included commonly used starter cultures in yogurt fermentation such as *Streptococcus thermophilus* and *Lactobacillus bulgaricus*, as well as *Lactobacillus plantarum* and *Lactobacillus fermentum*, both frequently used in yogurt processing. Additionally, two commonly detected bacteria in yogurt testing, *Escherichia coli* and *Staphylococcus aureus*, were used as templates. Detection primers were designed and used as amplification primers. qPCR detection was performed using 2×Talent qPCR Premix (SYBR Green). The amplification results are shown below. Figure 4 As shown in the figure, all 13 yeast strains tested exhibited fluorescent signals and clear amplification curves. These curves demonstrate that the method exhibits a good exponential growth phase and plateau phase, with a Cp value < 35, indicating a positive result. No false positives were observed with other non-yeast strains. These results demonstrate that the primers designed in this study have good specificity and can be used to specifically detect yeast in low-temperature yogurt.

[0028] 5. Standard curve and sensitivity detection The standard curve equation for yeast is y = -2.8313x + 38.226 (R²). 2 =0.9904), with a slope of -2.8313. As the yeast suspension concentration decreased, the Cp value also gradually decreased, and a good linear relationship was observed between the two. For example... Figure 5 As shown, yeast at 10 2 ~10 8 A clear amplification curve was observed across the entire CFU / mL concentration range, with the lowest detectable concentration being 10. 2 The CFU / mL value indicates that this method has high sensitivity.

[0029] 6. Method stability and repeatability The established qPCR method was used to detect yeast DNA of different batches and concentrations within and between groups, as shown in Table 2. Within the detected bacterial concentration range, the Cp value was between 15 and 35. On the other hand, the CV of the intra-batch and inter-batch repeatability of Cp value of this method was <2%, indicating that the method has good stability and reliability.

[0030] Table 1. Stability of qPCR method for detecting yeast

[0031] Example 2: Detection of yeast in yellow peach pulp milk Take 25 mL of yeast-free peach pulp milk, and add 10 mL of artificially contaminated milk (1.01 ± 0.02). 5 The yeast was incubated at CFU / mL with fruit milk and 225 mL of sterile pretreatment solution (100 μg / mL proteinase K, 1.0% EDTA and 3% polyethylene glycol 8000) for 20 minutes at 40°C to degrade proteins, break down colloidal structures and release the encapsulated bacterial cells. The pre-filter solution was then coarsely filtered through a 300 μm sterile filter to remove large particulate impurities such as fruit pieces. The coarse filtrate was then subjected to differential centrifugation to remove impurities and enrich bacterial cells. This differential centrifugation involved first centrifuging at 1000 × g for 5 min to remove impurities, followed by centrifugation at 6000 g for 5 min to enrich bacterial cells. The enriched bacterial cell pellet was resuspended, and DNA was extracted using a modified magnetic bead extraction kit. In the lysis step, 1.5% (w / v) polyvinylpyrrolidone (PVP) was added to the lysis buffer to specifically adsorb PCR inhibitors such as phenols and pigments commonly found in fruit pieces and dairy products, thereby improving DNA extraction efficiency and purity. Using the specific primer sequences of this invention—Forward primer Primer 1: 3'-CAGACACGGTTTTACCGGGC-5' and Reverse primer: 3'-GCTTGCAACCATTACGCCAG-5'—the extracted DNA was used as a template, and the DNA was extracted using Hieff UNICON® Universal Blue qPCR SYBR Green Master. Mix is ​​the amplification mixture. Quantitative PCR detection was performed on a real-time quantitative PCR-ZX-21. The SYBR Green I dye-real-time quantitative PCR reaction system was 25 µL, including 12.5 µL of 2×Talent qPCR Premix (SYBR Green), 1 µL each of forward and reverse primers (10 µmol / L), 1 µL of template, and molecular biology grade water (DNase and RNase free H2O) to make up the system.

[0032] The reaction conditions were: pre-denaturation at 95℃ for 2 min; denaturation at 95℃ for 30 s; annealing at 58℃ for 25 s with fluorescence signal acquisition; and extension at 68℃ for 25 s, for a total of 40 cycles.

[0033] The melting curve program is as follows: heat at 95℃ for 15 seconds, cool at 60℃ for 1 minute, gradually heat to 95℃ for 20 minutes, and hold for 15 seconds.

[0034] In the control group, peach yogurt samples were diluted directly with sterile saline, and artificial contamination was also measured at (1.01±0.02)×10⁻⁶. 5 25 g of a sample of *Saccharomyces cerevisiae* (CFU / mL) was thoroughly mixed with 225 mL of sterile pretreatment solution using fruit milk. DNA extraction was performed according to the kit instructions. Using the extracted DNA as a template, the specific primer sequences of this invention were used: Forward primer Primer 1: 3'- CAGACACGGTTTTACCGGGC-5' and Reverse primer: 3'- GCTTGCAACCATTACGCCAG-5'. Quantitative PCR was performed on a real-time quantitative PCR-ZX-21 using Hieff UNICON® Universal Blue qPCR SYBRGreen Master Mix as the amplification mixture. The reaction system for the SYBRGreen I dye-real-time quantitative PCR method was 25 µL, including 12.5 µL of 2×Talent qPCR Premix (SYBRGreen), 1 µL each of forward and reverse primers (10 µmol / L), 1 µL of template, and molecular biology grade water (DNase and RNase free H2O) to make up the volume.

[0035] The reaction conditions were: pre-denaturation at 95℃ for 2 min; denaturation at 95℃ for 30 s; annealing at 58℃ for 25 s with fluorescence signal acquisition; and extension at 68℃ for 25 s, for a total of 40 cycles.

[0036] The melting curve program is as follows: heat at 95℃ for 15 seconds, cool at 60℃ for 1 minute, gradually heat to 95℃ for 20 minutes, and hold for 15 seconds.

[0037] The amplification results of the two methods are shown below. Figure 6As can be seen from the figure, the amplification curve of this technique has a significantly higher amplification efficiency than that of directly extracting DNA from samples using magnetic beads. The Cp value of this invention is 12.34-12.70, and the coefficient of variation (CV) is 1.42%, less than 2%, indicating that this invention has good stability and reliability. The control group, using conventional extraction methods, has a Cp value of 25.80-29.00 and a CV of 5.06%, greater than 2%, indicating that the conventional DNA extraction method results in lower extraction efficiency and quality, leading to a wider qPCR amplification Cp value range (25.80-29.00) and a CV of 5.06%, greater than 2%. Therefore, compared to the conventional method, this invention has a higher detection efficiency.

[0038] Example 3: Comparative analysis of the detection of yeast in blueberry pulp milk using the present invention and conventional methods of extracting total microbial DNA from yogurt combined with qPCR. Take 25 mL of yeast-free blueberry pulp milk, and the artificial contamination level is (1.01±0.02)×10. 5 CFU / mL, (1.01±0.02)×10 4 CFU / mL and (1.01±0.02)×10 3Using the operating method of this technology, CFU / mL of brewing yeast is first thoroughly mixed with 225 mL of sterile pretreatment solution (150 μg / mL proteinase K, 1.5% EDTA and 4% polyethylene glycol 8000) containing fruit pulp milk with different yeast concentrations, and then incubated at 40℃ for 20 minutes to degrade proteins, break down colloidal structures, and release the encapsulated bacterial cells. Then, the pre-filter was coarsely filtered through a 400 μm sterile screen to remove large particulate impurities such as fruit pieces. The coarse filtrate was then subjected to differential centrifugation to remove impurities and enrich bacterial cells. This differential centrifugation included first centrifuging at 1200 × g for 5 min to remove impurities, and then centrifuging at 8000 g for 5 min to enrich bacterial cells. The enriched bacterial cell pellet was resuspended, and DNA was extracted using a modified magnetic bead extraction kit. In the lysis step, 2.0% (w / v) polyvinylpyrrolidone (PVP) was added to the lysis buffer to specifically adsorb PCR inhibitors such as phenols and pigments commonly found in fruit pieces and dairy products, thereby improving DNA extraction efficiency and purity. The specific primer sequences used were: Forward primer Primer 1: 3'-CAGACACGGTTTTACCGGGC-5' and Reverse primer: 3'-GCTTGCAACCATTACGCCAG-5'. Using the extracted DNA as a template, the Hieff UNICON® Universal BlueqPCR SYBR Green Master was employed. Mix is ​​the amplification mixture, which is used for quantitative PCR detection in a real-time quantitative PCR-ZX-21.

[0039] The reaction system for SYBR Green I dye-real-time quantitative PCR was 25 µL, including 12.5 µL of 2×Talent qPCRPremix (SYBR Green), 1 µL each of forward and reverse primers (10 µmol / L), 1 µL of template, and molecular biology grade water (DNase and RNase free H2O) to make up the system.

[0040] The reaction conditions were: pre-denaturation at 95℃ for 2 min; denaturation at 95℃ for 30 s; annealing at 58℃ for 25 s with fluorescence signal acquisition; and extension at 68℃ for 25 s, for a total of 40 cycles.

[0041] The melting curve program is as follows: heat at 95℃ for 15 seconds, cool at 60℃ for 1 minute, gradually heat to 95℃ for 20 minutes, and hold for 15 seconds.

[0042] In this embodiment, total DNA was extracted from the blueberry yogurt samples containing different concentrations of artificially contaminated yeast using conventional methods. First, 25 mL of fruit-flavored milk containing different yeast concentrations was diluted with 225 mL of sterile saline to extract DNA. DNA extraction was performed according to the kit instructions. Using the extracted DNA as a template, specific primers Forward Primer 1: 3'-CAGACACGGTTTTACCGGGC-5' and Reverse primer: 3'-GCTTGCAACCATTACGCCAG-5' were used. Hieff UNICON® Universal Blue qPCR SYBR GreenMaster Mix was used as the amplification mixture. Quantitative PCR detection was performed on a real-time quantitative PCR-ZX-21 instrument. The SYBR Green I dye-real-time quantitative PCR reaction system was 25 µL, including 12.5 µL of 2×Talent qPCR Premix (SYBR Green), 1 µL each of forward and reverse primers (10 µmol / L), 1 µL of template, and molecular biology grade water (DNase and RNase-free H2O) to make up the volume.

[0043] The reaction conditions were: pre-denaturation at 95℃ for 2 min; denaturation at 95℃ for 30 s; annealing at 58℃ for 25 s with fluorescence signal acquisition; and extension at 68℃ for 25 s, for a total of 40 cycles.

[0044] The melting curve program is as follows: heat at 95℃ for 15 seconds, cool at 60℃ for 1 minute, gradually heat to 95℃ for 20 minutes, and hold for 15 seconds.

[0045] The amplification results of the two methods are shown below. Figure 7 As can be seen from the figure, the 10 using the method of the present invention 5 CFU / mL, 10 4 CFU / mL 10 3 The CFU / mL amplification curves showed significantly higher amplification efficiency compared to directly extracting DNA from samples using magnetic beads. The Cp values ​​of the method in this invention were 17.27, 17.87, and 27.3, respectively, while the control's 10 5 CFU / mL and 10 4 CFU / mL Cp values ​​22.02, 31.4, 10 3 A Cp value greater than 35 for CFU / mL indicates a negative test result. In blueberry yogurt with the same yeast concentration, 10 samples prepared using the method of this invention... 3The Cp value of CFU / mL is 27.3, which is still detectable, but the detection result using conventional DNA extraction methods is negative. Therefore, the detection sensitivity of the present invention is higher, more than 10 times higher than that of conventional DNA extraction methods.

[0046] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0047] 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. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0048] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A rapid detection method of yeast contamination in fruit juice, characterized in that, The method comprises the following steps: S1, treating the fruit granule milk sample with a pretreatment solution containing protease K, EDTA and polyethylene glycol 8000; S2, filtering and centrifuging the sample treated in step S1 to remove impurities; S3, taking total DNA of the sample, and detecting yeast in the fruit granule milk by using a fluorescent quantitative PCR method.

2. The rapid detection method according to claim 1, characterized in that, In step S1, the pretreatment solution is a sterile mixture of normal saline containing 70-150 μg / mL of protease K, 1-2% of EDTA and 3-5% of polyethylene glycol 8000.

3. The rapid detection method according to claim 1, characterized in that, In step S1, the fruit granule milk sample is treated with the pretreatment solution at 37-42°C for 15-30 min.

4. The rapid detection method according to claim 1, characterized in that, In step S2, the filtering uses a 200-700 μm filter screen.

5. The rapid detection method according to claim 1, characterized in that, In step S2, the centrifugation comprises: first centrifuging at 500-1000 ×g for 3-5 min to remove impurities, and then centrifuging at 6000-8000 ×g for 5-8 min.

6. The rapid detection method of claim 1, wherein In step S3, 0.5%-2% (w / v) of polyvinylpyrrolidone is additionally added in the lysis system during the DNA extraction process.

7. The rapid detection method according to claim 1, characterized in that, In step S3, the specific primers used in the fluorescent quantitative PCR have the sequences of Forward primer Primer1: 3'-CAGACACGGTTTTACCGGGC-5' and Reverse primer: 3'-GCTTGCAACCATTACGCCAG-5'.

8. The rapid detection method according to claim 7, characterized in that, The yeast is absolutely quantified by drawing a standard curve, and the Cp value greater than 35 is set as negative, otherwise positive. The fruit granule milk sample is treated with a pretreatment solution containing protease K, EDTA and polyethylene glycol 8000;

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