CN114574568A - Dynamic change detection method for spoilage microorganisms in fresh-keeping process of fugu obscurus - Google Patents
Dynamic change detection method for spoilage microorganisms in fresh-keeping process of fugu obscurus Download PDFInfo
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Abstract
The invention discloses a dynamic change detection method of spoilage microorganisms in a fresh-keeping process of takifugu obscurus, in particular to the technical field of detection of the spoilage microorganisms of the takifugu obscurus, which comprises the following specific steps: step one, treating raw materials of takifugu obscurus, step two, preparing a PCA (principal component analysis) and CN (CN) culture medium, step three, determining the total number of colonies and the total number of pseudomonas, and step four, performing 16s rDNA sequencing analysis: placing the small fish slices in a freezing storage tube of 2ml in a refrigerator at the temperature of-80 ℃, grouping the fish slices according to the fish flesh of the day, and grouping the fish slices according to parallel comparison for storage; and (6) uniformly delivering samples. The invention shows the development process of the saprophytic microflora by selecting pseudomonas as an important indicator bacterium in the putrefaction process of the takifugu obscurus, researching the dynamic change conditions of the pseudomonas and other genera during refrigeration.
Description
Technical Field
The invention relates to the technical field of detection of spoilage microorganisms of takifugu obscurus, in particular to a dynamic change detection method of spoilage microorganisms in a fresh-keeping process of takifugu obscurus.
Background
With the continuous and deep research on the toxin carried by the puffer fish and the development of related culture technologies, the nutrition and safety of the puffer fish are gradually accepted by the society. The puffer fish is fresh and tender in meat quality, is popular with people and has quite wide market prospect. Particularly, the local market demand is continuously increased along with the general improvement of the living quality of people in recent years. However, the shelf life of the conventional refrigerated preserved globefish products after slaughtering in the current market is only 4-6 days, which causes a plurality of potential problems, including the tightening of food safety and sales cycle, the need for the construction and support of cold chain transportation for long-distance freight, the reduction of the preservation effect of globefish during transportation, and the like. Many enterprises strictly limit the sale and inventory period of the fresh globefish to be within 18 hours, and the requirements and difficulties of market promotion and logistics transportation also bother the development of the industry. Therefore, the problem that how to prolong the shelf life of the puffer fish, maintain the quality of the fish meat and reduce the preservation cost is still solved at present is explored.
At present, although certain knowledge is provided on the technical effect of freezing and fresh-keeping, the meat quality change in the middle and later stages before and after freezing and the growth curve of microorganisms and the main decay-causing microbial flora before and after decay, people still cannot comprehensively observe the specific transition process of the decay-causing microbial flora in the whole process and the quantitative relation of the decay-causing microbial flora in the whole flora.
Disclosure of Invention
Therefore, the invention provides a dynamic change detection method of spoilage microorganisms in the fresh-keeping process of fugu obscurus, which researches the dynamic change conditions of the spoilage microorganisms of the fugu obscurus and other genera by selecting pseudomonas as important indicator bacteria in the spoilage process of the fugu obscurus and displays the development process of the spoilage microorganism community so as to solve the problems in the background technology.
In order to achieve the above object, the embodiments of the present invention provide the following technical solutions: the method for detecting the dynamic change of spoilage microorganisms in the fresh-keeping process of the takifugu obscurus comprises the following specific steps:
step one, treating a raw material of takifugu obscurus: s1, selecting puffer fish: randomly taking 2-year-old fresh takifugu obscurus from the pond, wherein the average weight of the takifugu obscurus is 350-450 g; s2, killing puffer: hammering the selected globefish to death and cleaning; s3, carrying and initial bacteria reduction treatment of the puffer fish: intensively placing the fish meat on ice, storing the fish meat in a foam heat preservation box at a low temperature, and transporting the fish meat to a laboratory within 3 hours; after arriving at a laboratory, preparing a chopping board, a knife and scissors which are sterilized in advance, and carrying out initial sterilization treatment;
step two, preparing a PCA and CN culture medium: adding PCA culture medium dry powder into ultrapure water in equal proportion, stirring uniformly, subpackaging into conical flasks, then performing high-pressure sterilization at 121 ℃ for 15min, cooling, heating and melting with a microwave oven when in use, and pouring a flat plate for preparing a culture medium, wherein the culture medium comprises a PCA culture medium and a CN culture medium;
step three, determining the total number of colonies and the total number of pseudomonas: s1, taking out the fish bags to be measured and crushed, cutting again under aseptic operation table according to actual conditions, cutting the fish blocks into 2mm fish slices easy to homogenate, and weighing in advanceWeighing sterile homogenizing bag, placing 15g fish meat into sterile homogenizing bag containing 135ml sterile physiological saline, beating and pulverizing in beating type homogenizer for 8min, observing to mix well and beating into sample homogenate, if no mixing well, beating again for homogenizing; s2, igniting an alcohol lamp under an aseptic operation table, operating within a flame range, and reserving a small gap to prevent the juice from being poured out of the outer port of the homogenizing bag, wherein 10ml of the juice is poured into a centrifugal tube for later use; s3, adding 1ml sample homogenate into a test tube filled with 9ml sterile normal saline, mixing uniformly, diluting gradually according to 10 times coefficient, and selecting proper 3-grade dilution-10 according to storage time and freshness3-107Selecting 3 gradients, adding 1ml of diluent into a sterile culture dish, respectively pouring a proper amount of PCA culture medium and CN culture medium which are cooled to 45 ℃ and uniformly mixing, after agar of the culture medium is solidified, carrying out inverted culture, making 3 parallel and 3 dilution gradients for each sample, recording the dilution times and the corresponding total number of bacterial colonies, regulating aquatic products according to the national standard GB29921, culturing the total number of the PCA bacterial colonies at 30 ℃ for 3 days, culturing the bacterial colonies in the CN culture medium at 30 ℃ for 2 days, and during measurement, parallelly measuring and calculating the numbers of the pseudomonas and the total bacterial colonies according to the required concentration gradients;
step four, 16s rDNA sequencing analysis is carried out: placing the small fish slices in a freezing storage tube of 2ml in a refrigerator at the temperature of-80 ℃, grouping the fish slices according to the fish flesh of the day, and grouping the fish slices according to parallel comparison for storage; unified sample feeding, adopting an Illumina Hiseq high-throughput sequencing method to respectively carry out the following steps on 2 freshness protection bags under the condition of freshness protection at 4 ℃: the common polyethylene freshness protection package P and the air-conditioning freshness protection package M and 2 pretreatment means: the normal saline CK group and the chlorine dioxide tablets are prepared into a SAEW group of 10mg/L, 2 hypervariable regions V3-V4 of 16s rDNA genes of the fugu obscurus meat spoilage bacteria are subjected to sequencing analysis, a growth curve in the spoilage process of the fugu obscurus at the temperature of 4 ℃ is established, and gas components in the gas-conditioning fresh-keeping bag M comprise 60% of CO2, 5% of O2 and 35% of N2; constructing a small fragment library for sequencing by using a double-End sequencing Paired-End method, and revealing the species composition of a sample by splicing and filtering Reads, clustering OTUs, and performing species annotation and abundance analysis; further performing alpha diversity analysis, beta diversity analysis and significant species difference analysis, and mining differences among samples.
Further, in the step one, the method for killing the living takifugu obscurus by referring to the standard ' safe processing operating Specification of pollution-free domesticated takifugu obscurus ' (DB32/T543-2002) ', comprises the following specific steps: after washing the fish body with water, hammering the living body of the takifugu obscurus to death, and cutting off fish fins and fish tail; peeling from the upper neck as a cutting point; cutting the mandible along the center of the mandible, and taking out the eyes, gills and brain of the head; the abdomen of the fish is upward, the abdomen and the peritoneum are cut off from the cloaca without cutting off the viscera, and the viscera are taken out item by item; flushing with clear water to remove blood stasis, completely removing residual blood and mucus on trunk, and drying with dry cloth to remove body surface water.
Furthermore, in the transportation process of the first step, the tightness and heat preservation property are kept, so that the fish meat is not polluted.
Further, the initial sterilization process in step one includes the steps of: s1, under an ultra-clean workbench, taking back muscles along the middle spine by using sterile scissors and a sterile knife, washing the taken fish cleanly by using sterile water, and sucking water by using water absorption paper, wherein the fish needs to be subjected to sample mixing in order to ensure the stability of data of each test and eliminate the difference among fish individuals; s2, transversely cutting each slaughtered fish into 3 pieces, longitudinally cutting each piece into 2 pieces along the central line of the spine of the body, wherein each piece is 2cm multiplied by 3cm in size and 15g in mass, and grouping the mixed fish; s3, soaking and cleaning the fish blocks for 10min by respectively using 0.9 percent of sterile normal saline or 0.02g/L of chlorine dioxide sterilizing water prepared by tablet chlorine dioxide; then using 7cm multiplied by 10cm aseptic bags for subpackaging and using 7cm multiplied by 10cm self-aeration fresh-keeping bags MP30 for subpackaging, then placing in a refrigerator at 4 ℃ for refrigeration for 10day, and sampling for 0, 2, 4, 6, 8 and 10 days respectively to determine the total number of colonies and the total number of pseudomonas; grouping each type of packaged and stored fish, wherein each 3 fish are used as a small group for parallel comparison, writing detection date, storage conditions and packaging types according to specifications, and stacking; subsequently, the culture was stored at 4 ℃ for 10 days, and a group was periodically taken out for examination, and the inventive material on day0 was stored at least under the conditions of storageOpening the heat preservation box in advance to set the temperature before putting the container in the container for more than 2 hours, and waiting for the set temperature PV to be SV; the number of colonies on day0 should be 103To be determined to be present 107The experiment was ended at this time.
Furthermore, according to the GB29921, the colony count CFU/g of aquatic products such as fish can be divided into 3 grades, wherein<105Is the first grade freshness, and is also the highest safety standard M,<5×105the second grade freshness reaches 106-107At the moment, the fish is three-level, and the fish is rotten and can not be eaten at the moment.
Further, in the second step, the PCA culture medium is a plate counting culture medium, and the raw material ratio of the PCA culture medium is 5.0g of tryptone, 2.5g of yeast extract powder, 1.0g of glucose, 15.0g of agar, 1000ml of distilled water and the pH value is 7.0 +/-0.2.
In the second step, the CN culture medium is a pseudomonas agar basal culture medium, and the CN culture medium comprises 16.0g of gelatin peptone, 10.0g of acid hydrolyzed casein, 10.0g of anhydrous potassium sulfate, 1.4g of anhydrous magnesium chloride, 0.2g of hexadecyl trimethylamine bromide, 0.015g of nalidixic acid, 15.0g of agar, 1000ml of distilled water, and a pH of 7.1 ± 0.225.
Further, the equipment used for autoclaving in step two is a high pressure steam sterilizer, and the equipment used for weighing in step three is an electronic analytical balance.
The invention has the following advantages:
1. according to the invention, through 16s rDNA sequencing result analysis and colony counting, the main spoilage microorganisms are pseudomonas, and the main spoilage microorganisms are more relatively dominant under the conditions of SAEW water treatment in the early stage or self-gas-regulating freshness-keeping bags, and win in competition of flora participating in the later stage of spoilage, which indicates that the tolerance of the spoilage microorganisms is strong, and the spoilage microorganisms can better develop and grow under the conditions of 4 ℃ and even gas-regulating packaging and SAEW when other microorganisms are inhibited; viewed from subordinate and species, pseudomonas is always the most predominant microorganism, under common packaging conditions, the SAEW treatment is less than the CK treatment, the occupation ratio is increased firstly, then reduced and then increased, and the trend of the occupation ratio graph of the colony growth curve measured by CP and SP is matched with a certain degree, except that the proportion of pseudomonas in the total bacteria displayed on a culture dish is obviously more, and the existence of special non-culturable microorganisms can cause the reduction of the total bacteria at the early stage when the total bacteria PCA is cultured, so that the proportion of the pseudomonas is increased;
2. the present inventors found that it may not be dominant in the inhibition of the number of microorganisms by colony counting, which may be caused by the growth of Pseudomonas sp; the modified atmosphere packaging can well inhibit the growth of most microorganisms in the early, middle and later stages of the putrefaction of the takifugu obscurus to ensure that the abundance of the microorganisms is remarkably reduced, but the influence on pseudomonas is little or the adaptability of the pseudomonas is strong enough, so that the growth space of other inhibited microorganisms is occupied, the competitive internal consumption among the pseudomonas is lacked due to the modified atmosphere environment, and the pseudomonas can grow freely; it can also be observed that Shewanella and Proteobacteria are also involved in the later-stage putrefaction process, and the main genera of putrefaction may change with the increase of putrefaction degree of the meat and the change of substrate environment.
Drawings
FIG. 1 is a schematic diagram showing the change of the total amount of fresh-keeping colonies of Fugu obscurus at 4 ℃ according to the present invention;
FIG. 2 is a schematic diagram showing the change of the total amount of Pseudomonas bacteria at 4 ℃ in the Takifugu obscurus provided by the present invention;
FIG. 3 is a schematic diagram showing the change of the pseudomonad specific total community during the preservation of Takifugu obscurus at 4 ℃ provided by the present invention;
FIG. 4 is a histogram of OTU number frequency distribution according to the present invention;
FIG. 5 is a schematic view of a visualization diagram of the OTU number frequency distribution histogram provided by the present invention;
FIG. 6 is a schematic diagram of a dilution curve observed _ out provided by the present invention;
FIG. 7 is a schematic diagram of a dilution curve observed _ out provided by the present invention;
FIG. 8 is a schematic representation of the dilution curve Shannon index provided by the present invention;
FIG. 9 is a schematic representation of the dilution curve Shannon index provided by the present invention;
FIG. 10 is a schematic diagram of an exponential representation of the quantity of the observed _ otus reflecting species provided by the present invention;
FIG. 11 is a schematic representation of evenness uniformity provided by the present invention as affected by species and abundance;
FIG. 12 is a schematic diagram of the influence of the abundance and evolutionary relationship between species on the faith _ pd provided by the present invention;
FIG. 13 is a schematic representation of the Shannon community diversity provided by the present invention as affected by the abundance and uniformity of species in the community;
FIG. 14 is a schematic diagram of a Jaccard index distance matrix according to the present invention;
FIG. 15 is a schematic diagram of a bra-curves distance matrix provided by the present invention;
FIG. 16 is a diagram of NMDS-ANOSIM similarity analysis provided by the present invention;
FIG. 17 is a schematic diagram of a non-weighted uniform distance matrix according to the present invention;
FIG. 18 is a schematic diagram of a non-weighted uniform distance matrix PCoA according to the present invention;
FIG. 19 is a schematic diagram of a weighted unifrac distance matrix according to the present invention;
fig. 20 is a schematic diagram of a weighted unifrac distance matrix PCoA provided in the present invention;
FIG. 21 is a top30 family abundance table provided by the present invention;
FIG. 22 is a top40 genus abundance table provided by the present invention;
FIG. 23 is a Top50 abundance table provided by the present invention;
FIG. 24 is a PCA plot of genus grades based on sample abundance provided by the present invention;
FIG. 25 is a heatmap of the genus grade based on the sample abundance top50 provided by the present invention;
FIG. 26 is a heatmap of the genus grade based on the sample abundance top50 provided by the present invention;
FIG. 27 is a Upset graph based on sample abundance genus level provided by the present invention;
FIG. 28 is a graph of petals based on a sample abundance genus level provided by the present invention;
FIG. 29 is a box-plot representation of abundance of genus level top12 differential genus based on Metastat analysis provided herein;
FIG. 30 is a schematic diagram of a significant difference biomarker in a group analyzed by LEfSe analysis provided by the present invention;
FIG. 31 is a functional level PCA diagram of a KEGG database (L3) specific function provided by the present invention;
FIG. 32 is an inter-group community function Alpha diversity index analysis diagram provided by the present invention;
FIG. 33 is a graph of NMDS analysis based on beta diversity (euclidean distance) provided by the present invention;
fig. 34 is a drawing of significant difference biomarker analyzed and analyzed by LEfSe provided in the present invention;
fig. 35 is a picture of significant difference biorarker analyzed and analyzed by LEfSe provided by the present invention.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to the attached figures 1-35 of the specification, the dynamic change detection method of spoilage microorganisms in the preservation process of takifugu obscurus comprises the following specific steps:
step one, treating a raw material of takifugu obscurus:
(1) selecting the puffer fish:
randomly taking 2-year-old fresh takifugu obscurus from the pond, wherein the average weight of the takifugu obscurus is 350-450 g;
(2) fugu killing method
The living Fugu obscurus is killed with reference to standard ' nuisanceless domesticated Fugu obscurus ' safety processing operation Specification (DB32/T543-2002), ' and then the living Fugu obscurus is hammered and killed after the fish body is cleaned with water, so that the fins and the fish tail are cut off. Peeling from the upper neck as the incision point. Cutting the mandible along the center of the mandible, and taking out the eyes, gills and brain of the head; the abdomen and peritoneum of the fish are cut open from the cloaca with the belly facing upwards, and the viscera are taken out item by item without cutting the viscera. Flushing with clear water to remove blood stasis, completely removing residual blood and mucus on trunk, and drying with dry cloth to remove body surface water.
(3) Transportation and initial sterilization treatment of puffer fish
The fish meat is intensively placed on ice, stored at low temperature in a foam incubator and transported to a laboratory within 3 hours. The sealing property and the heat preservation property are preserved in the transportation process; after the laboratory, preparing the chopping board, the knife and the scissors which are sterilized in advance for subsequent treatment. During transportation, the fish meat is ensured not to be polluted as much as possible.
Under the superclean bench, use aseptic scissors and aseptic sword to get its back muscle along middle chine to wash clean with aseptic water the flesh of fish that takes off, the paper that absorbs water, in order to guarantee the data stability of experiment at every turn, eliminates the difference between the fish individual, need mix the appearance to above flesh of fish. Each of the slaughtered fish meat was cut into 3 pieces transversely and then cut into 2 pieces each having a size of about 2cm by 3cm and a mass of about 15g for each piece longitudinally along the midline of the spine of the body. The pooled fish flesh was then grouped.
Soaking and cleaning fish blocks with 0.9% sterile normal saline or 0.02g/L chlorine dioxide sterilizing water (SAEW) prepared from tablet chlorine dioxide for 10 min; then using 7cm multiplied by 10cm aseptic bags for subpackaging and using 7cm multiplied by 10cm self-aeration fresh-keeping bags MP30 for subpackaging, then placing in a refrigerator at 4 ℃ for refrigeration for 10day, and sampling for 0, 2, 4, 6, 8 and 10 days respectively to determine the total number of colonies and the total number of pseudomonas; grouping each type of packaged and stored fish, wherein each 3 fish is used as a group for parallel comparison, and stacking according to the specification, written detection date, storage conditions, packaging type and the like; then preserving at 4 deg.C for 10 days, periodically taking out a group for detection, preserving the inventive material at 0 deg.C for at least 2 hrBefore the placement, the incubator is opened in advance to set the temperature, and the temperature setting PV is waited for. The limit quantity of the raw fish blocks is moderately amplified by referring to other documents, and the colony count CFU/g of aquatic products such as fish can be divided into 3 grades, wherein<105Is the first grade freshness, and is also the highest safety standard M,<5×105the second grade freshness reaches 106-107At the moment, the fish is three-level, and the fish is rotten and can not be eaten at the moment. The number of colonies on day0 should be 103To be measured to 107Optionally, the experiment is ended when it occurs.
Step two, preparing a PCA and CN culture medium:
adding the PCA culture medium dry powder into ultrapure water in equal proportion, stirring uniformly, subpackaging into conical flasks, then performing autoclaving at 121 ℃ for 15min, cooling, heating and melting in a microwave oven during use, and pouring a plate for standby preparation of a culture medium, wherein the culture medium comprises a PCA culture medium and a CN culture medium, and the ratio of the two culture media is shown in tables 1 and 2.
TABLE 1 Plate Count (PCA) Medium
TABLE 2 Pseudomonas agar base (CN) Medium
Step three, determining the total number of colonies and the total number of pseudomonas:
(1) preparing the invented equipment reagents of sterilizing pot, knife and scissors, gun head (5ml, 2, 1ml, 1 box), centrifuge tube, sterilized normal saline, PCA culture medium, CN culture medium, etc.
Refer to GB4789.2-2016 food safety national standard-food microbiology test colony TotalNumber determination. Taking out the fish bag which needs to be measured and crushed on the day, carrying out secondary cutting under an aseptic operation platform according to actual conditions, cutting the fish blocks into 2mm fish slices which are easy to homogenate, weighing an aseptic homogenization bag in advance, putting 15g of fish into the aseptic homogenization bag containing 135ml of aseptic normal saline, beating and crushing for 8min in a beating type homogenizer, and observing that the fish is fully and uniformly mixed and is beaten into sample homogenate, if the fish is not uniformly mixed, beating and homogenizing again. And (3) igniting an alcohol lamp under an aseptic operation table, operating within the flame range, and leaving a small gap to ensure that the juice is poured out from the external port of the homogenizing bag by 10ml to a centrifugal tube for later use. Then taking 1ml sample homogenate, adding into a test tube containing 9ml sterile physiological saline, mixing, diluting gradually according to 10 times coefficient, selecting appropriate grade 3 dilution (10) according to storage time and freshness3-107) Selecting 3 gradients, adding 1ml of diluent into a sterile culture dish, respectively pouring a proper amount of PCA culture medium and CN culture medium, cooling to about 45 ℃, and mixing uniformly. After the medium agar is solidified, inverted culture is carried out, 3 parallel dilution gradients and 3 dilution gradients are made for each sample, and the dilution times and the corresponding colony counts are recorded. The specific culture method is shown in the following table 3, namely, according to the national standard GB29921, the total number of PCA colonies is cultured for 3 days at 30 ℃, and the number of CN colonies is cultured for 2 days at 30 ℃. During the determination, the number of pseudomonas and total colonies was determined and calculated in parallel according to the concentration gradient required previously.
TABLE 3 national Standard GB29921 aquatic microorganism culture Standard
Step four, 16s rDNA sequencing analysis is carried out:
placing the small fish slices in a-80 refrigerator by using a 2ml freezing tube, grouping the fish slices according to the fish flesh of the day, and storing the fish slices according to parallel comparison; uniformly delivering samples, and respectively performing high-throughput sequencing on 2 freshness protection packages (a common polyethylene freshness protection package P and a gas-conditioning freshness protection package M (gas component 60% CO) under the condition of freshness protection at 4℃ by adopting an Illumina Hiseq high-throughput sequencing method2，5％O2，35％N2) And2 pretreatment means (normal saline CK group, SAEW group prepared by 10mg/L chlorine dioxide tablets) 2 hypervariable regions (V3-V4) of the 16SrDNA gene of the fugu obscurus meat spoilage bacteria are used for sequencing analysis and establishing a growth curve in the spoilage process of the fugu obscurus under the condition of 4 ℃. Constructing a small fragment library for sequencing by using a method of double-ended sequencing (Paired-End). Species composition of the sample can be revealed by performing splicing filtration on Reads, OTUs (operational taxonomicunits) clustering, and performing species annotation and abundance analysis; further performing alpha diversity analysis (AlphaDiversity), beta diversity analysis (BetaDiversity), and significant species difference analysis, etc., differences between samples can be mined.
(1) Establishing a growth curve in the putrefaction process of the takifugu obscurus at the temperature of 4 ℃:
1) naming and grouping method
CK normal saline is abbreviated as 'C', SAEW hypochlorous acid water 10mg/L is abbreviated as 'S'; the self-gas-regulating fresh-keeping bag MP30 is abbreviated as 'M', the common PE fresh-keeping bag is abbreviated as 'P', so four groups of CP, CM, SP and SM are provided. Samples were analyzed every 0, 2, 4, 6, 8, 10 days; statistical analysis and profiling were performed using Origin 9.0 and SPSS software. Results of the invention the significance analysis and multiple comparisons of the same treatment group were performed using the Duncan's method as follows, with differences (P <0.05) in lower case for different storage times and differences between groups under different treatment conditions in upper case. (ABC represents the significance between groups on the same day, and acf represents the significance between different days of the same group). The statistical results are shown in fig. 1, 2 and 3.
From FIGS. 1, 2 and 3, it can be seen that the degree of spoilage of Fugu obscurus gradually deepens with the increase of the preservation time at 4 ℃ and gradually rises in CFU/g units as shown in the following tables, see tables 4 and 5.
TABLE 4 Total PCA in Fugu obscurus spoilage Process
TABLE 5 Total CN count in Fugu obscurus spoilage Process
The total microbial quantity and the pseudomonas quantity of the takifugu obscurus fish subjected to SAEW water treatment in the early stage are obviously reduced to 2.07526 +/-0.07526 even on day0, but gradually return to a higher level along with the increase of the preservation days, and the effect in the middle and later stages is not obvious relative to CK water treatment, has no obvious difference and is accompanied with the condition that the fish is sticky and greenish. For the common packaging PE and the modified atmosphere MP30, the two types of bacteria have obvious classification and data swing in the interval from the fourth day to the eighth day, the total number of the bacteria colony of the common packaging bag and the total number of the pseudomonas are well controlled, and the difference between the total number of the bacteria colony of the sixth day and the total number of the bacteria colony of the eighth day is 0.77(lgCFU/g) and 0.855(lgCFU/g) under CK water treatment with good sensory conditions; the difference between the total number of pseudomonas is 0.887(lgCFU/g) and 1.077 (lgCFU/g); and all have significant differences.
(2) Analysis of 16srDNA microbial diversity sequencing results
1) Sequencing grouping protocol
The invention sends three groups of SP, CM and CP sampling results for 0-8 days to analyze; the samples were named CK physiological saline abbreviated as "C", SAEW hypochlorous acid water 10mg/L abbreviated as "S"; the self-gas-regulating fresh-keeping bag MP30 is abbreviated as 'M', the common PE fresh-keeping bag is abbreviated as 'P', so four groups of CP, CM, SP and SM are provided. Every 0, 2, 4, 6, 8, day sample was analyzed, i.e., prefixed by "d 0" for "day 0", and 3 parallel samples were sent; respectively named A, B, C groups again, 1-5 means 0-8 days, and the grouping information is A1-d0sp1, d0sp2, d0sp 3; a2-d2sp1, d2sp2, d2sp 3; a3-d4sp1, d4sp2, d4sp 3; a4-d6sp1, d6sp2, d6sp 3; a5-d8sp1, d8sp2, d8sp 3; b1-d2cm1, d2cm2c2cm 3; b2-d4cm1, d4cm2 and d4cm 3; b3-d6cm1, d6cm2 and d6cm 3; b4-d8cm1, d8cm2 and d8cm 3; c1-d0cp1, d0cp2d0cp 3; c2-d2cp1, d2cp2, d2cp 3; c3-d4cp1, d4cp2, d4cp 3; c4-d6cp1, d6cp2, d6cp 3; c5-d8cp1, d8cp2, d8cp 3; basic group settings are: all + a1+ a2+ A3+ a4+ a5+ B1+ B2+ B3+ B4+ C1+ C2+ C3+ C4+ C5; setting a difference analysis group: A1-A2-A3-A4-A5; B1-B3-C1-B2-B4; C1-C2-C3-C4-C5; A1-C1; A2-B1-C2; A3-C3-B2; A4-B3-C4; A5-C5-B4.
2) Sequencing Overall data results
TABLE 6 sequencing Overall results Table
Continue the table
3) Evaluation results of sequencing quality
By shearing and filtering gene sequences, 102519 gene sequences are measured on average in each sample, 99450 effective reads are obtained through quality control average, and the average coverage of the samples is 96.98%, the maximum value is 97.94%, and the minimum value is 94.86%; the whole effect is better.
4) OTU number distribution and analysis
Figure 4 shows the OTU number frequency distribution histogram of the sample: the horizontal axis marks the total number of OTUs of each sample, and the vertical axis counts the number of the samples with the OTU number distributed in a certain range. The visual graph is shown in FIG. 5, which shows the number distribution of OTUs in a certain frequency range
5) Dilution Curve analysis
The statistical conditions of the dilution Curve (random cut) observed _ OUT and Shannon index are shown in fig. 6-fig. 9, and it can be seen that as the number of sequencing strips randomly drawn on the abscissa increases, the number of OTUs or Shannon index obtained by clustering the ordinate based on the number of sequencing strips tends to be flat at about 60000 depth, and the average of 99450 samples in the sample indicates that most samples have sufficient sequencing depth and reliable quality, and a small portion of samples may have pollution or other factors to cause many types of OUT, so that more OTUs can be found by increasing the data volume to some extent.
6) Results of diversity analysis
1. Alpha diversity analysis
As can be seen from FIGS. 10-13, A1, A2, C1 and C2 are the performances of the SP and CP groups at 0-2 days, the four indexes, namely evenness, Shannon, fast _ pd and observed _ otus, are all higher, the CP group tends to rise at 0-2 days, the SP group changes greatly, which indicates that the species are relatively abundant, but in the CM group of modified atmosphere package, the situation does not occur, and the early-middle stage is kept low all the time. Under the condition of modified atmosphere, the species diversity of the microorganisms is greatly inhibited in the early stage, and the SP, CP and CM groups have the condition that four indexes are less changed after being stabilized to a certain level along with the increase of time, so that a plurality of microorganisms are inhibited in a low-temperature environment for a long time.
2. beta diversity analysis
In FIGS. 14 and 15, 0 indicates that the OUT structures (including composition and abundance) of the two microbial communities are completely identical, and the darker the color between the two groups indicates the higher the degree of difference between the groups; it can be seen that Jaccard determines from the big trend that a certain main microorganism occupies relative advantages from beginning to end and participates in the putrefaction of Takifugu obscurus, and the microbial diversity between each parallel group has obvious difference, but the result algorithm is more direct, and the reference significance is not great for metagenome and 16 s. The great difference between the diversity of two days before preservation and the diversity of the subsequent microorganisms can be seen through the bra-curves distance, so that the method has practical reference significance, and the method proves that a plurality of microorganisms are inhibited in the low-temperature environment in the later period.
In addition, fig. 16 shows that the non-metric multi-dimensional scaling (NMDS) results show that stress values less than 0.2 indicate reliable results. The statistical value R is 0.334, which is larger than 0, indicating that the difference between groups is larger than the difference in groups. p is 0.001, and the quality is reliable. The differences among the SP, the CP groups and other groups at the early stage are large, the species richness is large, and the differences are not overlapped with other groups, which indicates that the sampling has large differences according to the sample conditions at the early stage.
It can be seen in FIGS. 17-20 that the weighted _ uniform matrices for the present invention show results similar to the break-curves matrices, and that the unweighted _ uniform matrices show results similar to the Jaccard index distance matrices. The left side of the scale 0 of the unweighted PCoA graph is provided with samples of 0-2 days in the early stage of the invention, and the distribution is more discrete; on the right side, the CK-treated group and the SAEW-treated group are also distributed separately from each other, but in the weighted monofa of unifrac, after considering the presence or absence of species and abundance of species, the figure shows a strong similarity, indicating that many microorganisms are inhibited and only partially similar microorganisms are grown in a low-temperature environment.
7) Colony structure analysis results
And comparing the representative sequence of the OTU with a microbial reference database to obtain the species classification information corresponding to each OTU. And (5) counting the community composition of each sample to generate an abundance table.
According to FIGS. 22-23, the genus Pseudomonas (Pseudomonas) is always the most predominant microorganism in terms of its dependent and species, with the SAEW treatment being less than the CK treatment under normal packaging conditions, and the occupancy ratio increasing first with progress, then decreasing last, then increasing, somewhat in a trend with the occupancy ratio of the colony growth curve previously measured by CP, SP, except that there is a significantly greater proportion of Pseudomonas to total bacteria present on the petri dish, and it is likely that the presence of non-culturable specific microorganisms in the culture of total bacteria PCA causes a reduction in the previous total bacteria, thereby increasing its proportion of Pseudomonas.
Secondly, it can be seen that in the early stage of pufferfish putrefaction, Acinetobacter, Aeromonas and Lactobacillus may also occur in a certain proportion, which indicates that the OUT structure in the early stage is complex, and different processing conditions and sample deviations can result in different results. Among them, Burkholderia-Kabalelia-Parabraholderia (Burkholderia-Caballeria-Parabourkii), Rhizobiaceae (Rhizobiaceae), and Lachnospiraceae (Lachnospiraceae) all have a certain small proportion of the three. In the middle stage, during the descent of Pseudomonas, the former site is replaced by Shewanella (Shewanella), Proteobacteria (Gamma), and the short-lived species Shewanella _ uncultred _ gamma and Enterobacteriaceae (Enterobacteriaceae) are also present.
8) Dimension reduction analysis based on species abundance
The principal component analysis PCA of the invention uses an AD method, and based on the distance of a gene-grade PCA-AD graph of the sample abundance in FIG. 24, the SP and CP samples are relatively far apart from each other in the 0 th-2 nd day of the early stage, which indicates that the species composition of the samples is greatly different. The results are relatively concentrated between later samples, indicating that many microorganisms are inhibited in a possibly low temperature environment.
1. Clustering analysis of species abundance
According to FIGS. 25 and 26, the sequences were clustered with 97% identity into OTUs based on the OTU sort operation units. The method is characterized in that the relative abundance table of different classification levels is used for showing, the genera of the abundance ranking Top50 and the abundance information of the genera in each sample are selected to draw a heat map, clustering is carried out on different layers, the dark colors at two ends indicate that the abundance is more or less in the whole, and the light colors indicate that the quantity tends to be a common level, and the result still indicates that the abundance of the microorganisms at the previous stage is larger, and the air-conditioned fresh-keeping bags have good inhibition effect.
2. Venn and petal plot analysis of species abundance
According to fig. 27, in the uppet diagram, the horizontal bar graph at the lower left shows the number of species in each group, each black dot at the lower right represents one group, the dots are connected with each other to indicate that the corresponding two groups intersect, and the bar graph at the upper right represents the number of species corresponding to each intersection. The species abundance was greatest in the first two days of the CP group, whereas the CM group fused well into the second cohort of species numbers under the modified atmosphere packaging conditions.
According to the figure 28, the central circle of the petal graph represents the number of species shared by all samples, the numbers on the petals represent the number of the species unique to the samples, the invention specially selects the group with the largest difference, namely the SP group at the 0 th day to make the petal graph, so that the invention has the advantages that the contingency and the specificity in the early stage are larger, the fish meat is selected and grouped, the environment of the puffer fish can cause certain influence, and the invention result is influenced by special unique microorganisms. Other groups have small differences, and the petal graphs have small reference significance, so that the species similarity can be only explained if the petal graphs are made.
3. Metastat analysis of species that differ between groups
According to species with differences among groups, principal component PCA analysis and abundance clustering heatmap Metastat analysis are carried out, box plot results with the abscissa as the group name and the ordinate as the relative abundance also show that in the early stage of preservation, species of SP and CP groups are rich and different in the early stage, and the comparison of the SP group and the CP group shows that after SAEW treatment, the species are obviously reduced in the next day, and the species treated by CK are increased.
4. Lefse analysis and analysis of difference biomuraker
Dimension reduction was achieved by the method of rank sum test and evaluation of the magnitude of the effect of the different species with LDA (linear discriminant analysis), LDA score 4 as shown in fig. 30; different colors represent the biomarkers identified in different groups, and the influence of the species of interest on the statistics (the LEfSe analysis results of the species with differences between groups include three parts, namely LDA value distribution histogram, cladogram (phylogenetic distribution) and abundance comparison graph of the biomarkers with statistical differences between groups in different groups).
9) Community function analysis
1. KEGG (L3) functional abundance dimensionality reduction PCA analysis
By utilizing the strong graphical function prediction of KEGG, the L3 level function classification is annotated as the following figure 31, and it can be seen that the distances between SP, CP and CM in the first two days are larger, which indicates that the microorganisms participating in the putrefaction process are more in variety, rich in gene function and more in channel, and indirectly reflects that a certain difference exists between the early CM group and the SP and CP groups.
2. Functional diversity analysis Alpha diversity analysis
Alpha diversity is mainly calculated by using Chao1, observed _ otus, Shannon and Simpson to the Alpha diversity index of the sample, and the significance of the difference is counted. As can be seen from FIG. 32, the functional diversity analysis is similar to the Alpha diversity results of species, but in CM group, it is more intuitive to see, and the abundance of functional genes tends to increase first and then decrease, indicating that there is a change in the metabolic pathway of Pseudomonas bacteria itself, the main microorganism, during the putrefactive process. In this aspect, species composition information obtained by comparing 16S sequencing data by using Tax4Fun software can be used to obtain KEGG family information corresponding to OTU, so as to calculate abundance of the KEGG, and Pathway, EC information, and OTU abundance are obtained from information in KEGG database, so as to calculate abundance and other contents of each functional class. Further understanding can be made after the sample volume is raised.
3. Functional diversity analysis Beta diversity analysis
The analytical results are shown in FIG. 33.
4. Lefse analysis of differential biorarker
According to fig. 34 and fig. 35, in order to screen the functional Biomarker with significant difference between groups, firstly, the difference function between different components is detected by a rank sum test method, and dimension reduction is realized by LDA (linear discriminant analysis) and the influence of the difference function is evaluated, i.e. LDA score is obtained; the LEfSe analysis results of the inter-group difference function include three parts, namely LDA value distribution histogram, cladogram (phylogenetic distribution) and comparison graph of abundance of biomarkers with statistical differences among groups among different groups. And carrying out biomaker identification among different groups, and screening from 2 to 4 by using LDA SCORE.
(3) Conclusion
Through analysis of a 16s rDNA sequencing result and colony counting, the main spoilage microorganism is pseudomonas, the main spoilage microorganism is more advantageous in SAEW water treatment in the early stage or under the condition of an automatic atmosphere-regulating fresh-keeping bag, and the main spoilage microorganism wins in competition of flora participating in the later stage of spoilage, which shows that the spoilage microorganism is strong in tolerance and can better develop and grow under the conditions of 4 ℃ and even under the conditions of atmosphere-regulating packaging and SAEW when other microorganisms are inhibited. From a subordinate and species point of view, pseudomonas is always the most predominant microorganism, which under normal packaging conditions is less after SAEW treatment than CK treatment and the occupancy increases first with progression, then decreases last and then increases, somewhat trending in comparison to previous occupancy maps of the colony growth curves measured by CP, SP, except that there is a significantly greater proportion of total bacteria present on the culture dish, and it is likely that the presence of non-culturable specific microorganisms in the culture of total bacteria PCA leads to a reduction in the former total bacteria, and thus to an increase in its pseudomonas occupancy.
Secondly, it can be seen that the cases of acinetobacter, aeromonas and lactobacillus in a certain proportion also occur in the early stage of the putrefaction of takifugu obscurus, which indicates that the OUT structure in the early stage is complex, and different processing conditions and sample deviations can lead to different results. Among them, Burkholderia-Carbarlotia-Paspaleaceae, Rhizobiaceae, and Lachnsoniaceae all have a certain small proportion. In the middle stage, during the descent of the pseudomonads, Shewanella, Proteobacteria, replaced their original position, while Shewanella _ uncultured _ gamma species and Enterobacteriaceae also had transient signs of appearance;
also in the case of the CK group treatment, the self-gassing bag MP30 may not be superior in the suppression of the microbial count, which may be due to the growth of pseudomonas, as compared to the ordinary PE bags, as found by colony counting. The spontaneous modified atmosphere packaging can well inhibit the growth of most microorganisms in the early, middle and later stages of putrefaction of takifugu obscurus to ensure that the abundance of the microorganisms is remarkably reduced, but probably has little influence on pseudomonas or has strong adaptability enough to occupy the growth space of other inhibited microorganisms, the modified atmosphere environment causes lack of competitive internal consumption among the pseudomonas and the pseudomonas can grow freely. It can also be observed that Shewanella and Proteobacteria are also involved in the later-stage putrefaction process, and the main genera of putrefaction may change as the putrefaction degree of the meat is deepened and the substrate environment is changed.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. The method for detecting the dynamic change of spoilage microorganisms in the fresh-keeping process of the takifugu obscurus is characterized by comprising the following steps of: the method comprises the following specific steps:
step one, processing raw materials of takifugu obscurus: s1, selecting puffer fish: randomly taking 2-year-old fresh takifugu obscurus from the pond, wherein the average weight of the takifugu obscurus is 350-450 g; s2, killing puffer: hammering the selected globefish to death and cleaning; s3, carrying and initial bacteria reduction treatment of the puffer fish: intensively placing the fish meat on ice, storing the fish meat in a foam heat preservation box at a low temperature, and transporting the fish meat to a laboratory within 3 hours; after arriving at a laboratory, preparing a chopping board, a knife and scissors which are sterilized in advance, and carrying out initial sterilization treatment;
step two, preparing a PCA and CN culture medium: adding the PCA culture medium dry powder into ultrapure water in equal proportion, stirring uniformly, subpackaging into conical flasks, then performing autoclaving at 121 ℃ for 15min, cooling, heating and melting with a microwave oven when in use, and pouring a plate for standby preparation of a culture medium, wherein the culture medium comprises a PCA culture medium and a CN culture medium;
step three, determining the total number of colonies and the total number of pseudomonas: s1, taking out the fish bags needing to be measured and crushed in the day, performing secondary cutting under an aseptic operating platform according to actual conditions, cutting the fish blocks into 2mm fish slices easy to homogenize, weighing the aseptic homogenization bags in advance, putting 15g of fish into the aseptic homogenization bags filled with 135ml of aseptic normal saline, beating and crushing for 8min in a beating type homogenizer, and observing that the fish slices are fully and uniformly mixed and beaten into sample homogenate, wherein if the fish slices are not uniformly mixed, beating and homogenizing again; s2, igniting the alcohol burner under the sterile operating platform, operating in the flame range, and leaving a small gap to allow juiceAvoiding an external port of the homogenizing bag, pouring out 10ml of the homogenizing bag into a centrifuge tube for later use; s3, adding 1ml sample homogenate into a test tube filled with 9ml sterile normal saline, mixing uniformly, gradually diluting according to a 10-fold coefficient, and selecting proper 3-grade dilution-10 according to storage time and freshness3-107Selecting 3 gradients, adding 1ml of diluent into a sterile culture dish, respectively pouring a proper amount of PCA culture medium and CN culture medium which are cooled to 45 ℃ and uniformly mixing, after agar of the culture medium is solidified, carrying out inverted culture, making 3 parallel and 3 dilution gradients for each sample, recording the dilution times and the corresponding total number of bacterial colonies, regulating aquatic products according to the national standard GB29921, culturing the total number of the PCA bacterial colonies at 30 ℃ for 3 days, culturing the bacterial colonies in the CN culture medium at 30 ℃ for 2 days, and during measurement, parallelly measuring and calculating the numbers of the pseudomonas and the total bacterial colonies according to the required concentration gradients;
step four, 16s rDNA sequencing analysis is carried out: placing the small fish slices in a freezing storage tube of 2ml in a refrigerator at the temperature of-80 ℃, grouping the fish slices according to the fish flesh of the day, and grouping the fish slices according to parallel comparison for storage; unified sample feeding, adopting an Illumina Hiseq high-throughput sequencing method to respectively carry out the following steps on 2 freshness protection bags under the condition of freshness protection at 4 ℃: the common polyethylene freshness protection package P and the air-conditioning freshness protection package M and 2 pretreatment means: the normal saline CK group and the chlorine dioxide tablets are mixed to 10mg/L SAEW group, 2 hypervariable regions V3-V4 of 16s rDNA gene of the takifugu obscurus meat spoilage bacteria are subjected to sequencing analysis, a growth curve in the spoilage process of the takifugu obscurus under the condition of 4 ℃ is established, and gas components in the air-conditioning fresh-keeping bag M comprise 60% CO2、5％O2And 35% N2(ii) a Constructing a small fragment library for sequencing by using a double-End sequencing Paired-End method, and revealing the species composition of a sample by splicing and filtering Reads, clustering OTUs, and performing species annotation and abundance analysis; further performing alpha diversity analysis, beta diversity analysis and significant species difference analysis, and mining the difference between samples.
2. The method for detecting the dynamic change of spoilage microorganisms in the fresh-keeping process of takifugu obscurus as claimed in claim 1, wherein: in the first step, the living Fugu obscurus is killed by referring to the standard 'pollution-free domesticated Fugu obscurus & safe processing operation Specification (DB 32/T543-2002)', and the killing specific steps are as follows: after washing the fish body with water, hammering the living body of the takifugu obscurus to death, and cutting off fish fins and fish tail; peeling from the upper neck as a cutting point; cutting the mandible along the center of the mandible, and taking out the eyes, gills and brain of the head; the abdomen of the fish is upward, the abdomen and the peritoneum are cut off from the cloaca without cutting off the viscera, and the viscera are taken out item by item; flushing with clear water to remove blood stasis, completely removing residual blood and mucus on trunk, and drying with dry cloth to remove body surface water.
3. The method for detecting the dynamic change of spoilage microorganisms in the fresh-keeping process of takifugu obscurus as claimed in claim 1, wherein: and in the transportation process of the step one, the tightness and heat preservation are kept, and the fish is ensured not to be polluted.
4. The method for detecting the dynamic change of spoilage microorganisms in the fresh-keeping process of takifugu obscurus as claimed in claim 1, wherein: the initial sterilization process in step one comprises the following steps: s1, under an ultra-clean workbench, taking back muscles along the middle spine by using sterile scissors and a sterile knife, washing the taken fish cleanly by using sterile water, and sucking water by using water absorption paper, wherein the fish needs to be subjected to sample mixing in order to ensure the stability of data of each test and eliminate the difference among fish individuals; s2, transversely cutting each slaughtered fish into 3 pieces, then longitudinally cutting each piece into 2 pieces along the midline of the spine of the body, wherein the size of each piece is 2cm multiplied by 3cm, the mass of each piece is 15g, and then grouping the mixed fish; s3, respectively soaking and cleaning the fish blocks for 10min by using 0.9% of sterile normal saline or 0.02g/L of chlorine dioxide sterilizing water prepared by tablet chlorine dioxide; then using 7cm multiplied by 10cm aseptic bags for subpackaging and using 7cm multiplied by 10cm self-aeration fresh-keeping bags MP30 for subpackaging, then placing in a refrigerator at 4 ℃ for refrigeration for 10day, and sampling for 0, 2, 4, 6, 8 and 10 days respectively to determine the total number of colonies and the total number of pseudomonas; grouping each type of packaged and stored fish, wherein each 3 fish are used as a group for parallel comparison, and writing detection date and storage according to the specificationCondition, packaging type, stacking; then preserving at 4 ℃ for 10 days, periodically taking out a group for detection, preserving the invention material on day0 for at least more than 2 hours under the preservation condition, opening the incubator in advance to set the temperature before putting in the incubator, and waiting for the set temperature PV (SV); the number of colonies on day0 should be 103To be determined to be present 107The experiment was ended at this time.
5. The method for detecting the dynamic change of spoilage microorganisms in the fresh-keeping process of takifugu obscurus as claimed in claim 4, wherein: according to the GB29921 regulation, the colony count CFU/g of aquatic products such as fish can be divided into 3 grades, wherein<105Is the first grade freshness, and is also the highest safety standard M,<5×105the second grade freshness reaches 106-107At the moment, the fish is three-level, and the fish is rotten and can not be eaten at the moment.
6. The method for detecting the dynamic change of spoilage microorganisms in the fresh-keeping process of takifugu obscurus as claimed in claim 1, wherein: in the second step, the PCA culture medium is a plate counting culture medium, and the PCA culture medium comprises 5.0g of tryptone, 2.5g of yeast extract, 1.0g of glucose, 15.0g of agar and 1000ml of distilled water, wherein the pH value is 7.0 +/-0.2.
7. The method for detecting the dynamic change of spoilage microorganisms in the fresh-keeping process of takifugu obscurus as claimed in claim 1, wherein: in the second step, the CN culture medium is a pseudomonas agar basic culture medium, and the CN culture medium comprises 16.0g of gelatin peptone, 10.0g of acid hydrolyzed casein, 10.0g of anhydrous potassium sulfate, 1.4g of anhydrous magnesium chloride, 0.2g of hexadecyl trimethylamine bromide, 0.015g of nalidixic acid, 15.0g of agar, 1000ml of distilled water and 7.1 +/-0.225 of pH.
8. The method for detecting the dynamic change of spoilage microorganisms in the fresh-keeping process of takifugu obscurus as claimed in claim 1, wherein: the equipment used for autoclaving in step two is a high-pressure steam sterilization pot, and the equipment used for weighing in step three is an electronic analytical balance.
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