阅读说明：本技术 一种快速区分水果罐头商业无菌的检测方法 (Detection method for rapidly distinguishing commercial sterility of canned fruits ) 是由 李正义 贾俊涛 祝素珍 姜英辉 唐静 黄小华 于 2021-09-26 设计创作，主要内容包括：本发明公开了一种快速区分水果罐头商业无菌的检测方法,它利用顶空气相色谱-质谱联用技术检测正常水果罐头与腐败水果罐头保温前后顶部空气中二氧化碳水平,根据二氧化碳水平是否发生显著变化(峰面积百分比差值超过10%),来区分水果罐头的商业无菌,达到区分水果罐头商业无菌的目的。本发明采用的方法中,比现有的传统检测方法时间缩短5～6天,该检测方法作为一种水果罐头商业无菌的初步筛选工具,不但可以降低检测成本,而且还适应快速发展的国际贸易需求。(The invention discloses a detection method for rapidly distinguishing commercial sterility of canned fruits, which is characterized in that a headspace gas chromatography-mass spectrometry technology is utilized to detect the carbon dioxide level in the headspace air before and after the normal canned fruits and the canned fruits are insulated, and the commercial sterility of the canned fruits is distinguished according to whether the carbon dioxide level is remarkably changed (the peak area percentage difference exceeds 10 percent) or not, so that the purpose of distinguishing the commercial sterility of the canned fruits is achieved. Compared with the conventional detection method, the method provided by the invention has the advantages that the time is shortened by 5-6 days, and the detection method is used as a commercial sterile primary screening tool for the canned fruits, so that the detection cost can be reduced, and the international trade requirement for rapid development is met.)

1. A detection method for rapidly distinguishing commercial sterility of canned fruits is characterized in that a headspace gas chromatography-mass spectrometry technology is used for detecting the carbon dioxide content in headspace air before and after heat preservation of normal canned fruits and canned rotten fruits, and commercial sterility of canned fruits is distinguished according to whether the carbon dioxide content is remarkably changed or not.

2. The method of claim 1, wherein the determination of whether the carbon dioxide content has changed significantly is based on a peak area percentage difference of more than 10%.

3. The method for detecting the commercial sterility of canned fruit as claimed in claim 1, comprising the steps of:

1) after the package is opened, taking out at least 5mL of contents into a sterilized headspace sample injection bottle by using a sterilization pipette in an aseptic operation, detecting the sealed headspace sample injection bottle before heat preservation, setting the conditions of a headspace automatic sample injector, and extracting 1mL of upper layer gas sample injection by using a headspace needle;

2) opening the package, simultaneously taking out at least 5mL of contents into a sterilized headspace sample injection bottle by using a sterilization suction pipe in an aseptic operation, sealing the headspace bottle, placing at 30-40 ℃ for 1-5 days, performing detection after heat preservation, setting the conditions of a headspace automatic sample injector, and extracting 1mL of upper layer gas sample injection by using a headspace needle;

the carbon dioxide content of the headspace gas before and after the normal fruit can is insulated is not obviously changed, the carbon dioxide content in the headspace gas before and after the putrefactive fruit can is insulated is obviously increased, and the peak area percentage difference exceeds 10%.

Technical Field

The invention relates to a detection method for rapidly distinguishing commercial sterility of canned fruits by utilizing a Headspace gas chromatography-mass spectrometry (HS-GC-MS) technology.

Background

It is important to control spoilage and toxin production caused by microorganisms during the production of canned food. Bad sealing or sterilization can cause the putrefaction and deterioration of the contents or the spoilage of the acid bacteria^[1]. The canned fruit is made up by using fresh fruit as main raw material through the processes ofThe commercial asepsis is achieved through the working procedures of processing, canning, exhausting, sealing, heating sterilization, cooling and the like, and the shelf life of the food is prolonged. The canned fruit belongs to acidic canned food, and if the thermal sterilization is insufficient, the preservation requirement of the food cannot be met, and the canned fruit is rotten and deteriorated due to the growth of spoilage bacteria such as various bacteria, mold, saccharomycetes and the like in an acidic environment, so that the commercial aseptic requirement cannot be met.

In order to standardize the safety of canned food in China, a special detection standard GB 4789.26-2013 'food safety national standard food microbiology inspection commercial sterility inspection' is established for the microbial detection of canned food. The principle of commercial sterility testing in the standard is: the canned food is kept warm at a specific temperature for ten days. During the heat preservation process, microorganisms which are not fully killed in the cans grow and reproduce by utilizing the nutrition of the cans, so that the change of pH value, the generation of gas and the change of product sense (appearance, color, smell and the like) are caused, and obvious microorganism proliferation conditions can be seen through smear dyeing microscopy. The canned food is non-commercial sterile when it is fat or leaked in heat preservation test, abnormal in sensory inspection, obvious in pH value change and obvious in proliferation of smear microscopy microorganism.

China is not only the largest can producing country in the world, but also the largest can exporting country in the world. The commercial sterility test of canned fruit adopts a traditional ten-day heat preservation microbiological test method, the detection period of the method is long, and the method cannot adapt to the rapidly developing trade demand, so that the establishment of a rapid method for detecting the commercial sterility of canned fruit is necessary.

Carbon dioxide is generated by the growth of microorganisms (spoilage bacteria such as bacteria, mold, yeast, etc.), and the generated carbon dioxide expands the container after the fruit can is rotten. Whether the fruit can is commercially sterile is judged by comparing the change of the carbon dioxide level in the air at the top of the fruit can before and after heat preservation: if the carbon dioxide level changes significantly, indicating that microorganisms are propagated in the sample, determining that the sample is non-commercial sterile; commercial sterility was judged if there was no significant change in carbon dioxide levels after the specified number of days. Further validation of non-commercial sterile canned fruit was carried out and recorded as annex B anomaly cause analysis in GB 4789.26-2013.

The method adopts a headspace gas chromatography-mass spectrometry combined method to detect gases such as carbon dioxide in the headspace of the fruit can, and judges whether the canned food is commercial aseptic or not by comparing the level change of the carbon dioxide in the headspace of the canned food before and after heat preservation.

Disclosure of Invention

The invention aims to provide a detection method for rapidly distinguishing commercial sterility of canned fruits, which overcomes the defects in the prior art, thereby providing scientific basis and guidance for food safety.

The main principle of the invention is as follows: detecting the carbon dioxide level in the Headspace air before and after heat preservation in normal canned fruits and canned rotten fruits by using a Headspace gas chromatography-mass spectrometry (HS-GC-MS) technology, and distinguishing the commercial sterility of canned fruits according to whether the carbon dioxide level is changed remarkably or not.

1. Pretreatment of canned fruits

After the package is opened, taking out at least 5mL of contents into a sterilized headspace sample injection bottle by using a sterilization pipette or other appropriate tools in a sterile operation, detecting the capped headspace sample before heat preservation, and extracting 1mL of upper layer gas by using a headspace needle for sample injection; and simultaneously, taking out at least 5mL of contents into a sterilized headspace sample injection bottle by using a sterilizing suction tube or other appropriate tools through aseptic operation, sealing the headspace bottle, standing at 30-40 ℃ for 1-5 days, detecting after heat preservation, setting the conditions of a headspace automatic sample injector, and extracting 1mL of upper layer gas sample injection by using a headspace needle.

2. HS-GC-MS analysis conditions

Headspace (HS) sample injection conditions: the temperature of the heating box is 30-50 ℃, and the quantitative ring temperature: 60 ℃, transmission line temperature: 60 ℃, sample equilibration time: 1-10 min; sample introduction volume: 1 mL; the split ratio is as follows: 70:1.

Gas Chromatography (GC) conditions: chromatography columns (30 m.times.250. mu. m.times.3 μm); sample inlet temperature: 220 ℃; carrier gas: high purity helium (He), flow rate: 1.2 mL/min.

Mass Spectrometry (MS) conditions: the ion source EI, the ion source temperature is 230 ℃, the quadrupole rod temperature is 150 ℃, the electron energy is 70eV, the transmission line temperature is 230 ℃, the full scanning of the collection mode is carried out, and the mass scanning range m/z is as follows: 20-300 u.

3. Air measurement of fruit can top

3.1 injecting the sample into a gas chromatography-mass spectrometer, and setting a chromatographic peak with an automatic identification signal-to-noise ratio of more than 100; and (3) carrying out qualitative analysis on an MS result by using a computer search spectrum library, and calculating by using an area normalization method to obtain the headspace gas content of the fruit can food sample, wherein the similarity of the mass spectrum is more than or equal to 80%. Air background, contents of headspace gas before and after incubation of normal and abnormal fruit cans see examples.

3.2 percentage of headspace gas in the canned food, Yi, calculated according to equation (1), the content of a given component i is calculated by determining the percentage of the corresponding peak area to the sum of the peak areas of all the components:

in the formula:

yi is the percentage of a certain headspace gas in the fruit can to the total headspace gas, and the unit is percentage (%);

As_i-peak area of a headspace gas i in the fruit can;

∑As_i-sum of peak areas of all headspace gas in the fruit can.

The result is a 3-bit significand.

The carbon dioxide content in the headspace gas before and after the heat preservation of the fruit can is not obviously increased, and the result is negative; the carbon dioxide content in the headspace gas before and after the fruit can is insulated is obviously increased (the peak area percentage difference exceeds 10 percent), which indicates that microorganisms propagate, and the result is positive.

The results of instrumental analysis were negative and normal sensory and pH measurements reported commercial sterility.

The result of the instrument analysis is positive, and no microorganism proliferation phenomenon exists through the verification test of national standard GB 4789.26-2013, and the result is reported as commercial sterility.

The result of the instrument analysis is positive, and the microbial proliferation phenomenon is verified by the national standard GB 4789.26-2013, and the result is reported to be non-commercial sterility.

As a primary screening tool for commercial sterility of canned fruits, the detection method shortens the time by 5-6 days compared with the conventional detection method, can reduce the detection cost, and is suitable for the international trade demand of rapid development.

Drawings

FIG. 1 is an air background Total Ion Current (TIC) mass spectrum chromatogram.

FIG. 2 is a mass spectrum chromatogram of Total Ion Current (TIC) of headspace gas before and after incubation of a commercial sterile yellow peach can.

FIG. 3 is a mass spectrum chromatogram of Total Ion Current (TIC) of headspace gas before and after incubation of a non-commercial sterile canned yellow peach.

Fig. 4 is a mass spectrum chromatogram of total headspace gas ion current (TIC) before and after incubation of a commercial sterile strawberry can.

Fig. 5 is a top air Total Ion Current (TIC) mass spectrum chromatogram of a non-commercial sterile strawberry can before and after incubation.

Detailed Description

The following examples further illustrate the invention but are not to be construed as limiting the invention.

Example 1 commercial sterility testing method for yellow peach canned food-headspace gas chromatography-mass spectrometry

(1) Pretreatment of yellow peach can

After the package is opened, taking out at least 5mL of contents into a sterilized headspace sample injection bottle by using a sterilization pipette or other appropriate tools in a sterile operation, detecting the capped headspace sample before heat preservation, and extracting 1mL of upper layer gas by using a headspace needle for sample injection; and simultaneously, taking out at least 5mL of contents into a sterilized headspace sample injection bottle by using a sterilization pipette or other appropriate tools through aseptic operation, sealing the headspace bottle, standing for 4 days at 36 ℃, performing detection after heat preservation, setting the conditions of a headspace automatic sample injector, and extracting 1mL of upper layer gas sample injection by using a headspace needle.

(2) HS-GC-MS analysis conditions

Headspace (HS) sample injection conditions: heating box temperature 36 ℃, quantitative loop temperature: 60 ℃, transmission line temperature: 60 ℃, sample equilibration time: 1 min; sample introduction volume: 1 mL; the split ratio is as follows: 70:1.

Gas Chromatography (GC) conditions: agilent CP7348PT PoraBOND Q PT column (30 m.times.250 μm.times.3 μm) or equivalent; sample inlet temperature: 220 ℃; carrier gas: high purity helium (He), flow rate: 1.2 mL/min.

Mass Spectrometry (MS) conditions: the ion source EI, the ion source temperature is 230 ℃, the quadrupole rod temperature is 150 ℃, the electron energy is 70eV, the transmission line temperature is 230 ℃, the full scanning of the collection mode is carried out, and the mass scanning range m/z is as follows: 20-300 u.

(3) Measuring top air of yellow peach can

3.1 injecting the sample into a gas chromatography-mass spectrometer, and setting a chromatographic peak with an automatic identification signal-to-noise ratio of more than 100; and (3) performing qualitative analysis on an MS result by using a computer search spectrum library, and calculating by using an area normalization method to obtain the content of headspace gas of the yellow peach can sample, wherein the similarity of the mass spectrogram is more than or equal to 80%. The air background, the contents of headspace gas before and after the normal and abnormal yellow peach cans were kept warm are shown in tables 1, 2 and 3.

3.2 percentage of headspace gas in the canned food, Yi, calculated according to equation (1), the content of a given component i is calculated by determining the percentage of the corresponding peak area to the sum of the peak areas of all the components:

in the formula:

yi is the percentage of a certain headspace gas in the yellow peach can to the total headspace gas, and the unit is percentage (%);

As_i-peak area of a headspace gas i in the yellow peach can;

∑As_i-sum of peak areas of all headspace gas in the yellow peach can.

The result is a 3-bit significand.

The carbon dioxide content in the headspace gas is not obviously increased before and after the heat preservation of the yellow peach can, and the result is negative; the carbon dioxide amount in the headspace gas before and after the heat preservation of the canned food is obviously increased (the peak area percentage difference exceeds 10 percent), which indicates that the microorganism is propagated and the result is positive.

The results of instrumental analysis were negative and normal sensory and pH measurements reported commercial sterility.

The result of the instrument analysis is positive, and no microorganism proliferation phenomenon exists through the verification test of national standard GB 4789.26-2013, and the result is reported as commercial sterility.

The result of the instrument analysis is positive, and the microbial proliferation phenomenon is verified by the national standard GB 4789.26-2013, and the result is reported to be non-commercial sterility.

Table 1 air background mass spectrum peak corresponding gas list

5mL of sterile water is added into a sterilized headspace sample introduction bottle, the composition of air background gas is detected after the headspace bottle is sealed, and the Total Ion Current (TIC) mass spectrum chromatogram (figure 1) of the air background is shown, so that the carbon dioxide content of the air background is lower than 2 percent (Table 1).

TABLE 2 gas tabulation of mass spectrum chromatogram peak correspondence before and after heat preservation of commercial sterile canned yellow peach

The national standard method detection shows that the commercial sterile canned yellow peaches are obtained, the content of the canned yellow peaches is 5mL to the sterile headspace sample injection bottle, the chromatogram of the headspace gas Total Ion Current (TIC) mass spectrum (shown in figure 2) before and after heat preservation of the commercial sterile canned yellow peaches shows that the difference of the carbon dioxide content (the difference of the percentage of the peak area) before and after heat preservation is 1.41 percent, the result is negative, and the results are consistent with the national standard method detection method and are commercial sterile (shown in table 2).

TABLE 3 list of corresponding gas in mass spectrum peaks before and after heat preservation of non-commercial sterile canned yellow peach

The national standard method detects that the canned yellow peaches are non-commercial aseptic canned yellow peaches, the content of the canned yellow peaches is 5mL to the sterilized headspace sample injection bottle, mass spectrum chromatograms (figure 3) of headspace gas Total Ion Current (TIC) before and after heat preservation of the non-commercial aseptic canned yellow peaches show that the difference (peak area percentage difference) of the carbon dioxide content before and after heat preservation is 59.8%, the carbon dioxide content is obviously increased, the result is positive, and the method is consistent with the national standard method detection method and is non-commercial aseptic (table 3).

The content of oxygen in food and a container is greatly reduced by filling nitrogen and carbon dioxide mixed gas and the like into the yellow peach can, so that the potential microorganism growth in the container is in a stagnation state, the putrefaction and deterioration of the can are effectively controlled, and the carbon dioxide in the top air of the can is detected. The fruit can is decayed and deteriorated due to air leakage or other reasons, microorganisms are propagated, and the content of carbon dioxide in the headspace air is changed.

Example 2 commercial sterility testing method for strawberry cans-headspace gas chromatography-mass spectrometry

(1) Pretreatment of strawberry can

After the package is opened, taking out at least 5mL of contents into a sterilized headspace sample injection bottle by using a sterilization pipette or other appropriate tools in a sterile operation, detecting the capped headspace sample before heat preservation, and extracting 1mL of upper layer gas by using a headspace needle for sample injection; and simultaneously, taking out at least 5mL of contents into a sterilized headspace sample injection bottle by using a sterilization pipette or other appropriate tools through aseptic operation, sealing the headspace bottle, standing for 4 days at 36 ℃, performing detection after heat preservation, setting the conditions of a headspace automatic sample injector, and extracting 1mL of upper layer gas sample injection by using a headspace needle.

(2) HS-GC-MS analysis conditions

Headspace (HS) sample injection conditions: heating box temperature 36 ℃, quantitative loop temperature: 60 ℃, transmission line temperature: 60 ℃, sample equilibration time: 1 min; sample introduction volume: 1 mL; the split ratio is as follows: 70:1.

Gas Chromatography (GC) conditions: agilent CP7348PT PoraBOND Q PT column (30 m.times.250 μm.times.3 μm) or equivalent; sample inlet temperature: 220 ℃; carrier gas: high purity helium (He), flow rate: 1.2 mL/min.

Mass Spectrometry (MS) conditions: the ion source EI, the ion source temperature is 230 ℃, the quadrupole rod temperature is 150 ℃, the electron energy is 70eV, the transmission line temperature is 230 ℃, the full scanning of the collection mode is carried out, and the mass scanning range m/z is as follows: 20-300 u.

(3) And measuring the air in the top of the strawberry can

3.1 injecting the sample into a gas chromatography-mass spectrometer, and setting a chromatographic peak with an automatic identification signal-to-noise ratio of more than 100; and (3) carrying out qualitative analysis on the MS result by using a computer search spectrum library, and calculating by using an area normalization method to obtain the headspace gas content of the strawberry can sample, wherein the similarity of the mass spectrum is more than or equal to 80%. Air background, contents of headspace gas before and after normal and abnormal strawberry can heat preservation are shown in table 4, table 5 and table 6.

3.2 percentage of headspace gas in the canned food, Yi, calculated according to equation (1), the content of a given component i is calculated by determining the percentage of the corresponding peak area to the sum of the peak areas of all the components:

in the formula:

yi is the percentage of a certain headspace gas in the strawberry can to the total headspace gas, and the unit is percentage (%);

As_i-peak area of a headspace gas i in the strawberry can;

∑As_i-sum of peak areas of all headspace gas in the strawberry can.

The result is a 3-bit significand.

The carbon dioxide content in the headspace gas before and after the heat preservation of the strawberry can is not obviously increased, and the result is negative; the carbon dioxide amount in the headspace gas before and after the heat preservation of the canned food is obviously increased (the peak area percentage difference exceeds 10 percent), which indicates that the microorganism is propagated and the result is positive.

The results of instrumental analysis were negative and normal sensory and pH measurements reported commercial sterility.

The result of the instrument analysis is positive, and no microorganism proliferation phenomenon exists through the verification test of national standard GB 4789.26-2013, and the result is reported as commercial sterility.

The result of the instrument analysis is positive, and the microbial proliferation phenomenon is verified by the national standard GB 4789.26-2013, and the result is reported to be non-commercial sterility.

Table 4 air background mass spectrum peak corresponding gas list

5mL of sterile water is added into the sterilized headspace sample introduction bottle, the composition of air background gas is detected after the headspace bottle is sealed, and the Total Ion Current (TIC) mass spectrum chromatogram (figure 1) of the air background is that the carbon dioxide content of the air background is lower than 2 percent (Table 4).

TABLE 5 gas List corresponding to mass spectrum chromatographic peaks before and after incubation of commercial aseptic strawberry cans

The national standard method is used for detecting that the strawberry can is commercially sterile, the content of the strawberry can is 5mL to the sterilized headspace sample bottle, the chromatogram of the headspace gas Total Ion Current (TIC) mass spectrum (shown in figure 4) before and after heat preservation of the commercially sterile strawberry can is 0.3 percent, the difference of the carbon dioxide content (the difference of the percentage of the peak area) before and after heat preservation is not obviously increased, the result is negative, and the method is consistent with the national standard method and is commercially sterile.

TABLE 6 list of gas corresponding to mass spectrum chromatographic peaks before and after incubation of non-commercial aseptic strawberry cans

The national standard method detects that the strawberry can is non-commercial aseptic, the content of the strawberry can is 5mL to the sterilized headspace sample bottle, the headspace gas Total Ion Current (TIC) mass spectrum chromatograms (figure 5) of the non-commercial aseptic strawberry can before and after heat preservation are shown in the figure, the result is that the difference value of the carbon dioxide content (peak area percentage difference) before and after heat preservation is 54.1%, the carbon dioxide content is obviously increased, the result is positive, and the detection method is consistent with the national standard method and is non-commercial aseptic.

The method is characterized in that the content of oxygen in food and a container is greatly reduced by filling nitrogen and carbon dioxide mixed gas and the like into the strawberry can, so that the growth of potential microorganisms in the container is in a stagnation state, the putrefaction and deterioration of the can are effectively controlled, and the carbon dioxide in the air at the top of the can is detected. The fruit can is decayed and deteriorated due to air leakage or other reasons, microorganisms are propagated, and the content of carbon dioxide in the headspace air is changed.